An isolated dna molecule encoding a glucagon receptor, dna structure, cell line, the method of producing glucagon receptor, an isolated peptide glucagon receptor, an isolated antibody probe, the method of detecting the presence of antagonists of glucagon

 

(57) Abstract:

The invention concerns a glucagon receptor. Proposed an isolated DNA molecule encoding a glucagon receptor or peptide glucagon receptor. DNA design provides the expression of the isolated DNA molecule and is embedded in the cell line COS or KSS. Also, a method of producing glucagon receptor, providing for the cultivation of cell lines COS or KSS and isolation of the target product. The invention also presents an isolated monoclonal antibody that specifically bind to the glucagon receptor and blocks the binding of glucagon to its receptor glucagon. Another aspect of the invention is a method for detection of glucagon antagonist, involving the binding of agonist isolated glucagon receptor. The invention can be used to encourage the selection of glucagon, which is associated with stimulation of glycolysis and glycogenolysis and explore the role of glucagon in such a disease as diabetes. 9 C. and 4 h.p. f-crystals, 5 Il., 8 table.

The technical field

The invention relates in General to receptors on the cell surface and, more specifically, relates to a glucagon receptor.

Interaction of glucagon and insulin is very important to maintain glucose levels within the body. Believe that the imbalance of glucagon or insulin plays a role in various diseases, such as diabetes mellitus and diabetic ketoacidosis. According to one theory, hyperglycemic condition of diabetes can be caused not only by the reduction of glucose utilization (due to reduced insulin levels), but also the excessive formation of glucose due to higher concentrations of glucagon (see Under, "Diabetes and the alpha cell, Diabetes 25:136-151, 1976; Unger and Orci, "the essential role of glucagon in the pathogenesis of diabetes mellitus". Lancet 1:14-16, 1975).

An important factor in the study of glucagon and its role in such diseases as diabetes, is a glucagon receptor, which upon binding to glucagon transfers the signal to the cell, thereby triggering the glycogenolysis (the hydrolysis of glycogen) and glukopovirat intracellular levels of cyclic adenosine monophosphate (camp). In particular the binding of glucagon to its cellular receptor activates adenylate cyclase to produce camp, thereby increasing the intracellular levels of camp. Believe that this increase in intracellular levels of camp results in glycogenolysis and gluconeogenesis and ultimately to improve the education of glucose by the liver (see Unson et al., "Biological Activities of des-HisI[Glu]9Glucagon Amide, a Glucagon Antagonist", Peptides 10:1171-1177, 1989).

However, there have been suggestions about additional ways to stimulate glycogenolysis and gluconeogenesis. In particular, it was reported that glucagon binds to receptors in the membrane of hepatocytes (liver cells), which are connected via a G-protein with phospholipase C. promote this protein causes the breakdown of phosphatidylinositol-4,5-phosphate with the formation of second messengers (intermediaries) Insectivora and 1,2-diacylglycerol (see Wakelam et al. , "Activation of two signal-transduction systems in hepatocytes by glucagon", Nature 323: 68-71, 1986; Unson et al., Peptides 10:1171-1177, 1989; and Pittner and Fain, Btochem. J. 277:371-378, 1991). Stimulation of glucagon metabolism of insectophobia may be an additional way by which glucagon can stimulate glycogenolysis and gluconeogenesis.

This invention describes a receptor (quick summary of the invention

According to one aspect of the present invention proposed a selected DNA molecule encoding a glucagon receptor. Used here, the term "isolated DNA refers to DNA molecules or DNA sequences, which are separated, placed separately from other cellular components. For example, the DNA molecule is isolated if it is separated from other DNA molecules, including other chromosomal sequences with which it is associated natural in the genome and, in particular, free from other structural genes. An isolated DNA molecule can contain 5'- and 3'- noncoding sequence, with whom she associated natural. In one embodiment of the invention the glucagon receptor selected from the group consisting of rat and human glucagon receptor. In another embodiment, the DNA molecule contains a nucleotide sequence SEQ ID 14 from nucleotide 145-nucleotide 1599. In another embodiment, the DNA molecule encodes glucagon receptor containing the amino acid sequence of SEQ ID 15, from methionine, a number of amino acids 1 to threonine, room 485 amino acids. In the following embodiment, the DNA molecule contains a sequence of nucleotides SEQ ID 24, from nucleotide 53 to nucleotide 1486. ED 25, from methionine, a number of amino acids 1 to phenylalanine, room 477 amino acids. Also provided design DNA containing the first segment of DNA encoding a glucagon receptor operatively linked to additional DNA segments required for the expression of the first DNA segment, cell host containing such constructs DNA, and methods for producing glucagon receptor to which the stages of culturing the host cell under conditions conducive to expression of the DNA segment that encodes the receptor for glucagon.

According to another aspect of the invention proposed isolated peptides of the glucagon receptor. According to one variant of the proposed isolated peptide glucagon receptor containing the amino acid sequence of SEQ ID 15, glutamine, amino acids 28 to tyrosine, room 142 amino acids.

According to another aspect of the invention proposed isolated antibodies that specifically bind to the receptors of glucagon. In one embodiment, these antibodies are monoclonal antibodies. In an additional embodiment, provided monoclonal antibodies capable of blocking the binding of glucagon to its receptor glucagon. Also provided hybridoma, baked method of detecting the presence of antagonists of glucagon, providing stage (a) the exposure of the compounds in the presence of agonist with recombinant glucagon receptor glucagon related by the reaction, under conditions and for a time sufficient to cause binding of the compound to the receptor and has been associated response through the pathway metabolism and (b) detecting a decrease in the stimulus response paths caused by binding of the test compound with the receptor, glucagon, compared with the stimulation of the response pathway one agonist of glucagon, and determine from these data the presence of a glucagon antagonist.

In various embodiments of the invention the response is the response of the membrane-bound adenylate cyclase and phase detection provides a measure of the reduction of the production of cyclic AMR in the return path, mediarama the membrane-bound adenylate cyclase. In another embodiment of the invention the response includes a luciferase reporter system.

In another aspect of the present invention provided with the probes of at least 12 nucleotides capable of gibridizatsiya with nucleic acids encoding the receptor for glucagon.

Brief description of drawings

Fig.1 illustrates the structure of a typical receptor glucagon. Following symbols are used: D (the extracellular aminobenzoic domain); ED (effector domain), surrounded by a dotted line; 1ID, the first intracellular loop domain; 2lD, the second intracellular loop domain; 3lD, the third intracellular loop domain; C-ID, carboxykinase intracellular domain; IELD, the first domain of the extracellular loop domain; 2ELD, the second extracellular loop domain; 3ELD, the third extracellular loop domain; TMDI, the first transmembrane domain; D2, the second transmembrane domain; D3, the third transmembrane domain; D4, the fourth transmembrane domain; TMD5, the fifth transmembrane domain; D6, the sixth transmembrane domain and D7, the seventh transmembrane domain.

Fig.2 graphically depicts a hydrophobicity of rat glucagon receptor.

Fig. 3 graphically depicts the binding125I-with glucagon receptor glucagon.

Fig. 4 presents an analysis of Scatchard is Ceptor glucagon with lines above transmembrane domains.

A detailed description of the invention

As indicated above, the present invention provides an isolated DNA molecule encoding a glucagon receptor. Consider that in their natural configuration glucagon receptors exist as membrane-bound proteins consisting of the extracellular aminoanisole domain, as well as several external and internal domains of smaller size (see Fig.1). In the context of this invention the glucagon receptor" refers to proteins and similar derivatives. Derivatives may be allelic variants and genetically engineered variants that contain conservative amino acid substitutions and / or minor additions, substitutions or deletions of amino acids. The glucagon receptor of the present invention are able to bind glucagon and implement the transduction of the signal into the cell. Preferably, the glucagon receptor of the present invention are able to bind glucagon with d 100 nm or less, more preferably 50 nm or less, and most preferably 33 nm or less. Typical tests that can be used to determine the binding of glucagon receptor glucagon, described in more detail in examples 3 and 6.

Typically, signal transduction, PRSA with membrane-bound receptor. Responding to the stimulus path usually induce a cellular response, such as extracellular matrix secretion of susceptible cell lines, hormone secretion, chemotaxis, differentiation, or the initiation or inhibition of cell division susceptible cells. Mate receptors with susceptible to the analyzed signal paths is called here the direct activation of the answering signal path or signal transduction through a second messenger, such as G-protein to activate the path of the cellular response.

Many ways cell response can be used by the glucagon receptor for signal transduction of the binding of glucagon to the cell, including, for example, the path of adenylate cyclase response and reply path intracellular calcium.

Tests determination of adenylate cyclase activity are well known in this area of research, for example, described by Lin et al. (Biochemistry 14: 1559-1563, 1975). This invention also provides a measurement of the biological activity of glucagon receptor on the basis of the concentrations of intracellular calcium (see Grynkiewicz et al., J. Biol.hm. 260:3440-3450, 1985), and by applying luciferase reporter system described in greater detail below. In addition, Biologicheskaya of Insectivora, as described in Subers and Nataanson (J. Mol.Cell.Cardiol. 20: 131-140, 1988) or Pittner and Fain. (Biochem.J. 277:371-378, 1991). It should be noted that in the context of the present invention is not all respond to the signal path must be present in order for the glucagon receptor endured the signal into the cell. For example, some cellular responses, such as increased levels of intracellular calcium may be initiated by the binding of glucagon to its receptor in the absence of camp or Iosifovich signals.

Isolation (separation) of cDNA clones receptor glucagon

As indicated above, the present invention provides an isolated DNA molecule encoding a glucagon receptor. Briefly, genomic or cDNA molecule encoding a glucagon receptor, can be obtained from libraries prepared from cells and tissues according to the procedures described below and in the examples. Cells and tissues that can be used in this invention, can be obtained from a variety of mammals, including, for example, from human, macaque, cattle, pigs, horses, dogs, rats, and mice. Preferred cells and tissues are adipose tissue, kidney, pancreas, heart and liver.

In one and the ü procedures. Briefly, poly(A)+-RNA was isolated from Srgu Dwl rats and used as template for synthesis of cDNA essentially as described umd et al. (Sin 252:1318-1321, 1991), to obtain the full length cDNA. Then the library that contains approximately 1106clones, designed in expressing plasmid in mammalian cells by directional cloning of cDNA larger than 800 N. p. Then the plasmid DNA obtained from pools containing 5000 clones were transfusional in COS-7 cells were selected and grown on slides for the microscope. Transfetsirovannyh cells were analyzed after 72 hours by linking with1251-glucagon followed by the emulsion radiography (McMahan et al., EMBO J. 10:2821-2832, 1931). Positive pools were consistently broken, until it was isolated individual clone. A plasmid obtained from this clone, designated as LJ4, contains approximately 2.0, etc., ad insertions, which encodes a protein of 485 amino acids with the predicted mol. mass 54962 daltons (see SEQ ID 15).

In other aspects of the present invention are provided methods of isolation and cloning of the human glucagon receptor. Many technologies can be used to provided here way, including, capriglione (example 4), which can then be used to identify libraries that contain sequences encoding human glucagon receptor with subsequent cloned this receptor (example 5). Especially preferred strategies for cloning of the human glucagon receptor are the strategies described in examples 4 and 5. Alternative expressing a library of human cDNA, can be obtained from the appropriate sources RNA as described in example 1 and skanirovaniya as described in example 3 for clones expressing functional receptors for glucagon.

The preparation of recombinant glucagon receptor

This invention provides a preparation of recombinant glucagon receptor by culturing host cells containing the construct DNA containing the first segment of DNA encoding a glucagon receptor, operatively connected to additional DNA segments required for the expression of the first DNA segment. As noted earlier, in the context of the present invention under the glucagon receptor imply and their derivatives, largely similar to the receptors. In addition, receptors for glucagon can be encoded sequences is a RAM similar", if: (a) the DNA sequence produced from the coding region of the native gene receptor glucagon (including, for example, allelic variations of the following sequences); (b) the DNA sequence is capable of gibridizatsiya with DNA sequences of the present invention under high or low stringency (see Sambrook et al., lulr Cloning: A Lbrtr nul, 2d Ed., ld Sring rbr Laboratory rss, V, 1989); or (c) DNA sequences are degenerate as a result of degeneracy of the genetic code to the DNA sequences described in (a) or (b).

Mutations in nucleotide sequences constructed for expression of the variant receptor glucagon should keep reading frame of the coding sequences. In addition, mutations preferably should not create complementary regions that could gibridizatsiya with the formation of secondary mRNA structures such as loops or hairpins, which can adversely affect translation of the mRNA of the receptor. Although the site of the mutation can be given, not necessarily that the nature of the mutation per Se need to be pre-defined. For example, for selection of the optimum characteristics of mutants at a given site mutagenesis can PRT.

Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. After ligating the resulting sequence encodes a derivative having the desired amino acid insertions, replacement or division.

Alternative treatments oligonucleotides sitespecific mutagenesis can be used to ensure that the modified gene that has changed certain codons in accordance with the desired substitution, deletion or insertion. Examples of making the above changes are described Walder et al. (Depe 42:133-1986); Bauer et al. (Depe 37:73, 1985); Craik (SIV Techniques, Jnury 1985, 12-19); Smith et al. (Gnti Advenged: rinils and Methods, Plenum Press, 191); and Smbrook et al. (Supra).

The primary amino acid structure of the glucagon receptor may also be modified by formation of a covalent or aggregate conjugates with other chemical parts of the molecule, such as glucosamine groups, lipids, phosphate, acetyl groups or with other proteins or polypeptides. In the following embodiment, the glucagon receptor may be fused with draino get in the form of a fused protein with FLAG-polypeptide sequence (see U. S. tnt 4851341; see also Knorr et al., Bio/Technology 6:1204, 1988). FLAG polypeptide sequence has a high antigenicity and provides an epitope to bind specific monoclonal antibody, enabling rapid purification of expressed recombinant protein. This sequence is also specifically cleaved by enterokinase bullish mucosa at rest, following directly after a couple s-Lys. For convenience can be prepared numerous design DNA comprising the complete sequence or portion of sequence of a native or variant receptor glucagon discussed above. In the context of this invention the structure of DNA is called DNA molecule, or a clone of such a molecule (single-stranded or double-stranded), which was modified so that it contains segments of DNA combined and placed in such a way that the molecule is formed, which did not exist in nature. The design DNA of the present invention contain a first DNA segment encoding a glucagon receptor, operatively connected to additional DNA segments required for the expression of the first DNA segment. In the context of this invention Eome, to contain enhancers and other elements.

Design DNA, also known as expressing the vectors may also contain segments of DNA that is necessary to control the secretion of the target polypeptide. These segments of DNA can contain at least one secretory signal sequence. Preferred secretory signals are secretory signal sequence of glucagon (pre-proposedvalue), signal sequence of the alpha-factor pre-proposedvalue; Kurian and Herskowitz, Cell 30:933-943, 1982; Kurian et al. , U. S. tnt 4546082; Brake, EP 116 201), signal sequence RN (VESK et al., WO 86/00637), the secretory signal sequence BAR I Maskow et al., U. S. tnt 4613572; MacKay WO 87/002670), signal sequence, SUC2 (Carlson et al., Mol. Cell. Biol. 3:439-447, 1983), signal sequence-1-antitrypsin (urhi et al., The OEWG. Ntl. Acad. Sci. USA 78:6826-6830, 1981), signal sequence of an inhibitor of plasmin -2 (Top et al., J. Biochem. (Tokyo) 102:1033-1042, 1987), signal sequence of tissue plasminogen activator (Ripps et al. , Nature 301:214-221, 1983) the signal sequence of the PhoA E. Li (Weap et al. , J. Biol. hem. 265:13528-13552, 1990), or any of the bacterial signal sequences, an overview of which is given ü synthesized according to the rules, for example, Heinje (Eur. J. Biochem. 133:17-21, 1983; J. Mol. Biol. 184: 99-105, 1985; Nuc. Acids Res. 14:4683-4690, 1986).

The secretory signal sequence may be used individually or can be combined. For example, the first secretory signal sequence can be used with a sequence encoding the third domain Wagger (described in U. S. tnt 5037243, to which full reference is given). The sequence encoding the third domain Wagger can be placed in the appropriate reading frame 3' of the target DNA sequence or 5' with respect to a segment of DNA in the appropriate reading frame as the secretory signal sequence and the target DNA segment.

For the expression of a DNA molecule encoding a glucagon receptor is inserted into a suitable design DNA, which in turn is used for the transformation or transfection of suitable host cells for expression. Cell hosts for use in the practice of this invention are cells of mammals, birds, plants, insects, bacterial and fungal cells. Preferred eukaryotic cells are cultured cell line pleocytosis (for example, cell lines rodents Il or Kluyveromyces spp.) or filamentous fungi (e.g., Aspergillus spp., Neurospora spp.). Particularly preferred strains of the yeast Saccharomyces cerevisiae. Methods of production of recombinant proteins in many prokaryotic and eukaryotic host cells known to specialists in this field (see "Depe Expression Technology, Methods in nzymlg, Vol. 185, Goeddel (ed.), dmic Press, San Diego, Calif., 1990; see also "Guide to Yst Genetics and Molecular ilgy", Methodk in Enzyrmology, Guthrie and Fink (ed.) Academic Press, Sn Diego, Calif., 1991). Typically, the cell host is chosen based on its ability to produce the target protein at a high level or on the basis of its ability to carry out at least some of the stages of processing that are required for biological activity of this protein. In this way the number of cloned DNA sequences, which must be transfetsirovannyh in a cage-the owner, can be minimized, and the overall yield of biologically active protein can be optimized.

Suitable yeast vectors for use in this invention are vectors YR7 (Struhl et al., The OEWG. Ntl. Acad. Sci. USA 76:1035-1039, 1978), War (rh et al., Gene 8:121-133, 1979), POT (Kawasaki et al. , U. S. Patent 4931373, which is incorporated herein by reference) pJDB249 and pJD219 (Beggs, Nature 275:104-108, 1978) and their derivatives. Such vectors typically contain a breeding marker, which can be dnopause conduct the selection of transformants. Preferred are such breeding markers that complement auxotrophic host cell, provide antibiotic resistance or make the cell is able to utilize specific carbon sources, and include LU2 (Broach et al., ibid.), UR 3 (Botstein et al., Gene 8:17, 1979), HIS 3 (Struhl et al., ibid.) or NOT (wsi et al., ibid.). Other suitable breeding marker is the CAT gene, which tells yeast cells resistant to chloramphenicol.

Preferred promoters for use in yeast include promoters from yeast glycolytic genes (it Zeman et al., J. Biol. hm. 255: 12073-12080, 1980; lbl and wsi, J. Japan. l. Gnt. 1:419-434, 1982; wsi., U. S. Patent 4599311) or alcohol dehydrogenase genes (Young et al. in Genetic Engineering of Microorganisms of hmil, Hollaender et al., (ads. ), p.355, Plenum, Nw Wagc, 1982; Amegah, Meth. nzymol. 101:192-201, 1983). In this regard, particularly preferred promoters are TPLI promoter (Kawasaki, U. S. tnt 4599311, 1986) and ADH2-4Cthe promoter (Pussel et al. , Nature 304:652-654, 1983; Irani and Kilgore, U. S. tnt lition Serial 07/764653, which is incorporated herein by reference). Units of expression may also contain a transcription terminator. It is preferable TPLI transcription terminator (Alber and Kawasaki, ibid.).

In addition to yeast proteins of the present invention is in the references). Examples of applicable promoters are promoters produced from glycolytic genes of Aspergillus nidulans, such as D3 promoter (night et al., EMBO J. 4:2093-2099, 1989) and the tpiA promoter. An example of a suitable terminator is D3 terminator (Kight et al., 1985). Units of expression, using such components, cloned into a vector capable of integration into the chromosomal DNA of Aspergillus.

Methods of transformation of fungi are well known in the literature and are described, for example, Beggs (ibid.), innt et al., (OEWG.Ntl. Acad. Si. USA 75: 1929-1933, 1978) Yltn et al., (OEWG. Ntl. d. Sci USA 81:1740-1747, 1984) and Russel (Ntur 301:167-169, 1983). The genotype of the host cell usually contains a genetic defect, which is complemented by a breeding marker present on expressing vector. The choice of a specific host and breeding token accessible to people with normal skills in this area. In order to optimize production of heterologous proteins in yeast, for example, it is preferable that the host strain carrying a mutation, such as RER mutation of yeast (Jones, Genetics 85:23-33, 1977), leading to reduced proteolytic activity.

In addition to the fungal cells in this invention as the host cells can be used cultured cells mlda cell line COS-I (ATSS CRL 1650), COS-7 (ATSS CRL 1651), KSS (ATSS RL 1632) and 293 (ATSS RL 1573; Graham et al., J. Dept. Virol. 36:59-72, 1977). The preferred cell line KSS is a cell line KSS 570 (deposited in ATSS under Accession CRL 10314). In addition, a number of other cell lines mammals can be used in this invention, including cell lines Rat ner I (ATSS CRL 1600), Rat Hep II (ATSS RL 1548), TCMK ADS CCL 139), Human lung (lung cells) (ATSS CCL 75.1), Human hepatoma cells human hepatoma) (ATSS NTV-52), ner G2 (ATSS HB 8065). Mouse liver (liver cells of mice) (ATSS CL 29.1), N STS 1469 (ATSS CCL 9.1), SP2/O-Ag-14 (ATSS 1581), HIT-T15 (ATSS CRL 1777) and RINm 5AHT2B (Orskov and Nielson, FEBS 229 (1): 175-178, 1988).

Expressing the vectors mammals for use in this invention contain a promoter capable of directing transcription of a cloned gene or cDNA. Preferred promoters are viral promoters, cellular promoters. Viral promoters are the early promoter of cytomegalovirus (Boshart et al., ll 41:521-530, 1985) and the SV40 promoter (Subramani et al., Mol. ll. iol. I: 854-864, 1981). Cellular promoters include the mouse promoter metallothionein-1 (Palmiter et al., U. S. Patent 4579821), murine Vkthe promoter (Vegmap et al., The OEWG. Ntl. Acad. Sci. USA 81: 7041-7045, 1983; Grnt et al., Nu. Acids Res. 15:5496, 1987) and mouse VHis adenovirus 2 (ufmn and Shr, Japan. ll. il. 2:1304-1319, 1982). Such expressing vectors can also contain a number of sites of RNA splicing, localized in the direction of 5' --> 3' from the promoter and 3' -- > 5' DNA sequence encoding the target peptide or protein. Preferred sites of RNA splicing can be obtained from the genes of adenovirus and (or) of immunoglobulin genes. In expressing vectors is also a signal polyadenylic simulation, located towards the 5' --> 3' from the coding target sequence. Suitable polyadenylation signals are early or late polyadenylation signals from SV40 (ufmn and Sharp, ibid.), the polyadenylation signal from EV region of adenovirus and the terminator gene of human growth hormone (DeNoto et al., Ni. Acids Res. 9:3719-3730, 1981). Expressing the vectors may contain non-coding viral leader sequence, such as consisting of three parts, the leader of the adenovirus 2, located between the promoter and the sites of RNA splicing. Preferred vectors may also include enhancer sequences, such as the SV40 enhancer and enhancer mice (Gillies, Cll 33:717-728, 1983). Expressing the vectors may also contain sequences encoding RIVERS adenovirus VA. Suitable vectors of the successive DNA can be introduced into cultured mammalian cells, for example, by mediaremote calcium phosphate transfection (Wigler et al., ll 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603, 1981; Graham and Vn der Eb, Virology 52:456, 1973), by electroporation (Numnn et al., EMBO J. 1:841-845, 1982), or by mediawindow DEAE-dextran transfection (Ausubel et al., (eds.), Currnt Protocols in lulr ilgy, Jhn Wily and Sons, Inc., NY, 1987), which are incorporated by reference. To identify cells, stably integrated cloned DNA, usually injected into the cells of breeding token together with the target gene or cDNA. Preferred breeding markers for use in cultured mammalian cells are genes that give the cells a resistance to drugs, such as neomycin, hygromycin and methotrexate. Breeding marker may be amplificare breeding marker. Preferred amplificatoare breeding markers are gene DHRR and the gene of resistance to neomycin. Breeding markers considered Thily (Mammalian Cell Technology, Butterworth ublishers, Stoneham, MA, which is included in the references). The choice of breeding markers readily available at the level of ordinary skill in this field.

Breeding markers can be introduced into the cell on a separate vector simultaneously with the serial the breeding marker and the sequence of the glucagon receptor may be under control of different promoters or the same promoter, the latter option will give dicistronic mRNA. The design of this type known in the art (for example, Lvinsn and Simonsen, U. S. tnt 4713-339). Preferred is the addition of extra DNA, known as DNA carrier, to the mixture, which is introduced into the cell.

Transfitsirovannykh the mammalian cells to grow during the period of time is usually 1-2 days, after which they begin to Express the target sequence (target sequence) DNA. Then apply the selection using drugs for selection in the growth of cells that Express stable breeding marker. For cells transfected amplificare breeding marker, the concentration of drugs can be increased stepwise to selection for increased number of copies of the cloned sequences, i.e., to increase the level of expression. Cells expressing the introduced sequence is selected and subjected to screening for the production of the target protein in the desired form and at the desired level. Cells that meet these criteria, can then be cloned and evaluated for performance.

Preferred prokaryotic cells-hosts for the AMB Baccillus and other genera. The technique of transforming these hosts and expression of foreign DNA sequences are well known in this area of research (see for example Maniatis et al., Molecular lning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982, which is included in the reference; or Smbrk et al., sur). The vectors used for the expression of cloned DNA sequences in bacterial hosts, usually contain breeding marker such as a gene for resistance to the antibiotic, and a promoter that functions in the cell host. Preferred promoters are the promoter of the trp system (Nichols and Yanofsky, Meth. Enzymol. 101: 155-164, 1983), lac (Casadaban et al., J. Bacteriol. 143:971-980, 1980), and phage (Qun, J. Mol. l. Genet 2:1-10, 1983). The plasmid used for transformation of bacteria are PBP 322 (Bolivar et al., Gene 2:95-113, 1977), rice (Messing, Meth. nzyml. 101:20-78, 1983; Viir and Messing, Depe 19:259-268, 1982), pCQV2 (Oaep, ibid.) and their derivatives. Plasmids can contain both viral and bacterial elements. After the data here explanations promoters, terminators and methods introduction expressing vectors encoding a glucagon receptor of the present invention in cells of plants, birds, insects, will be clear to experts in the field. The use of baculoviruses, for example, as vectors for the heterologous expression of th the e grbtriumrhizogenes as vectors for gene expression in plant cells was considered in the review Sinkar et al. (J. isi. (nglr) 11:47-58, 1987).

Cell host containing the design DNA of the present invention, cultured for expression of the DNA segment that encodes the receptor for glucagon. Cells were cultured according to standard methods in culture medium containing nutrients required for growth of the selected host cells. There are many suitable environments, which typically contain a carbon source, a nitrogen source, essential amino acids, vitamins and mineral salts, as well as other components such as growth factors or serum that may be needed for certain host cells. Growth environment will be discriminates cells containing the design (design) DNA, for example, by means of selective application of the drug or selection when failure of the primary nutrient that is complemented by a breeding marker on the structure of DNA or other vector, cotransfected together with design DNA.

Suitable conditions for the growth of yeast cells include, for example, culturing in a chemically defined medium containing a nitrogen source, which may be different than the amino acid, or yeast extract, is preferably at 30oC. the pH of the environment support preferably more than 2 and less than 8, more preferably 5-6. Ways to maintain a stable pH include the use of buffers and constant pH control. A preferred agent for pH control is sodium hydroxide. Preferred buffering agents are succinic acid and Bis-Tris (Sigma Chemical Co., St.Louis, MO). Due to the tendency of yeast host cells to hyperglycosylated heterologous proteins, it may be preferable to Express the glucagon receptor of the present invention in yeast cells having a defect in a gene required for the associated with asparagine glycosylation. These cells preferentially grow in a medium containing an osmotic stabilizer. Preferred osmotic stabilizer is sorbitol added to the medium at concentrations between 0.1 M and 1.5 M, preferably 0.5 M or 1.0 M Cultured mammalian cells are typically cultured in commercially available serum and serum-free media. The choice of medium and growth conditions suitable for a particular cell line, is within the knowledge of specialists of ordinary skill in this field.

The glucagon receptor may also expresslane transgenic animals, including mice, rats, rabbits, sheep and pigs, known in the art and described, for example, Hammer et al. (Nature 315:680-683, 1985), Palmiter et al. (Science 222:809-814, 1983), rinster et al. (OEWG. Ntl. Acad. Sci. USA 82:4438-4442, 1985), Palmiter and Brinster (Cell 41:343-345, 1985) and U. S. Patent 4736866, incorporated herein by reference. Briefly, the unit expression that contains the DNA sequence that you want to Express, together with the properly placed controls the expression sequences, is introduced into the pronuclei of fertilized oocytes. Introduction DNA is usually performed by microinjection. Integration inyecciones DNA detected by blotting DNA from tissue samples, usually samples of fabric tails. Preferably, the introduced DNA was incorporated into the germ line of the animal, so it was passed down to the offspring of the animal.

In a preferred embodiment of the invention the transgenic animal, such as a mouse, was created by targeted mutations to destroy the sequence of the glucagon receptor (see Mansour et al., "Disruption of th rtngn int-2 in mus em bryo-derived stecells: a general strateqy for targeting mutations to non-selectable genes", Nature 336:348-352, 1938). These animals can easily be used as a model to study the role of glucagon receptor in metabolism.

Peptization. In the context of this invention, the peptides of the glucagon receptor contain parts of the receptor glucagon or its derivative, discussed above, which do not contain transmembrane domains and have a length of at least 10 amino acids. Briefly, the structure of the glucagon receptor, as well as possible transmembrane domains can be predicted from the primary products broadcast using hydrophopicity plot function, for example, P/S Depe or Intelligenetics Suite. (Intelligenetics, t. Viw, CA) or in accordance with the methods described by Kyte and Dlittl (J. Japan. il. 157:105-132, 1962). Schedule hydrophobicity of rat glucagon receptor is depicted in Fig.2. Believe that the glucagon receptor have the structure shown in Fig.1. In particular believe that these receptors containing the extracellular aminobenzoic domain, three extracellular domain in the form of loops and four intracellular domain in the form of loops separated transmembrane domains.

In one aspect of the present invention is provided with the selected peptide glucagon receptor containing the extracellular aminobenzoic domain of the receptor. In the preferred embodiment is provided with the selected peptide glucagon receptor containing the amino acid sequence of SEQ ID 15, glutamine, room 18 amino acids, which may be selected from extracellular and intracellular domains in the form of loops of the receptor glucagon (see Fig. 1 and 5). In one embodiment, the peptides of the glucagon receptor selected from the group consisting of 1 ID (SEQ ID 15, lysine, room 169 amino acids to histidine, room 178 amino acids), 1ELD (SEQ ID 15, tyrosine, number of amino acids 203 to isoleucine, room 231 amino acids), 2 ID (SEQ ID 15, phenylalanine, room 259 amino acids, to serine, room 266 amino acids), 2LD (SEQ ID 15, valine, the number of amino acids 293 to isoleucine, room 307 amino acids), 3 ID (SEQ ID 15, leucine, room 334 amino acids to lysine, number of amino acids 345), and 3ELD (SEQ ID 15, aspartic acid, room 371 amino acids, to serine, room 380 amino acids).

Peptide glucagon receptor of the present invention can be produced using recombinant techniques, as discussed above, or synthetic methods and can be further purified as described below.

Purification of the peptides of the glucagon receptor

Isolated peptides of the glucagon receptor can be obtained, among other methods, the cultivation of suitable systems owner/vector to obtain a recombinant products broadcast of this invention. Supernatant of these cell lines can then be processed using various cleaning procedures for the selection of peptide receptor glucantime proteins, such as Amicon or Millir lln ultrafiltration unit. After concentration, the concentrate can be applied to suitable cleansing matrix, such as, for example, antibodies against the receptor for glucagon or glucagon associated with a suitable carrier. An alternative can be used for purification of the receptor or peptide anyone - or cation-exchange resin. Finally, stage liquid chromatography high-resolution reversed-phase (RP-HPLC) can be used for further purification of the peptide glucagon receptor.

Peptide receptor glucagon is considered "isolated or purified in the context of this invention, if it detects only one lane in the analysis in PAG-ordinator, followed by staining using Kumasi brilliant blue.

Antibodies to receptors of glucagon

In one aspect of the present invention the glucagon receptor, including their derivatives, as well as portions or fragments of these proteins, such as discussed above, the peptides of the glucagon receptor, can be used to produce antibodies specifically bind to the receptors of glucagon. In the context of this invention the term "antibody" encompasses polyclonal antibodies, monoclonal anti These binding partners include variable regions of the gene, encoding the specific binding of monoclonal antibody. Antibodies believe specifically bind if they bind to the receptor glucagon with Kagreater than or equal to 107M-1. The affinity of monoclinal antibodies or binding partner can be easily determined by the expert in this area (see Scatcharcd, Ann. N. Y. Acad. Sci. 51:660-672, 1949).

Polyclonal antibodies can be obtained easily qualified from many warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice or rats. Briefly, the receptor for glucagon is used to immunize the animal through intraperitoneal, intramuscular, intraocular, or subcutaneous injections. The immunogenicity of the glucagon receptor or peptide glucagon receptor may be increased by the use of adjuvant, such as beta-blockers or incomplete adjuvant. After repeated immunization take a small serum samples and test them on the reactivity with the glucagon receptor. You can use a variety of tests to detect antibodies that specifically bind to the glucagon receptor. Examples of tests are described in detail in Antibodies: A Laboratory Manual, Harbow and Lane (eds.), Cold Sping Harbor Laboratory , is radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot-blot tests, inhibitory or competitive tests and "sandwich"tests (see. U. S. tnt 4376110 and 4486530; see also Antibodies: A Laboratory Manual, supra). Particularly preferred polyclonal antisera will give a signal that is at least three times the background. Once the titer of the animal reaches a plateau from the viewpoint of the reactivity of the antibodies to the receptor glucagon, large quantities of polyclonal antibodies can be obtained easily either weekly removing blood or after slaughter of the animal.

Monoclonal antibodies can also be easily obtained using well known methods (see U. S. Parent RE 32001, 4902614, 4543439 and 4411993; see also nlnl Antibodies, Hybritdomas: A New Dimension in Biological Analyses, Plenum Press, Kennet, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harobor Laboratory Press. 1988). Briefly, in one embodiment, an animal, such as rat or mouse is injected form of the glucagon receptor, suitable for generating an immune response against glucagon receptor. Typical examples of suitable forms include, among others, are cells that Express the receptor for glucagon, or peptides,

based on the sequence of the glucagon receptor. receptor or peptide of the receptor with another protein, such as ovalbumin or hemocyanin fissurella (KLH), or by application of adjuvants, such as complete or incomplete beta-blockers. The initial immunization may be intraperitoneal, intramuscular, intraocular, or subcutaneous.

Between one and three weeks after the initial immunization the animal can be remunerat re-immunization. Then the blood and serum of the animal tested for binding to the glucagon receptor by using the above-described tests. Additional immunizations may be held up until the animal will not leave the plateau on its reactivity with the glucagon receptor. Then give the animal the final injection of glucagon receptor or peptide receptor glucagon and 3-4 days to kill. At this time the animal take the spleen and lymph nodes and crushed in cell suspension conducting bodies through a sieve or destruction of the membranes of the spleen or lymph nodes surrounding cells. In one embodiment, the red blood cells then are lysed by adding hypotonic solution with subsequent immediate return to isotonicity.

In another embodiment, suitable for obtaining monoclonal antibodies cells with the help of technology immuniza ucaut suspension of individual cells, and these cells are placed in culture, which contains a form of glucagon receptor, suitable for generating an immune response as described above. Then collect the lymphocytes and merge, as described above.

Cells obtained with the use of immunization or immunized animal, as described above, can be immortality by transfection with a virus, such as Epstein-Barr (EBV) (see Glasky and Reading, ybridm 8 (4): 377-389, 1989). Alternatively, in a preferred embodiment, the cell suspension collected spleen and (or) lymph nodes were merged with suitable myeloma cells to obtain "hybridoma", secreting monoclonal antibodies. Suitable myeloma lines are preferably defective in design or expression of antibodies lines, which, moreover, are syngeneic with cells from the immunized animal. Known many such myeloma cell lines, and they can be obtained, for example, from American Type Culture Collection (ATCC), Rockville, Maryland (see Catalogue of Cell Lines and Hybridomas, 6-th ad., ATCC, 1938). Typical myeloma lines are: for a man of IP 729-6 (ATCC RL 8061), MC/CAR-Z2 (ATCC CRL 8147) and SKO-007 (ATCC CRL 8033); for mouse SP2/O-Ag 14 (ATCC CRL 1581) and Rad 8 (ATCC TIB 9); and in rats, Y3-AD1.2.3 (ATCC CRL 1631) and Y2/0 (ATCC CRL 1662). Especially preferred are merged Fox or person. Fusion between a myeloma cell line and cells from immunized animals can be performed in various ways, including the use of polyethylene glycol (ED) (see Antibodies: A Laboratory Manual, rlw and Lane (eds.), Cold Spring rbr Laboratory Press, 1988), or electroline (see Zimmerman and Vienken, J. Membrane Biol. 67:165-182, 1982).

After fusion, the cells are placed in a culture Cup containing a suitable environment, such as RPM1 1640 or DMEM (modified by way of Dulbecco Wednesday Needle) (JRH Biosciences, Lenexa, Kan.). This medium may also contain additional ingredients, such as fetal bovine serum (FBS, i.e., from Hyclone, Logan, Utah or JR Bioscience), thymocytes, which can be taken from the baby animal of the same species used for immunization, or agar for solidification of the medium. In addition, the environment must contain a reagent that selectively allow us to grow spleen cells and myeloma. In particular, it is preferable to apply the HAT (gipoksantin, aminopterin and thymidine) (Sigma Chemical Co., St. Louis, Missouri). After approximately 7 days the obtained fused cells or hybridoma can be subjected to screening to determine the presence of antibodies that recognize the receptor for glucagon. Following several clonal dilutions and re-tests can be other ways to construct monoclonal antibodies (see William D. Hues et al., "Generation of and Lrg Combinational Library of the Immunoglobulin Repertoire in Phage Lambda", Sin 246: 1275-1281, December 1989; see also L. Sastry et al., "Cloning of the Immunological Repertorie in Escherichia Li for Generation of Monoclonal Catalytic Antibodies: Construction of a Heavy Chain Variable Region-Specific with DNA Library," Proc. Natl. Acad. Sci. USA 86:5728-5732, August 1989; see also Michelle Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas", Strategies in Molecular Biology 3: 1-9, January 1990; these references describe a commercial system available from Stratacyte, La Jolla, California, which produces antibodies using recombinant methods). Briefly, mRNA is isolated from the population of b-cells and is used to create gene-expression cDNA libraries of the heavy and light chains of immunoglobulin in the vectors IMMUNOZAP(H) and IMMUNOZAP(L). These vectors can be skanirovat individually or coexpression with the formation of Fab fragments or antibodies (see. Huse et al., supra; see also Sastry et al., supra). Positive stains can then be turned into neoliticheskoy plasmid, which makes possible a high level of expression of fragments of the monoclonal antibodies from E. Li.

This way can be constructed binding partners using techniques of recombinant DNA to enable the variable regions of the gene encoding the specific binding of antibodies. The design of these lymerase Chain Rtin Using Mixed Primers: Cloning of Nimap nlnal Antibody Variable Region Genes From Single Hybridoma Cells, Biotechnology 7:934-938, September 1989; Riechmann et al., "Reshaping Human Antibodies for Therapy", Nature 332:323-327, 1988; Roberts et al., "Generation of an Antibody with Enhanced Affinity and Specificity for its Antigen by Protein Engineering, Nature 328:731-734, 1987; Verhoeyen et al., "Reshaping Human Antibodies: Grafting an Antilysozyme Activity," Science 239:1534-1536, 1988; Director et al., "A Recombinant Immunotoxin Consisting of Two Antibody Variable Domains fused to Preudomonas Exotoxin", Nature 339:394-397, 1989; see also U. S. Patent 5132405 called "Biosynthetic Antibody Binding Sites"). Briefly, in one embodiment, the segments DICK encoding antigennegative domains specific for glucagon receptor, amplified from hybridomas producing specific binding receptor monoclonal antibodies and injected directly into the genome of cells producing human antibodies (see Verhoeyen et al., supra; see also Rihmn et al., supra). This technique allows you to transfer antigennegative site having a specific binding of rat or mouse antibodies in the antibody man. Such antibodies are preferred for therapeutic applications in man, because they have a lower antigenicity than the rat or mouse antibodies.

Alternative antigennegative site (variable region) can either be connected to another, completely different protein, or incorporated into such protein that lead to the formation of a new protein is to elistan, antigennegative site or binding domain of the receptor glucagon these antibodies can be detected in the variable region of antibodies. In addition, the DNA sequence encoding a small portion of these antibodies or variable regions that specifically bind to the receptor glucagon mammals, can also be applied in the context of the present invention. These parts can easily be tested for specificity of binding by using the following tests.

In a preferred embodiment, the genes encoding the variable region of hybridoma producing targeted monoclonal antibody, amplified using oligonucleotide primers for the variable region. These primers can be synthesized by experts or can be purchased from commercially available sources. Stratacyte (La Jolla, lif.) sends the primers for mouse and human variable regions including primers for VLand CLareas. These primers can be applied for amplification of variable regions of heavy and light chains, which can then be incorporated into vectors, such as IMMUNOZAP* (H) or IMMUNOZAP(L)* (Stratacyte), respectively. Then these vectors can be introduced into E. Li for expression. UB> and VLdomains (see Bird et. al., Science 242:423-426, 1988).

In other embodiments, the binding partner is poured inside expressing vector with another protein, such as a toxin. Cells connected in such a binding partner, can be killed as a result of the inclusion of the toxin (see Director et al., supra). After obtaining the appropriate antibodies or binding partners, you can isolate or purify various well-known methods (see Antibodies: A Laboratory Manual, supra). Such methods are peptide or protein affinity columns, HPLC or RP-HRLC, purification on columns with A - or G-proteins, or a combination of these methods. In the context of this invention, the term "isolated" refers to antibodies or binding partners, generally do not contain other blood components.

Antibodies and binding partners of the present invention have many applications. For example, they can be used in flow cytometry for sorting (sort) bearing the glucagon receptor cells or for histochemical staining of tissues bearing the glucagon receptor. Briefly, in order to detect the glucagon receptor on the cells, cells are incubated with the labeled monoclonal antibody which specifically binds to receivedit with additional stages, such as washing to remove unbound antibodies. Labels suitable for use in this invention are well known labels such as fluoresceinisothiocyanate (FI), phycoerythrin (RE), horseradish peroxidase (HRP) and colloidal gold. In flow cytometry is particularly preferred FI, which can be anywhereman with purified antibody according to the method Keltkamp in "Conjigation of Fluorescein Isothiocyanate to Antibodies. I. Experiments of the Conditions of Conjuhation, Immunology 18:865-873, 1970. (See also Ketkamp, "Conjugation of Fluorescein Isothiocyanate to Antibodies. II. A Reproducible Method", Immunology 18:875-881, 1970; and Goding, "Conjugation of Antibodies with Fluorochromes: Modification to the standard Methods", J. Immunol. Methods 13:215-226, 1970). For histochemical staining preferred R, which can be conjugated with purified antibody according to the method of the Nn and Kawaoi ("Buffer-Labeled ntibd: Nw Method of njugtion", J. isthem. Cytochem. 22:1084-1091, 1974; see also Tijssen and urst, "Highly ffiit and Simple Methods of rrtin of Peroxiodase and Active Buffer Antibody Conjugates for Enzyme Immunoassays", Anal. Biochem. 136:451-457, 1984).

In addition, purified antibodies or binding partners may also be used therapeutically to block the binding of glucagon to its receptor glucagon in vitro and in vivo. Briefly, blocking antibodies are those antibodies that bind to epitopes rect action of glucagon on signal transduction. As noted above, you can use a variety of tests to detect antibodies that block or inhibit the binding of glucagon to its receptor glucagon, including inter alia, inhibitory and competitive tests mentioned above. In one embodiment, monoclonal antibodies (obtained as described above) are tested for binding to the glucagon receptor in the absence of glucagon, as well as in the presence of different concentrations of glucagon. Blocking antibodies or binding partners are those that, for example, bind to the glucagon receptor and in the presence of glucagon block or inhibit the binding of glucagon to its receptor glucagon.

Antibodies or binding partners, which should be applied therapeutically, preferably provide in the form of a therapeutic composition comprising the antibody, or binding partner and a physiologically acceptable carrier or diluent. Suitable carriers or diluents among others are buffered to neutral pH saline solution or saline solution, which may also contain additional excipients or stabilizers, such as buffers, sugars such as glucose, sucrose or dextrose, gelatinous, this invention provides methods of detecting antagonists of glucagon. In the context of this invention, the antagonist is called a molecule able to bind to the receptor but do not stimulate or reduce stimulation respond to glucagon path within the cell. In particular antagonists of glucagon usually identified by their ability to bind with receptor glucagon and consequently to reduce the stimulation respond to glucagon route of metabolism inside the cell.

In one aspect of the present invention are provided methods for detecting the presence of antagonists of glucagon, providing stage (a) the exposure of the compounds in the presence of agonist with recombinant glucagon receptor glucagon conjugate to respond to glucagon by, under conditions and for a time sufficient to cause binding of the test compound to the receptor and was obtained reaction path, and (b) detection of reduction incentive respond to glucagon path caused by the binding of compounds to the receptor glucagon, compared with stimulation of this pathway one agonist of glucagon, with subsequent determination of the received data presence is Isla and glucagon), able to contact the glucagon receptor and stimulate sensitive to glucagon path inside the cell.

With the help of such methods can be skanirovat a lot of connections. Typical examples include blocking antibodies discussed above, the peptides of the glucagon receptor and analogues of glucagon (including peptide and ones ligands). U. S. Serial 07/741931, for example, provides methods for producing large amounts of analogues of glucagon using pools of DNA sequences encoding such analogs. Such pools of DNA sequences encoding analogs of glucagon, you can get saturating mutagenesis of DNA sequences encoding glucagon (e.g., Little, Depe 88: 113-115, 1990; Hembers et al., Depe 88:143-151, 1989), segmentarity by mutagenesis (e.g., Shortle et al., The OEWG. Ntl. Acad. Sci. USA 77:5375-5379, 1980), increased the incorrect inclusion of nucleotides (e.g., Liao and Wise Gene 88:107-111, 1990) or by applying a randomly mutated oligonucleotides (Hutchinson et al. , Proc. Natl. Acad. Sci. USA 85: 710-714, 1986). Individual transformants expressing the analogue of glucagon, can then be cloned, as described above, or accumulate.

The compounds exhibit with recombinant glucagon receptor in the presence of what atoron and an associated response through the metabolic pathway. In this invention, the time and conditions sufficient for binding of glucagon antagonist to the receptor, will range depending on the source of the receptor, however, are usually suitable conditions are the 4oC-55oWith in a buffer solution with a 0-2 M Nl, preferably 0-0,9 m Nl, most preferably from 0.1 M NCl, and in the range of pH 5-9, preferably between 6.8 and 8. Sufficient time for communicating and receiving feedback - 5-15 minutes after the start of exposure.

After exposure of the test compound with a recombinant glucagon receptor in the presence of agonist glucagon under conditions and time sufficient to complete the binding of the compound to the receptor, decreasing stimulation respond to glucagon way you can detect if this compound competes with the agonist for glucagon recombinant glucagon receptor. In one embodiment of the invention, respond to glucagon by, is the response of adenylate cyclase reaction. In this case, the phase detection provides a measure of the reduction education cAMP the membrane-bound adenylate cyclase compared to the formation of cyclic AMR in the presence of one agonist of glucagon. For the purposes of this invention predpochtitel what is1-glucagon. Tests of adenylyl cyclase activity can be performed, for example, using the method described by Lin et al. (ihm. 14: 1559-1563, 1975) and in the examples. These methods measure the stimulation of camp compared to native glucagon and typically involve exposure of the drug cells, expressroute biologically active recombinant glucagon receptor, with a mixture of glucagon and test compounds in the presence of radioactively labeled ATP.

Alternative education camp can easily be measured using methods well-known in this field, including, for example, by methods described by Salomon et al. (nl. Biohem. 58:541-548, 1976) or Krishna et al. (J. Phrmacol. Exp. hr. 163:379, 1968) or preferably with the use of commercially available whales, such as Scintillation Proximity Assay it from Amersham rrtin. This kit measures the formation of camp by using the competition itinerating camp with anti-camp antibodies. Particularly preferred receptor glucagon have biological activity in these tests ED50(effective dose for 50% response) less than 1 nm, more preferably from ED50less than 0.7 nm, and most preferably with ED50less than 0.25 nm.

In the next version izobretatelya catalyzes the release of photons by luciferion and therefore can be easily detected in the presence of luciferin (lm and Cooke, nl. Biochem. 188:245-254, 1990). As described in more detail below, in a particularly preferred variant of the invention provide design DNA that contains the element that responds to cyclic AMR, such as proenkephalin meets at camp element, operatively connected to the luciferase cDNA. This design DNA containing the luciferase cDNA, stable transfairusa in a cage of a host. Then this cell the owner transferout second design DNA containing the first segment of DNA encoding a glucagon receptor, operatively connected to additional DNA segments required for the expression of the receptor. By linking the receptor agonist of glucagon increased levels of camp induce the expression of luciferase. The luciferase exhibit with luciferine and measure the photons released during the oxidation of luciferin luciferase.

In other embodiments of the invention, the activation response leads to an increase in the intracellular concentration of free calcium. To determine the concentration of free intracellular calcium can be applied a variety of tests, such as how Calcim fluor Quinz described Charest et al. (J. Biol. Chem. 259: 8769-8773, 1983) or quorin photoprotein - described Nakajima - Shimada (Proc. Natl. Acad. Sci. USA 88:6878-8882, the example 6. Briefly, in one embodiment, the cells transformed with the plasmid expressing the glucagon receptor, and grown for three days under normal culture conditions. Then the growth medium removed and replaced with a solution containing 10 μm fura-2AM (see Grynkiewicz et al. . , J. Biol Chem 260:3440-3450, 1985). The cells are then incubated for 30-120 minutes. Photoisomerization (imaging) can be performed using an inverted fluorescent microscope Nicon Diht equipped with a lamp with a mercury arc. Cells first watch at least 60 seconds to establish the line of the ion, and then stimulated with buffer containing glucagon. Images are usually recorded for at least 3 minutes after stimulation. For processing and quantitative image you can use software such as Inovision (Research Triangle Park, N. C.).

Antagonists of glucagon that can be detected, as described above, can be cleaned by ion-exchange and distribution chromatography, as described, for example, Coy et al. (Peptides Structure and Function, Pierce hmil Company, Rkfrd IL., pp.369-372, 1982), chromatography with reversed phase (Andreu and rrifield, Eur. J. Biochem. 164:585-590, 1987) or by HPLC (for example, fd et al. , Int.expert cleaning, such as liquid chromatography, gradient centrifugation and gel electrophoresis, and others. Methods of protein purification known in the art (see Scopes, R., Protein Purification, Springer-Verlag, NY, 1982) and can be used to described here recombinant antagonists of glucagon. Alternatively, antagonist glucagon can be synthesized by the solid phase method of Barany and rrifield (in The Peptides, Vol. 2A, Gross and Meienhofeer, eds., Academic Press, NY, pp.1-284, 1979) or using an automated peptide synthesizer.

Preferred of practically pure antagonists of glucagon with homogeneity at least 50%, more preferably 70-80%, and most preferably 95-99%, in particular for pharmaceutical use. In purified to homogeneity as antagonists of glucagon can be used in therapy. Typically, the antagonists can be administered parenterally or by infusion. In a typical case, the antagonists are present in the form of free bases or as salts with acids. Suitable salts must be pharmaceutically acceptable. Typical examples are metal salts, salts of alkaline and alkaline-earth metals such as potassium and sodium. Other pharmaceutically acceptable salts are salts lim is established with representation in the form of an aqueous isotonic solution with a pH of between about 5.6 and 7.4. Suitable isotonic solutions may be solutions of sodium chloride, dextrose, boric acid-sodium tartrate and polyethylene glycol. Therapeutic doses of antagonists can be administered concomitantly with insulin or in the same composition or in separate compositions.

Diagnostic use of probes of receptor glucagon

In another aspect of the present invention provided with the probes and primers for detection of glucagon receptor. In one embodiment of the invention are provided probes that are capable of gibridizatsiya to DNA or RNA of the glucagon receptor. For the purposes of this invention, the probes are capable of gibridizatsiya" with DNA receptor glucagon, if they hybridize or at high stringency, or low stringency (see Sambrook et al., supra). Preferably, the probe could be used for hybridization with the appropriate nucleotide sequences in the presence of 6SSC, 1Denhardt's (Sambrook et al., supra), 0.1% sodium dodecyl sulfate at 65oWith at least one washing to remove excess probe in the presence of 2SSC, 1Denhardt'S, 0,1% DS PA at 65oC. the Sequences of the probes are preferably constructed so that it was possible hybridization with DNA sequences Redmon.

The probes of this invention can consist of either DNA or RNA and may have a length of only about 12 nucleotides, usually 14 to 18 nucleotides, but can be as large as the entire sequence of the glucagon receptor. The choice of the size of the probe depends on its application. For example, to determine the presence of different polymorphic forms of the receptor glucagon within the individual, the preferred probe containing virtually the entire length of the coding sequence of the glucagon receptor. Probes of receptor glucagon can be used to identify polymorphisms associated with the gene of the receptor glucagon (see, for example, Wbr, Gnomis 7:524-530, 1990; Weber and May, AMEG. J. Hum. Gen. 44:388-396, 1989). Such polymorphisms may be associated with inherited diseases such as diabetes.

Probes can be designed and labeled using methods that are well known in this field of knowledge. Short probes, for example, from 12 bases can be obtained synthetically. Longer probes, approximately 75 bases to less than 1.5 KB (etc., ad) are preferably, for example, R amplification in the presence of labeled precursors such as32R-d, digoxigenin-dU or Biotin-d. the sootvetstvujushij probe, cultivation of transfected cells to large quantities and purification of the corresponding sequence of the transfected cells.

Probes can be labeled with different markers, including, for example, a radioactive marker, a fluorescent marker, an enzyme and a chromogenic markers. The use of32R particularly preferably for tagging specific probes.

The probes of this invention can also be used to detect the presence of mRNA or DNA receptor glucagon inside the sample. However, if the glucagon receptor are present only in limited quantities, or if it is desirable to detect the selected mutant sequence, which is present only in limited quantities, or if it is desirable to clone the receptor glucagon from the selected warm-blooded animal, it may be advantageous to amplify the corresponding sequence so that it can be more easy to find or get.

For amplification of selected sequences can be applied in various ways, including, for example, amplification of RNA (see. Lizardi et al., Bio/Technology 6: 1197-1202, 1988; Kramer et al., Nature 339:401-402, 1969; Lomeli et al., Clinical hm. 35(9): 1826-1831, 1989; U. S. Patnt 4786600 the tnt 4876187 and 5011769, which describe an alternative system for the detection/amplification involving the use of cutting ties).

In a particularly preferred variant of R amplification is used to detect or obtain DNA receptor glucagon. Briefly, as described in detail below, the sample DNA is denatured at 95oWith to generate single-stranded DNA. Then specific primers, as discussed below, hybridized at 37oC-70oWith depending on the ratio of AT/GC in the primers. The primers extend at 72oWith q polymerase to get the chain opposite the matrix. These steps constitute one cycle, which can be repeated for amplification of selected sequences.

Primers for the amplification of selected sequences must be selected from sequences that vysokospetsifichnymi and form stable duplexes with the sequence of the target. Primers should be complementarily, in particular at the 3'-end, must not form dimers with each other or with other primers and must not form a secondary structure or a duplex with other regions of DNA. As a rule preferred primers of the 18-20 of nucleotidase in table 1 and include degenerate oligonucleotides ZC4715 and ZC4701 (SEQ ID 9 and 8, respectively), as well as oligonucleotides ZC4758 and ZC4778 (SEQ ID 10 and 11, respectively).

Additional use of the nucleotide sequences of glucagon receptor

In another aspect of the present invention provided with viral vectors that can be used to treat diseases in which the glucagon receptor (or mutant receptor glucagon) redundantly expressed or not expressed receptor glucagon. Briefly, in one embodiment of the invention, viral vectors are provided which direct the formation of antisense RNA of the glucagon receptor and thereby prevent excessive formation of glucagon receptor or expression of mutant receptor glucagon. In another embodiment, viral vectors are provided which direct the expression of the cDNA of the receptor for glucagon. Viral vectors suitable for use in this invention, among others, are recombinant vaccinia virus vectors (U. S. tnt 4603112 and 4769330), recombinant poxvirus vectors (PCT Publication WO 89/01973) and preferably recombinant retroviral vectors ("Recombinant Retroviruses with Anphotropic and Ecoptropic Host Ranges", PCT Publication WO 90/02806; "Rtrvirl Raschid ll Lines and Processes of Using Sm", PCT Publication WO 89/07150; and "Antisense RN for rtm which may be used in the treatment of painful conditions, when either redundantly expressed glucagon receptor, either expressed mutant receptor glucagon or when not to Express the receptor for glucagon.

The following examples are presented to illustrate but not to limit the invention.

EXAMPLES

Example 1

Synthesis of cDNA and obtaining cDNA libraries

A. Synthesis of cDNA of rat liver

The liver of 30 g of female rats of Sprague-Dawley (Simonsen Labs, Gilroy, CA) were removed and immediately placed in liquid nitrogen. Total RNA was obtained from liver tissue using guanidinoacetate (hirgwin et al., Biochemistry 18:52-94, 1979) and centrifugation in CSCI. Poly(A)+RNA was isolated using oligo d(T)-cellulose chromatography (Aviv and Leqer, the OEWG. Ntl. Acad. Sci. USA 69:1408-1412, 1972).

The first chain cDNA was synthesized from poly(A)+RNA liver, twice selected using poly d(T). 10 μl of a solution containing 10 μg of poly(A)+RNA liver was mixed with 2 μl of 20 nmol/ál primer first circuit ZC3747 (SEQ ID 7) and 4 µl of the treated diethylpyrocarbonate water. This mixture was heated at 65oC for 4 minutes and cooled on ice.

The synthesis of the first chain cDNA were initiated by addition of 8 ál of 5x SUPERSCPIRT buffer (GIBCO BRL, Gaithersburg, Md.), 4 μl of 100 mm Dimitri LKB iothnlg Inc., Piscataway, N. J.) to the RNA-primerno mixture. The reaction mixture was incubated at 42oC for 3 minutes. After incubation were added to 6.0 ál 200 U/ál reverse transcriptase SUPERSCRIPT (GIBCO BRL). The efficiency of synthesis of the first circuit analyzed in a parallel reaction by adding 10 µci32P-dCTP to the aliquot 10 ál of the reaction mixture for labeling the products of the reaction. The reaction mixture for synthesis of the first chain incubated at 45oC for 45 minutes followed by 15-minute incubation at 50oC. the Reaction was stopped by adding water to a final volume of 100 μl, followed by extraction twice with a mixture of phenol/chloroform (1:1) and once with a mixture of chloroform/ISO-amyl alcohol. Not included nucleotides were removed from each reaction double precipitation of the cDNA in the presence of 6 μg glycogen as a carrier, 2.5 M ammonium acetate and 2.5 volumes of ethanol. Unlabeled cDNA resuspendable in 50 μl water and used for synthesis of the second chain. The length of the first chain cDNA was assessed by resuspending labeled cDNA in 20 µl of water and determining the amount of cDNA using gel electrophoresis in agarose.

The synthesis of the second chain was performed on a hybrid RNA-DNA from the reaction of synthesis of the first circuit when the conditions conducive to the use of the thus, what it contained 20,0 4 ál of polymerase buffer I (100 mm Tris-HCl, pH 7.4, 500 mm KCl, 25 mm MgCl250 mm (NH4)2SO4), of 4.0 μl of 100 mm dithiothreitol, 1,0 μl of a solution containing 10 mm each of deoxynucleotidase, 3,0 cells / ml NAD, 15,0 μl of 3 U/μl DNA ligase E. Li (NL Enzymes Ltd., Cramlington., Northumbri, nglnd, 5,0 ál 10 U/ál DNA polymerase I of E. coli (GIBCO RL) and 50.0 μl of the unlabeled first DNA chain. A parallel reaction in which an aliquot of 10 µl of the mixture for synthesis of the second circuit pointed out by adding 10 µci32P-dCTP was used to monitor the efficiency of the synthesis of the second chain. The reaction mixture was incubated at room temperature for 4 minutes followed by the addition of 1.5 μl 2 U/μl RNase E (GI BRL) to each reaction mixture. Reactions were incubated at 15oC for 2 hours followed by incubation for 15 minutes at room temperature. Each reaction was stopped by adding 4 μl of 500 mm EDTA, followed by extraction with a mixture of phenol/chloroform and a mixture of chloroform/isoamyl alcohol as described above. DNA from each reaction was besieged in the presence of ethanol and 2.5 M ammonium acetate. DNA from unlabeled reactions resuspendable in 50 ál of water. Labeled DNA resuspendable and subjected to electrotrash as the discription mixture contained 10 µl of 10x buffer for nucleases Masha (Stratagene lning Systems, La Jolla, Calif.), 4 μl of 200 mm dithiothreitol, 34 μl water, 50 μl of the second chain cDNA and 2 μl of 1:10 dilution nucleases Masha (Promega Corp. , Madison. , Wis. in the buffer for cultivation Stratagene MB (Stratagene Cloning Systems). The reaction was incubated at 37oC for 15 minutes and stopped by adding 20 μl of Tris-HCl, pH 8.0 followed by successive extraction with a mixture of phenol/chloroform and a mixture of chloroform/isoamyl alcohol as described above. After extraction the DNA was besieged in ethanol and resuspendable in water.

Resuspending cDNA "small mistake" by using T4 DNA polymerase. This cDNA resuspendable in volume 192 μl of water was mixed with 50 ál of 5x buffer for T4 DNA polymerase (250 mm Tris-HCl, pH 8.0, 250 mm KCl, 25 mm MgCl2), 3 μl of 100 mm dithiothreitol, 3 μl of a solution containing 10 mm each of deoxynucleotidase and 2 ál of 6.7 U/μl T4 DNA polymerase (Pharmacia LKB Biotechnology Inc.). After incubation at 15oC for 30 minutes the reaction was stopped by adding 2 μl of 500 mm EDTA followed by successive extraction with a mixture of phenol/chloroform and a mixture of chloroform/isoamyl alcohol as described above. Based on32P-dCTP was determined that the yield of cDNA was 4 µg of the original matrix 10 μg of mRNA.

C. Obtaining bibliotekos, as described above, cDNA was added Eco R1 adapters. An aliquot of 10 µl of cDNA and 800 rmala adapter (12 ml) was mixed with 4.0 μl of 10x ligase buffer (Stratagene Cloning Systems), of 4.0 μl 10 mm ATP, 6,0 μl of water and 16 E of T4 DNA ligase (4,0 μl; Stratagene lning Systems). The reaction was incubated 16 hours at a temperature gradient of 4oC-15oC. the Reaction was stopped by adding 185 μl of water, 25 μl R 2 buffer (GI BRL) followed by incubation at 65oC for 30-60 minutes. After incubation the reaction was extracted with a mixture of phenol/chloroform, then with a mixture of chloroform/isoamyl alcohol and precipitated with ethanol as described above. After centrifugation of the precipitated DNA was washed with 70% ethanol and dried in air. Sediment resuspendable in 180 μl of water.

To facilitate directional embedding expressing the cDNA in the vector mammalian cDNA were digested using Xho I, getting cDNA with 5'-Eco R1 sticky end and 3'Xho I sticky end. The restriction site Xho I at the 3' end of the cDNA was injected through primer ZC3747 (SQ ID 7). Restriction cleavage was stopped by sequential extraction with mixtures of phenol/chloroform and chloroform/isoamyl alcohol. cDNA precipitated with ethanol, and the precipitate was washed with 70% ethanol and air-dried. Sediment resuspen suspendirovanie cDNA was heated to 65oC for 10 minutes, cooled on ice and subjected to electrophoresis on a 0.9% agarose gel with a low melting point (Seaplaque GTG Low Melt Agarose, FMC Corp., Rockland, Me.) using RL I kb ladder(GiBCO BRL) and Pharmacia 100 bp ladder (Pharmacia LKB Biotechnology Inc.) as markers of sizes. Impurity adapters and fragments by-products size below 800 N. p. cut out of the gel. The electrodes were changed places and perform electrophoresis of cDNA as long as she is not concentrated near the beginning of the track. This area, containing the concentrated DNA was cut out, placed in a microcentrifuge tube and determine the approximate volume of the segment of the gel. In a test tube was added an aliquot THOSE equivalent to half of the volume of the gel and the agarose was melted by heating to 65oC for 15 minutes. After equilibration of the sample to the 42oWith added approximately 5 units-Agarase I (New nglnd Biolabs, Beverly, Mass.). The sample is incubated for 90 minutes to break down the agarose. After incubation the sample was added 0.1 x volume of 3 M sodium acetate and the mixture incubated on ice for 15 minutes. After incubation the sample was centrifuged at 14000 g for 15 minutes at 4oC to remove undigested agarose. Then besieged cDNA in the supernatant by ethanol. The precipitated cDNA was washed with 70% e the p ZCEP E. coli, derived p CDANAI (invitr ogen) in which breeding marker SupF replaced-lactamase cassette from pUC18. Plasmid Z, which translated into a linear form by cleavage using Eco RI and Xho, ligated with Eco RI-Xho I-cDNA. The resulting plasmid was introduced by electroporation into cells of the strain E. Li DH10 ELECTROMAX (GIBCO BRL).

C. the cDNA Synthesis cells of pancreatic islets man

Cells of the islets were isolated from the pancreas of a person obtained from donors of transplantable organs, for which no suitable recipient. After perfusion with cold UW solution (DuPont, Boston, Mass.) each pancreas was carefully cut, pancreatic duct was Coulibaly and poured in a solution of collagenase 4 mg/ml (Type V, Sigma, St.Louis, Mo.) at constant speed, first at 4oAnd then, when the 39oC. Gland was dismantled into pieces and the released fragments were washed by centrifugation, crushed through a needle with a smaller caliber and were purified by centrifugation with continuous gradienta density ficoll (Warnock Diabeles 35 (Suppl. I): 136-139, 1989). The material collected from the upper interfaz were combined and considered after determining the purity of islets using staining ditiazem. The Islands used the purity of more than 40%. The average diameter of the Islands was 175 μm. In addition, isolated islets were detected both the first and second phase function of insulin secretion after perfusion or high glucose concentration, or isobutylmethylxanthine (VMH).

Poly(A)+RNA was isolated using FSR China to highlight mRNA (Invitrogen) according to the manufacturer's instructions. Briefly, 30000 purified islets were quickly literally in the buffer for lysis, homogenized using needles of decreasing caliber and were digested in the presence of proteinase K and Rcasino, then was isolated poly(A)+RNA by chromatography on oligo-d(T)-cellulose. The concentration and purity buervenich fractions was determined at OD 260/280.

Approximately 2.5 μg of poly(A)+RNA from human pancreatic islets were used to create a cDNA library using system constructing cDNA library LIBRAPIAN R11 (Invitrogen) and cells of E. coli DH10B ELSCTROMAX (GIBCO BRL) according to the manufacturer's instructions. Briefly, approximately 2.5 μg of poly(A)+RNA isolated from human islets were converted into double-stranded cDNA and then adding st X I nepalindromnoi of linkers (Invitroqen). This cDNA was fractionally in size and the main chain DNA greater than 600 N. p.

Example 2

The selection of cDNA of rat glucagon receptor using amplification using polymerase chain reaction (PCR amplification)

CDNA of rat liver was used as template for amplification of the nucleotide sequences of glucagon receptor using degenerate oligonucleotides (ZC4715 and ZC4701; SEQ ID 9 and 8, respectively), corresponding to areas of high conservatism among the members of the secretin gene family. 50 µl reaction mixture contained 5 ng matrix cDNA (example 1A); 100 pmoles each of the oligonucleotides ZC4715 (SEQ ID 9) and ZC4701 (SEQ ID 9); 0.25 mm each deoxynucleotidase (Cetus, Emeryville, CA); 1x Promega 10x buffer (Promega Corp.); 1.25 units q polymerase (Promega). PCR reaction was performed in 40 cycles (1 minute at 95oC, 1 minute at 42oC and 2 min at 72oC) followed by incubation for 7 minutes at 72oC.

PCR product 650 N. p. were isolated using gel electrophoresis and ligated with R1000 (Stratagene Cloning Systems). The obtained plasmid was used to transform cells XL-I E. Li. Plasmid DNA was obtained from selected transformants, designated G 13/R1000, and sequenced (SEQ ID 14). Sequencing of the clone revealed that the insertion of Kony rat glucagon receptor

CDNA of full length rat glucagon receptor was obtained by screening libraries described in example 1B, with the help of test binding of glucagon. The library was sown, receiving one million independent clones. Colonies transformant from each Cup was viscerale in 10 ml of LB-Amp (Sambrook et al., supra). Cells were unscrewed and the environment would be thrown out. Precipitation cells resus-Bandarawela in 4 ml L-AMR, 15% glycerol, and aliquots of 1 ml were stored at -80oC. First glitserinovyi the original solution was titrated and seeded 100 pools of 5000 colonies per Cup. After growth of colonies of each Cup was viscerale in 10 ml LB-AMR. Take an aliquot of cells from each pool for use in the preparation of plasmid DNA. The remaining cell mixture was brought to a final concentration of glycerol 15%, divided into aliquots and frozen at -80oC. Plasmid DNA was obtained from each pool of cells and DNA were digested Sncaso (Boehringer Mannheim, Indianopolis, Ind.) in accordance with the manufacturer's instructions. RNA asnow reaction was stopped by extraction with a mixture of phenol/chloroform/isoamyl alcohol (24:24:1) and the DNA precipitated with ethanol.

Clsmenu DNA from each pool was transfusional in COS-7 cells (ATSC RL 1651) and the transfectants were skanirovali the presence Rotz is Ino 105cells COS-7 were sown on sterile glass slides with one camera (NUNC AS Roskil de, Denmark), which were covered with 10 µg/ml human fibronectin (table 1) for 30 minutes at room temperature and washed suboceana phosphate saline (PBS, Sigma Chemical Co. St. Louis, Mo.). Two micrograms of plasmid DNA from each pool was used for transfection of cells, growing on a separate glasses with camera, using the method described by McMahan et al. (EMBO J. 10:2821-2832, 1991, which is incorporated by full references). After transfection cells were grown for 72 hours at 37oWith 5% CO2.

Dried powder was dissolved in a buffer solution. Then was added ammonium sulfate to a concentration of 25% and the solution was allowed to precipitate for 2 hours at 4oC. Fibronectin besieged by centrifugation at 1000 rpm in a centrifuge Bench Top (Beckman Instruments, Inc. Irvine, Calif.) within 15 minutes. The supernatant was discarded and the precipitate was dissolved in 10 ml of NaPO4-buffer solution (see above).

Fibronectin in the final volume of 16.9 ml were dialyzed overnight against 1 l N4buffer solution (described above). Cialisovernight material was diluted 3 times with 1 mm phosphate buffer, pH 7.4, receiving solution in 1 mm N4, rmali glass rod.

Fibronectin was subjected to FPLC through a 50 ml column D Sepharose FF (Pharmacia LKB iothnlg Inc., is cataway, N. J.), which was equilibrated with three volumes of 10 mm Tris-HCl, pH 8.1, 50 mm Nl. After the column was washed with 10 mm Tris, pH 8.1, 50 mm Nl to obtain samples of background, fibronectin was suirable salt gradient up to 10 mm Tris, pH 8.1, 300 mm NaCl. Fractions were collected, aliquots of the fractions were subjected to electrophoresis on polyacrylamide gels and the gels were analyzed by staining Kumasi blue and the analysis of Western. Fraction peaks were combined and were dialyzed against 10 mm CAPS (3-(cyclohexylamino)-1-propanesulfonate, Sigma), pH of 11.0, 10 mm CaCl2, 150 mm NaCl. The solution was stored at -80oC.

To obtain transfectants to test binding125I-glucagon depleted medium was aspirated from the cells and cells were washed three times with cold (4oC) S. After the final wash the cells were plated medium for binding (table 2) and incubated 10 minutes at room temperature. The medium was replaced with 0.5 ml of medium for binding containing 0.5 nm125I-glucagon (mrsham receptor grade, specific activity 2000 CI/mmol;

Amersham). The cells were then rocked at 30oC for 1 hour. Medium was aspirated from the cells, was added a cold (oC) environment for linking without g what does 3 times with cold (4oC) PBS. After the last washing, the cells were fixed with 1 ml of 2.5% glutaraldehyde in PBS at room temperature for 20 minutes. Glutaric aldehyde was removed and cells were rinsed 3 times with PBS. Slides were air-dried for 1 hour at room temperature, immersed in liquid photographic emulsion (Estman dak Co., Rochester, N. Y.) according to the manufacturer's instructions and dried at room temperature in the dark for at least 30 minutes. The glass was then placed in a light-tight box for 72 hours at 4oC. Cells are able to bind glucagon was detected at 2.5 X magnification under bright field illumination. Some pool, 57, was identified as the pool containing cells, are able to bind glucagon.

1 M sodium bicarbonate

of 8.4 g of solid NaCO3< / BR>
Sodium bicarbonate was poured into a measuring flask of 100 ml and was added 80 ml of distilled water. The solution was stirred to dissolve the solids, distilled water was added to 100 ml. of the Solution is again stirred and kept at 4oWith in a closed tube bottle.

Environment

1 ml of 1 M sodium bicarbonate was added per liter of distilled water. 4 liters was prepared in advance and the solution was cooled during the course of the Sabbath.
69 g of sucrose

Sucrose was dissolved in 31 ml of distilled water by heating. The concentration of the solution was measured using a Refractometer. Added solid sucrose or water (if necessary) to obtain the concentration 690,5%.

42,3% sucrose solution

42 g of sucrose

Sucrose was dissolved in 57 ml of distilled water by heating. The concentration of the solution was measured using a Refractometer. Added 69% sucrose solution or water (if necessary) to obtain the concentration 42,31%.

2x buffer for binding

100 mm HEPES, pH 7.3

300 mm NaCl

2 mm EDTA

2% bovine serum albumin

1.6 mg/ml bacitracin

A buffer for receiving image

140 mm Nl

10 mm HEPES

5.6 mm glucose

5 mm KCl

1 mm MgSO4< / BR>
1 mm CaCl2< / BR>
A solution of Fura-2 AM

50 mg fura-2 AM (Molecular Probes)

50 ml of DMSO

5 ml of the buffer for receiving the image

50 mg fura-2 AM was dissolved in 50 ml DMSO. After dissolution of the solids, the solution was mixed with 5 ml of buffer to receive the image.

An aliquot of plasmid DNA from a pool of 57 was subjected R amplification using oligonucleotides ZC4701 and ZC4715 (SEQ ID 8 and 9, respectively). The reaction mixture of 50 μl was prepared so the D 8 and 9, respectively); 50 mm Kl; 10 mm Tris-HCl, pH 9,0 (at 20oC); 1.5 mm MgCl2; 0.01% of gelatin; 0.1% Triton X-100; 0.2 mm each of deoxynucleotidase (Pharmacia LKB Biotechnology Inc.) and 1 unit of Taq polymerase (Promega). PCR reaction was performed in 30 cycles with the following 7-minute incubated at 72oC. the Reaction mixture was kept at 4oC. Analysis R product using gel electrophoresis revealed the presence of bands 700 N. p., approximately the same size as the product described in example 2.

The original solution of glycerol from a pool of 57 was titrated and seeded 2% cups of 500 colonies. Colonies were pooled and the original solution and plasmid DNA was prepared as described above. Plasmid DNA was transfusional in COS-7 cells and the transfectants were skanirovali using test binding of glucagon as described above. One pool, 57-18, was identified as the pool containing cells, are able to bind glucagon.

An aliquot of plasmid DNA from the pool 57-18 were subjected to PCR amplification using oligonucleotides ZC4701 and ZC4715 (SEQ ID 8 and 9, respectively), as described above. Analysis of the PCR product by gel electrophoresis revealed the presence of bands 700 N. p., confirming the presence of DNA sequences of glucagon receptor.

The outcome is zeinali source and glycerol solutions and plasmid DNA was prepared, as explained above. An aliquot from each pool of plasmid DNA was transfusional in COS-7 cells and the transfectants were skanirovali using test binding of glucagon described above. In addition, an aliquot from each pool of plasmid DNA was subjected to PCR amplification using oligonucleotides Z4701 and ZC4715 (SEQ ID 8 and 9, respectively), as described above. 4 positive pool ( 57-18-16, 57-18-18, 57-18-36 and 57-15-48) were identified as pools containing cells, are able to bind glucagon, and was shown by PCR amplification, that they contain the same band 700 N. p.

To highlight cDNA two cups with a diameter of 150 mm were seeded at 2500 colonies (each) pool 57-18. Filter lifts were prepared according to the method described by Hanahan and Meselson (Depe 10:63, 1980) and Sambrook et al (ibid), which provides full references. Hybridization probe was obtained by PCR amplification of plasmid DNA from a pool of 57 with the use of oligonucleotides and methods described above. CR product was gel purified from the agarose with a low melting point and used for arbitrary primers from China MGRIME (Amersham, Arlingtn Heights, III.) in accordance with the manufacturer's instructions. Filters are hybridized in a solution containing 6x SSC, 5x Denhardt's, 5% LTOs, 200 μg/ml treated with ultrasound DNA salmon sperm Excess label was removed by three washes using 1x SSC, 1% - ordinator at 65oC. the Filters were exposed to film for 4 hours at -80oWith two screens. A positive clone containing a plasmid pLJ4, was identified and sequenced. Plasmid pLJ4 was deposited with ATS (12301 Parklawn Dr., Rovill, MD 20852) as the transformant E. coli under the entry number 69056 21 August 1992. Restricciones analysis and sequencing showed that pLJ4 contains an insertion of approximately 2 KB (etc., ad), encoding a protein of 485 amino acids with the predicted mol.mass 54962 daltons. This sequence of nucleic acid and deduced from it the amino acid sequence shown in SEQ ID 14 and 15. The hydropathy analysis according to the method of Kyte and Doolittle (J. Mol. Biol 157:105-132, 1982, including here in the form of links) identified 8 clusters of hydrophobic amino acids corresponding to the amino-terminal signal sequence and 7 transmembrane domains (Fig.2). In addition, the analysis indicated aminosilanes sequence showed the presence of 4 potentially N-United glycosylation sites in the extended hydrophilic sequence and the presence of 6 cysteines in the same area.

Example 4

The allocation of the cDNA of the human glucagon receptor using R amplification

CDNA cells pancreatic acute the glucagon receptor by using degenerate oligonucleotides ZC4715 and ZC4701 (SEQ ID 9 and 8 respectively). 50 µl reaction mixture contained 5 ng matrix cDNA (example 1 (C); 100 pmoles each of the oligonucleotides ZC4715 (SQ ID 9) and ZC4701 (SEQ ID 8); 0.25 mm each deoxynucleotidase (Cetus, Emeryville, Calif); 1x Promega 10x buffer (Gomeda) and 1.25 units q polymerase (Gomeda). CR the reaction was carried out in 40 cycles (1 minute at 95oC, 1 minute at 45oC and 2 min at 72oC) followed by incubation for 7 minutes at 72oC.

Using gel electrophoresis was selected R product size approximately 750 N. p. One-tenth allocated R product was used as a matrix for other CR reaction using oligonucleotides Z4758 and Z4778 (SEQ ID 10 and 11), which were designed to embed website restriktsii Bam H1 at the 3'-end of the website restriktsii Eco R1 at the 5'-end R product for sublimirovanny. The reaction mixture of 50 μl was prepared as described above. PCR, the reaction was carried out in 40 cycles (1 minute at 95oC, 1 minute at 50oC and 1.5 minutes at 72oC) with the following 7 minute incubation at 72oC.

For screening of transformants on the nucleotide sequence of the glucagon receptor the insertion of DNA present in each transformant, amplified with primariily sequencing and joined the pUC sequences, flanking built-in R product. 48 transformants were added (each) in 25 µl reaction mixture containing 20 picomoles of each oligonucleotide; 0.125 mm of each deoxynucleotide (Cetus, Emeryville, lif.); 1x Promega 10x buffer (Promega); and 1.25 units q, polymerase (Promega). R the reaction was carried out in 30 cycles (1 minute at 95oC, 1 minute at 45oC and 1.5 minutes at 72oC followed by 7 min incubation at 72oC.

R products were then analyzed by hybridization to Southern (Southern, J. Mol. Biol. 98: 503, 1975; Sambrook et al., ibid; included here in the form of full references) using the 1.9 KB Eco R1-Xho 1 fragment pLJ4, which was marked by arbitrary inclusion of nucleating using whale Amersham MEGAPRIME (Amersham) as a probe. It was shown that one clone, G30, hybridisable with a full-size cDNA probe rats. This nucleotide sequence G30 shown in SEQ ID 16.

Example 5

The cDNA cloning of the full length receptor glucagon man

To identify the library containing the sequence encoding human glucagon receptor, a number of libraries have skanirovali using PCR using oligonucleotide primerov ZC5433 and Z5432 (SEQ ID 13 and 12, respectively), which was the military and cooked human genomic and cDNA libraries from human liver, cells of the islets, G2 cells, brain and human placenta were subjected to screening (table 3). Individual 50 µl reaction mixture for PCR reactions were prepared so that they contained DNA from each library in the proportions indicated in table 4, 20 pmole/µl of each ZC5433 and ZC5432 (SEQ ID 13 and 12, respectively), 0.25 mm of each deoxynucleotidase, 5 ál of 10x Taq 1 buffer (Promega), 15 mm MgCl2only 19.5 ál of distilled water and 0.5 μl of 5 u/ál Taq 1 polymerase (Promega). In addition, a separate reaction mixture contained LJ4 as a positive control, or did not contain DNA as a negative control.

R reaction was performed in 30 cycles (1 minute at 94oC, 1 minute at 50oC and 1.5 minutes at 72oC) with subsequent 10-minute incubation at 72oC. Then PCR products were analyzed using agarose gel electrophoresis. Only NI library liver generated strip 320-410 N. p., i.e. of size equal to the size of the bar that is visible in the case of the positive control (pLJ4).

Library KJHK human liver, cloned in plasmid D2 (Chen and Okayama, Mol. ll. Biol. 7:2745-2752, 1987), obtained from Dr.Rgr Bertolloti (National Institutes of Health, Bethesda, Md.), was used to obtain cDNA clone full the century Column transformants from each Cup was viscerale in 10 ml of LB-Amp (Sambrook et al. ibid. ). Cells were unscrewed and the environment would be thrown out. Precipitation cells resuspendable in 4 ml L-AMR, 15% glycerol and 4 aliquots of 1 ml were stored at -80oC. First source glycerin solution was titrated and 100 pools 5000 colonies were planted on the Cup. After growth of the colony each Cup was viscerale in 10 ml of LB-Amp. Take an aliquot of cells from each pool for use in obtaining plasmid DNA. The remaining cell mixture was brought to a final concentration of 15% glycerol, divided into aliquots and frozen at -80oC. Plasmid DNA was prepared from each pool of cells and DNA were digested with RNase (Boehringer Mannheim, Indianapolis, In.) according to the manufacturer's instructions. Recusou reaction was stopped by extraction with a mixture of phenol/chloroform/isoamyl alcohol (24:24:1) and the DNA precipitated with ethanol.

Aliquots resuspending plasmid DNA from each pool were combined into groups (i.e. 1-10, 11-20, 21-30, 31-40, and so on). Plasmid DNA was diluted 1: 20 and 1 ál of DNA from each pool was used R. the reaction mixture identical to the reaction mixture described above. The reaction mixture was subjected to amplification under the conditions described above. Analysis of the products R using agarose gel ptx2">

Plasmid DNA from the original pools 31-40 diluted 1:20 and 1 μl of each pool was used in the reaction mixture, identical mixture described above. R amplification reaction mixtures were performed using conditions described above. Analysis R products using agarose gel electrophoresis showed that a pool of 40 gave the band size is approximately 310-420 N. p.

The concentration of plasmid DNA was roughly estimated as approximately 70 ng/ál with agarose gel electrophoresis 1 μl of a pool of 40, which was diluted 1: 10. 70 ng pool 40 plasmid DNA was introduced by electroporation into cells of strain DIOB E. coli. when is 2.3 volts, 400 ohms, 25 microfarad and resuspendable in 1 ml SOC (Smbrk et al., ibid.). Prepared three cultivation of cells: 10-2, 10-3and 10-4. 100 µl of each dilution were sown on 4 cups. It was determined that there were approximately 10,000 colonies per Cup to 4 cups containing cells from breeding 10-2and approximately 1000 colonies per Cup to 4 cups containing cells from breeding 10-3. 2 filter-lift was prepared from each of the 4 cups dilution 10-3and one of the cups with dilution 10-2that have been designated as pools 1 through 5. One filter from each of the two replicates was placed on telecine DNA for PR amplification. In addition, the three remaining cups with dilution 10-2was viscerale and from these cells was obtained plasmid DNA.

The remaining pre-filters were washed 3x SSC+0.5% of LTOs at 65oWith stirring for 12 hours to remove residual bacteria. Then the filters were prehybridization buffer Ulrich (Ulrich, EMBO J. 3:361-364, 1984)+50% formamide, 1% LTOs overnight at 37oC. Clone DNA G30, which were marked by means of a set mrshm MGRIM (Amersham) according to the proposed factory conditions, boiled and added to the solution for hybridization (buffer Ulrich + formamid) to a final concentration of 6105pulse/million ml. Filters were incubated overnight at 37oWith stirring. After incubation overnight, the probe solution was removed and the filters were washed for 5 minutes at 65oC in 2x SSC + 0.1% of LTOs. After the first washing, the filters were washed in the same solution for 15 minutes at 65oC, then 5 minutes with rocking at room temperature. Minutes of the last wash was repeated 2 more times. Filters were exposed to film overnight at room temperature. One colony on plate 2, which corresponded to the pool 2 were positive hybridization with 30 G. Plasmid DNA obtained from an individual, s and deposited with ATS (12301 Parklawn Dr., Rockville, D 20852) as the transformant E. Li. under input 69055 21 August 1992 Partial sequence of a DNA clone 40-2-2 and derived from it the amino acid sequence shown as SEQ ID 17 and 18.

To confirm the presence of the nucleotide sequences of glucagon receptor in hybridization of the filters was performed R amplification for each preparation of plasmid DNA (plasmid DNA obtained from each re-filter, and plasmid DNA obtained from each of the 3 cups of 10-2). United plasmid DNA was diluted 1:20 in water and 1 μl of each DNA was used as a matrix in R reaction performed as described above. Agarose gel electrophoresis of the obtained PR products showed that a pool of 2 and three for the 4 pools of 10000 contained generated R strip between 310 and 420 N. p. the Presence of generated R strip in pool 2, corresponding to the Cup 2, confirmed the presence of DNA sequences of glucagon receptor.

F. Strategy for cloning the 5' sequence of the human receptor for glucagon

Analysis of the partial cDNA sequence of clone 40-2-2 and sequencing of full length cDNA of rat glucagon receptor showed that clone 40-2-2 had no amino-terminal sequence of the RA glucagon was obtained by adaptation of the method, described by Frohman et al. (OEWG. Ntl. Acad. Sci. USA 85:8998-9002, 1988). Briefly, was designed oligonucleotide primer so that he hybridisable sequence near the 5'end of the coding sequence 40-2-2. This primer is hybridized with the G-tail first chain cDNA-matrix from human liver and the primer is extended in the direction of the 5'-end using q 1 DNA polymerase. The second poly d(C) primer hybridized with the G-tail cDNA-matrix, which made possible the polymerase chain reaction for amplification, followed cloned, sequencing and splicing before the formation of the coding sequence present in the clone 40-2-2.

Received three cDNA-matrix. The first matrix was the first commercially available chain cDNA of a human liver. The second and third matrix cDNA was obtained by synthesis of the first chain cDNA from commercially available mRNA human liver (Clontech) using either of the oligonucleotide containing sequences specific for the human glucagon receptor, or traditional use as a primer oligo d(T), respectively.

The second cDNA-matrix was obtained by synthesis from mRNA of human liver using the oligonucleotide ZC5 is on. The reaction mixture containing 2 μl of liver mRNA (1 μg/μl), 8 μl of 20 pmol/ál ZC5453 (SEQ ID 13) and 0.5 μl of 10 mm Tris, pH 7.4, 0.1 mm EDTA, incubated for 7 minutes at 68oWith and stood then 2 minutes on ice. After incubation to the reaction mixture were added 4 μl of 5X SUPERSCRIPT buffer (GIBCO-BRL), 1 μl of 200 mm dithiothreitol, 1 μl of a solution containing 10 mm each of dNTP, 1 μl of 0.25 µci/µl32P-dCTP and 5 μl of the reverse transcriptase SUPERSCRIPT. The reaction was incubated for 1 hour at 45oC. After incubation the reaction was incubated for 10 minutes at 45oC. the Reaction was stopped by adding 80 μl of TE. RNA is hydrolyzed by adding 1 μl of 0.5 M EDTA and 1 μl of the CON. The hydrolysis reaction was carried out at 65oC for 5 minutes. After incubation the sample was diluted to 1 ml of 50 mm KOH, 0.1 mm EDTA and conducted through the hub ENTRICON 100 (AMAP, Danvers, Mass.). The column was washed with 1 ml of 50 mm KOH, 0.1 mm EDTA. Concentrated cDNA was collected and neutralized 1/2 volume of 100 mm Hcl. The neutralized sample cDNA precipitated with ethanol and the precipitate resuspendable in 26 μl of distilled water.

The third cDNA-matrix was obtained by synthesis from mRNA of human liver using purchased oligo d(T) primer. Preparing the reaction mixture, with the 7,4, 0.1 mm EDTA. Synthesis of cDNA was performed using the conditions described for the synthesis of the above.

For cDNA first chain was attached G-tail. Prepared reaction tubes containing 4 μl of cDNA first chain of a human liver (QUICK CLONE; Clontech, Palo Alto, Calif. ), 4 μl of cDNA first value of human liver from primer ZC5433 (SEQ ID 13) or 4 µl of cDNA first chain of human liver from oligo d(T) primer. Each reaction mixture received 22 μl water, 8 μl 5X buffer (Gomeda), 4 μl of 20 mm d-GTP and 2 μl of 15 U/μl terminal transferase, and the reaction was carried out for 30 minutes at 37oC followed by a 10-minute incubated at 65oC. the Reaction mixture was then diluted with 90 μl of 10 mm Tricom, pH 7.4, 1 mm EDTA and precipitated with ethanol.

The synthesis of the second chain with all G-the end (tail) of cDNA was performed in the same way. Each cDNA is first suspended in 50 µl of distilled water. Then, for each cDNA were added 100 pmole ZC4814 (SEQ ID 20) in 5 μl. Then cDNA hybridized primer by heating each mixture to 68oC for 5 minutes followed by curing for 2 minutes on ice. After the primer was hybridisable with cDNA, each mixture received 20 ál of 5x buffer for polymerase 1 (example 1), 1 ál of 100 mm dithiothreitol, 2 μl of the solution, stnk polymerase 1 E. coli (Amersham). The reaction was conducted for 5 minutes at 22oC followed by the addition of 1.5 μl 2 U/μl RNase H (GIBCO-BRL), and then incubation was continued at 16oC for 2 hours. The reaction was stopped by adding 200 μl of 10 mm Tris, pH 8.0, 1 mm EDTA, followed by extraction with 150 ál of water-saturated phenol and 150 μl of chloroform. The mixture was stirred on a vortex and then centrifuged for 3 minutes at 22oWith phase separation. The aqueous phase was removed and extragonadal phenol and chloroform as described above. After the second extraction with phenol and chloroform, the aqueous layer was extracted with chloroform. Then cDNA in a water layer was besieged by adding 5 μg of glycogen mussels, 200 ál of 8 M ammonium acetate and 300 ál of ISO-propanol for the selective deposition of large nucleic acids, whereas uninvolved oligonucleotide primers remain in the supernatant. Then cDNA was besieged by centrifugation and the precipitate was washed with 70% ethanol and air-dried. The precipitated cDNA resuspendable in 15 ál of bidistilled water.

Double-stranded cDNA amplified in 5 parallel reactions using oligonucleotide primers homologous to the 5'-end Z4814 (SEQ ID 20) and contains sites of restricts to facilitate subtle resultsa in clone 40-2-2. Each reaction mixture contained 5 µl of 10x buffer for q, 1,3 ál of 25 mm MgCl25 µl of a solution containing 2.5 mm of each dNTP, 1 μl of double-stranded cDNA and 0.5 ál q 1 polymerase (Gomeda). distilled water was added to a final volume of 50 µl. The reaction was denaturiruet for 5 minutes at 98oC before adding 1 μl each of 10 nmol/ál Z5624 (SEQ ID 21) and 20 nmol/ál ZC4812 (SEQ ID 19). Each sample was layered 70 μl of mineral oil and the sample was kept at 90oC. Amplification was performed in 30 cycles (95oC 60 seconds, 57oC for 40 seconds, 72oWith 60 seconds) and then the 7-minute extension at 72oC.

Each product PR were subjected to agarose gel electrophoresis and amplificatoare fragments were cut out separately and was subcloned into the PCR-1000 with the use of a kit for cloning (Linvitrogen). Three clones from each ligation reaction were collected and analyzed for the presence of insertions using oligonucleotide primers ZC5624 (SEQ ID 21) and ZC4812 (SEQ ID 19) independent R reaction mixtures, each of which contained the inoculum from the clone as DNA templates. R the reaction was carried out as described above, except that spent only 30 cycles of amplification. One insertion-polatera no erroneous nucleotides 5'-coding sequence of the glucagon receptor, he was selected one clone to obtain the 5'-coding sequence of the human glucagon receptor. Clone 9A uncoupled Eco R1 and the PKK 1 for fragment 551 N. p., containing the 5'coding sequence of the glucagon receptor. the 3'coding sequence of the glucagon receptor was obtained in the form of the PKK 1-You N1 fragment 561 N. p. from 40-2-2. Eco R1 - PKK 1 fragment and the PKK 1-You N1 fragment 561 N. p. was Legerova in the presence of Eco R1 and H1 You to prevent concatemeric. The product of ligation of the fragment 1112 N. p. was purified by gel-electrophoresis and identified as A. For convenience, the fragment A ligated into the cleaved with Eco R1-Bam H1 plasmid pUC18. Legirovannoi mixture was transformed into cells of strain D10b E. Li. and the selected clones were analyzed for the presence of insertions. One clone, A contained insertions.

The coding sequence of the glucagon receptor was built in expressing vector mammals using plasmids RA and clone 40-2-2 to get pornogalereya sequence of the glucagon receptor. elazkudu RA were digested with restrictase vu 11 and Bam H1 for marquee 967 N. p. Clone 40-2-2 split You H1 and Sac 1 for marquee 828 N. p., containing the 3'coding sequence of chelovecheskaia with Eco R1 and it was a small mistake by using T4 DNA polymerase. Then the linearized vector with blunt ends were digested using Sac I. Pvu 11-Batn H1 fragment 967 N. p. and Bam H1 - Sac 1 Fragment 828 N. p. ligated with splitting S 1 vector with blunt ends pHZ1.

Plasmid pHZ1 represents expressing vector, which can be used for protein expression in mammalian cells or in the system broadcast soltow frogs of mRNA, which was transcribability in vitro. pHZ1 unit expression contains the murine promoter metallothionein-1, promoter of bacteriophage T7, flanked by multiple cloning fragments containing unique restriction sites for insertion of coding sequences, terminator human growth hormone and terminator of bacteriophage T7. In addition, Z1 contains the seed of the replication of E. coli; bacterial gene-lactamase; unit expression breeding marker mammals, containing the SV40 promoter and the seed, the gene of resistance to neomycin and terminator of transcription of SV40.

Plasmid Z1 containing the cDNA sequence of the glucagon receptor in the correct orientation relative to the promoter, was named pLJ6'. The connecting sections insertions and vector were sequenced to confirm the presence correcto January 1993 This DNA sequence and deduced from it the amino acid sequence of glucagon receptor shown in SEQ ID 24 and SEQ ID 25.

Example 6

Cloning of cDNA of the receptor glucagon cells of pancreatic islets man

In addition to the cloning of the receptor for glucagon from the liver cells human cDNA of the receptor glucagon was obtained from a cDNA library of cells of pancreatic islets person. An aliquot of the cDNA library of cells of the islets (example 1) was subjected R amplification using the oligonucleotide ZC5763 (SEQ ID 22), which is the sense oligonucleotide containing the website Eco R1, flanked by sequences from the 5'-noncoding sequence of the cDNA of the human glucagon receptor, and the oligonucleotide ZC5849 (SEQ ID 23), which is antimuslim the oligonucleotide containing the Xho site 1, flanked by sequences from the 3'-noncoding sequence of the cDNA of the human glucagon receptor. The inclusion of restriction sites in the oligonucleotide primers facilitate directional cloning products R into a suitable plasmid vector. Preparing the reaction mixture for R containing 4 µl cDNA library of cells of the islets (example 1), 8 ál of 10x Gomeda PCR of bufuralol and 46.5 μl of water. The mixture was heated to 95oC for 3 minutes, then the temperature was lowered to 80oC for 3 minutes. The mixture was stirred at 80oWith until it was ready for use. To start the reaction, to the reaction mixture were added 20 μl of enzyme mixture containing 2 μl of 10x Promega buffer for R (Gomeda), 2 μl of 5 u/µl Taq, 1 polymerase (Cetus) and 16 μl of water. The mixture was layered with 50 ál of mineral oil (Sigma) and the reaction mixture was subjected to 30 cycles of PCR (95oC for 1 minute, 55oC for 1 minute, 72oC for 2 minutes and 15 seconds, during which incubation at 72oWith held for 3 seconds longer in each cycle) followed by a 10-minute incubation at 72oC. then the reaction mixture is kept at 4oC. Agarose gel electrophoresis of 10 µl-aliquot of the product R showed the presence of a fragment of approximately 1.8 to 1.9 KB. On the basis of presence in pLJ6', cDNA of the human glucagon receptor waited for the detection of a fragment of approximately 1845 N. p.

R the reaction mixture was extracted with chloroform, followed by two extraction with a mixture of phenol/chloroform and a final extraction with chloroform. After the final extraction was added 5 μl of 4 μg/μl glycogen carrier (Boerhi were washed in 70% ethanol. Precipitate DNA resuspendable in water and digested using Xh 1 and Eco R1. DNA was purified by gel-electrophoresis and subcloned into the plasmid pBLUESCRIPT SK+(Stratagen Cloning Systems), which was linearized using Xho 1 and Eco R1. Mixture for ligation was transformed into cells of strain DH10B E. coli. (GIBCO-BRL). Selected transformants were obtained plasmid DNA and subjected to its action restrictase and blotting on the Southern. The clones were compared by insertion of cDNA of the human glucagon receptor, present in pLG6'. Selected on the basis of diagnostic splitting restrictase clones were subjected to sequencing. Sequencing showed that one clone, pSLIGR-1, contained the coding sequence of the glucagon receptor. Coding region SLIGR-1 contained three nucleotide substitutions in the encoding region of the glucagon receptor compared to liver cDNA present in pLJ6'. One of nucleotide substitutions was silent mutation, the remaining two led to changes in conservative amino acids, as shown in table 5. Analysis of changes suggests that they may be the result of allelic variation, representing polymorphic differences.

Example 7

Cloning of the receptor for glucagon-televizoriem cDNA of the human glucagon receptor as a probe. Amplificatory genomic library of human placenta FIX II and amplificatory genomic library of the human lung FIX (both libraries obtained from Stratagene Cloning Systems, catalog numbers 946203 and 944201 respectively) were subjected to screening for gene receptor glucagon person.

Amplified genomic library of the human lung was titrated and approximately 4104plaque-forming units (pfu) were sown with cells of strain LE392 E. Li (Stratagene Cloning Systems) on each of the 30 cups with a diameter of 150 mm, an Additional 10 cups seeded by cells of strain LE392 E. coli, approximately 6104pfu per Cup. The cups were incubated overnight at 37oC and 30 cups were selected for screening.

For each of the 30 cups were prepared by two filters. Each filter was prepared by overlaying a Cup YND nylon membrane (Amersham) according to the procedure recommended by the manufacturer. The filters were lifted from the plates, cells were literally 1.5 M NaCl, 0.5 M Paon 5 minutes at room temperature. The filters were neutralized for 5 min in 1 M Tris-Hcl (To 7.5), 1.5 M NaCl and recorded using UV (1200 microjoules) in a STRATALINKER (Stratagene lning Systems). After fixing their pre-filters were washed three times in 0.25 × SSC, 0.25% of LTOs, 1 mm EDTA at 65oC. Pulsational solution (5x SSC, 5x solution Dnhrdt, 0.2% of LTOs, 1 mm EDTA), which was filtered through a 0.45 µm filter and to which was added 100 μg/ml (final concentration) denatured by heating DNA salmon sperm directly before use. Filters were prehybridization at 65oWith during the night.

cDNA of the human glucagon receptor from R40-2-2 were combined with random primers using the kit Amersham MEGAPRiME (Amersham) according to the method recommended by the manufacturer. Prehybridization solution of each series of filters was replaced with fresh prehybridization solution containing 28,5105pulse/min probe. Filters are hybridized at 65oC for 20 hours. After hybridization, the hybridization solution was removed and the filters were washed 4 or 5 times in the washing solution containing 0.25 x SSC, 0.2% of LTOs, 1 mm EDTA at room temperature. After rinsing, the filters were washed 8 times consecutively, with 65oC in the washing solution with subsequent final rinse at 70oC. After washing, the Filters were exposed to film for autoradiography (R-5; Eastman dk.; NY) for 4 days at -70oC with intensifying screen.

Research radioautography revealed the presence of 4 districts hybridise soaked overnight in 1 ml of SM (Maniatis et al., eds., Molecular Cloning: A Laboratory Manual, Cold Spring rbr, NY, 1982; here are the full references), 1% chloroform. After incubation over night phage from each tube was diluted 1:1000 S. Aliquots of 5, 25 and 50 ál were sown with cells of the strain E. coli LE392. The cups were incubated and one filter was prepared from each Cup of crops 5 and 25 ál. Filters were prepared, pregnanediol and hybridized and washed as described above. Filters were exposed to film for autoradiography.

The study autoradiographs found positively labeled regions from each of the 4 clones. In General, 10 agar plugs were taken from the positive areas representing at least two positive zone for each of the original clones. Agar plugs were processed as described above. Phage from each agar plugs were diluted 1: 10000 in SM. Aliquots of 2.5 and 10 µl were sown with cells of the strain E. coli LE392. The cups were incubated and one filter was prepared from cups with well-separated plaques, filters were prepared and hybridized as described above. Radioautography found areas corresponding to the individual plaques. 12 positive plaques were removed, and they were represented by at least one clone from each of the 4 initial positive clones. One plaque from each Cup of ubirajara above. The phage were diluted 1:1000 in S and inoculable in cell culture strain LE392 E. coli. Double-stranded DNA was obtained as described (C) Grossberger (Nu. Acids Res. 15:6737, 1987; included here by reference). Double-stranded DNA was digested using b 1 to release genomic insertions. Agarose gel electrophoresis showed that the clones 2-2-1 and 11-2-1 contained insertions b 1 9 KB (etc., ad), clone 14-2-1 contained an insertion of 15 KB and 3-3-1 contained an insertion of 13 KB. The blot on the Southern split b 1 and Xb-You H1 clones showed that the cDNA of the human glucagon receptor was hybridities fragments are shown in table 6.

Clones 11-2-1 and 14-2-1 were selected for further analysis. For convenience, the names of the clones were changed as shown in table 6. Double-stranded DNA was obtained from each phage clone to sublimirovanny in the plasmid vector according to the method described by Maniatis. DNA was digested b 1, was purified on gel and subcloned into the plasmid pBLUESCRUPT SK+(Stratagene lning Systems), which was linearized by cleavage with the help of b 1 and treated with alkaline phosphatase calf to prevent short circuits in the ring. Mixture for ligation was introduced by electroporation into cells D10 LROM (GIBCO-BRL) in a Biorad GENEPULSER (Biorad Laboratories; Richmond, CA) at 400 ohms, 25 microfarad and 2.3 kinow 6 and 2 were named pSLHGR 6 and SLHGR 2, respectively. These clones were sequenced. Sequencing and comparison with the coding region of the cDNA of the human glucagon receptor revealed the presence of 12 exons containing the coding region. Analysis of localization in the chromosomes was performed on chromosomal threads in metaphase as described Durnamet et al. (Mol. Cell. Biol. 8:1863-1867, 1988, incl. full reference) using biotinylated probe gene of the human glucagon receptor and specific for chromosome 17 centromeric probe. Denaturation of chromosomes, hybridization and detection of color was performed basically as described by Pinkel et al. (OEWG. Natl. d. Sci. USA 83:2334-2938, 1386, incorporated by full reference) modified by Kievits et al. (Cytogenet. Cell Genet. 53: 134-136, 1990, incorporated by full reference), except that hybridization was performed in a mixture of 65% (vol./about.) formamide/10% dextran sulfate/2x SSC and postliberalization washing was performed with a mixture of 65% formamide/2x SSC at 42oAnd then 0.1 x SSC at 55oC, as described by Palmiter et al. (Proc. Natl. Acad. Sci. 89: 6333-6337, 1932, included as full references). The location of q25it was confirmed by staining D1 some hybridized metaphase threads, as described Testa et al. (Cytogenet. Cell. Genet. 60: 247-243, 1932; incl. here in the form of full references). Staining D1 gave Q-band on gene receptor glucagon using the above described method was unsuccessful. Briefly, the library was titrated and cells stamp LE392 E. coli and approximately 5104fu were sown on each of the 30 cups with a diameter of 150 mm. in Addition, 11 of the same cups seeded, about 105 pfu and cells of ptma E. coli LE392. The cups were incubated overnight at 37oC and selected 38 cups for screening.

Filter lifts were prepared, washed and prehybridization, as described above. The cDNA fragment of the receptor glucagon pancreatic islets man, 30 G (example 4) were randomly primiraly using whale Amersham MEGAPRIME (mrshm) according to the method recommended by the manufacturer. Prehybridization solution for each series of filters was replaced with fresh prehybridization solution containing 1,110 land only6pulse/min probe.

Filters are hybridized at 65oWith during the night. After hybridization, the hybridization solution was removed and the filters were washed 4 or 5 times in the washing solution containing 0.25 SSC, 0.25% of LTOs, 1 mm EDTA at room temperature. After rinsing, the filters were washed 8 times consecutively, with 65oWith in the washing solution. After washing with 70oThe filters were exposed to film for autoradiography (XAR-5; Eastman Kodak Co.) within 4 days at -70oC with intensifying screen.

Example 8

Expression of the cDNA of the receptor glucagon in mammals

A. Expression of rat glucagon receptor in cells VNC

Plasmid LJ4 was cotranslationally with plasmid LJ1 cells BHK570 (deposited in ATSS under Accession 10314) using the calcium-phosphate method, mainly described, Graham and Vander Eb (Virology 52:456, 1973, incl. here in the form of full references). Plasmid pLJ1 was produced from plasmid R, which contains the seed Adenovirus 5, the SV40 enhancer, the major late promoter of Adenovirus 2, consisting of three parts, the leader of the Adenovirus 2, 5'- and 3' splicing sites, cDNA DFR, the SV40 polyadenylation signal and RM-1 vector (Lusky and Botchan, Nature 293:79-81, 1981). The unit of expression of the Eco R1-b 1 DFR of R ligated into pUC18, which was linearized by cleavage with Eco R1 and b to construct plasmids pLJ1. Transizione cells were grown in growth medium (modified by way of Dulbecco environment Needle) containing 10% fetal calf serum, 1x PSN mixture of antibiotics (GIB BRL 600-EU medium (growth medium, containing 250 mm methotrexate (MTX)). Cells were destroyed and diluted 1:20 and 1:50 in selective media on 10-cm plates. After 7-10 days of selection in 250 mm MTX colonies were picked using cloning cylinders in cell 24-cell plates. The resulting colonies were tested for the ability to bind to the glucagon as described above (example 3). Binding of glucagon were also carried out on whole cells by planting 2105cells of each clone in the cells of a tablet with 24 cells. The plates were incubated for 72 hours at 37oWith 5% CO2. Binding of glucagon were performed as described in example 3, except that after the final washing cycle, PBS, the cells were removed from the cells in the test tube by trypsinization. The tubes were counted for radioactivity in the counter. Cells with higher pulse count and, therefore, are able to bind most of glucagon, were selected for further characterization. Selected transformants were also tested on Mediaroom glucagon response of intracellular calcium, as described in example 8E, and mediawindow glucagon response of camp as described in example 8D. Tests of binding of glucagon were also carried out on the membrane preparations from transfectants as described below.125I-glucagon. Membranes were obtained essentially as described by Rodbell et al. (J. Biol. Chem. 246:1861-1871, 1971, incl. here in the form of full references). Two series of membranes of the liver received approximately 80 g of liver from 12-16 beheaded female rats. The liver was quickly removed and placed in ice chemical glass. The liver was divided into part 10, Part crushed scissors and connective tissue was removed during the grinding procedure.

Part 10 g separately homogenized in the homogenizer Dun. For each part was added 25 ml of medium (table 2) to the crushed tissue homogenizer and the tissue homogenized in a glass of ice with the help of 8 vigorous blows loose pestle. After homogenization, the homogenates were collected in 450 ml of cold medium (table 2). Joint homogenates was stirred for 3 minutes and filtered through 2 layers of gauze, followed by filtration through 4 layers of gauze. Then the homogenates were centrifuged for 30 minutes at 1500 g at 4oC. Supernatant threw, and precipitation were combined in a clean Dounce homogenizer. Precipitation resuspendable three soft blows loose pestle. Resuspendable precipitation, decantation in 250-ml volumetric vessel containing 62 ml 69% solution of sugar is on cold. The concentration of the solution was brought up to 44.0%0,1% through 69% sucrose or water, depending on the measurement Refractometer (Bausch and Lomb, Rochester, N. Y. ). Sucrose suspension was distributed equally in ultracentrifuge tubes 2589 mm On each suspension was layered on 20 ml of 42.3% sucrose solution (table 2) and the suspension was centrifuged for 150 minutes at 24000 rpm in a SW28 rotor (Sorval, Dupont Company, Wilmington Dl.) at 4oC.

After centrifugation floating material from each tube was removed by suction in a 10-ml syringe through a needle of 18 size. The material of each tube is connected and resuspendable approximately 10 ml of medium (table 2) by retracting and releasing the mixture through a needle in a centrifugal tube. The tube was filled environment (table 2) and was centrifuged at 15,000 rpm in a rotor SS-34 (Sorval).

The supernatant carefully decantation and discarded. Sediment resuspendable in the environment (table 2) and was diluted 1:1000 with distilled water. The absorption was measured in a cuvette of 1 cm to determine the concentration of protein. The protein concentration was determined by the formula

< / BR>
The preparation of the membranes was divided into aliquots, quickly frozen in a bath of dry ice/ethanol and stored at -80oC.

Membranes were obtained from Unkasa of transfectants were rinsed 2 times with cold buffered using phosphate buffer saline (PBS; Sigma Chemical Co., St.Louis, Md.) and each Cup was added 10 ml of PBS containing 1 mm PMSF. Cells from each Cup was scraped in a solution of PBS and cells from each Cup was transferred into a fresh tube. Each Cup was rinsed with 5 ml PBS containing 1 mm PMSF and washings were combined with the corresponding cells. Cells were centrifuged at 2000 rpm in a tabletop centrifuge at 4oC. Supernatant was discarded, and the cells resuspendable in 30 ml of 5 mm HEPES 1 mm SF, pH 7.5. Cells were incubated on ice for 15 minutes followed by centrifugation at 47800 g at 4oC. Each sediment resuspendable in a solution of PBS containing 1 M PMSF, was divided into aliquots and frozen at -80oC.

Competitive analysis tests the binding of glucagon were performed on membrane preparations of rat liver and KSS transformed. Briefly, prepared reaction tubes containing 20 μl of glucagon from solutions of 10-11M - 10-6M, dissolved in 10 mm SPLA, or 20 ál of BSA. To each tube was added 100 μl of 2 buffer for binding; 20 ál125I-glucagon (mrsham); 20 μl of 1 mm NaHCO3; 20 μl of 10 mm HOAc, and 0.5% BSA (Novo Nordisk N/A, Bagsvaerod, Dnmrk); and 40 μl of distilled water. The binding reaction was initiated by addition of 20 μl of the preparation of the membranes to the reaction PR is at high speed for 10 minutes at 4o0. Supernatant of each sample was aspirated and precipitation counted for radioactivity. Competition with it glucagon gave approximately the same sigmoidal curves for the binding of glucagon (Fig.3). Analysis Of Scatchard (Scatchard, Ann. N. Y. Acad. Si. 51: 660-672, 1949, inclusive. here in the form of full reference) showed that the visible d equal to 50 nm for the cloned receptor and 49 nm for membranes of rat liver (Fig.4).

The specificity of the receptor encoded pLJ4, was determined by the ability of the related peptide hormones compete for binding125I-glucagon. Micromolar amounts of glucagon and related peptides (table 7) was added with125I-glucagon to the membrane preparations pLJ4 of transfectants in the test link above. Only native glucagon and antagonist amide des-His1[Glu9] glucagon were able to compete with125I-glucagon for binding to the membranes.

C. Expression of rat glucagon receptor in COS-7 cells

The ability of COS-7 cells transfected pLJ4, be stimulated by glucagon to increase the level of camp was assessed using Amersham SPA kit (Amersham), as described in the thread (example 8). The analysis showed that stimulated glucagon LJ4 the transfectants Naka is ery of related peptides such as secretin, VIP, PTH, GLP and calcitonin were added to the transfectants at concentrations of 100 nm - 1000 nm and evaluated their ability to increase levels of camp. The results of this test showed that none of these pentelow was not able to induce significant levels of camp.

D. determination of luciferase and adenylyl cyclase activity in intact cells

cDNA of rat glucagon receptor expressed in the cell line VNC, stable transtitional Z6, unit expression containing the promoter, which was at least one meeting at camp element luciferase cDNA and hGH terminator. This cell line makes possible the measurement of the luciferase activity of adenylyl cyclase activity and intracellular calcium concentrations in response to the binding of glucagon to its receptor.

Proenkephalin meets at camp element (CRE) in the plasmid Z6 received from Zem233. Zem233 was produced from the plasmid Zem67 and Zem106. Plasmid Zem106 designed from predecessor Zm93. To construct Zem93 PKK I-Bam HI fragment containing the MT-1 promoter, was isolated from MTRGHIII (Palmiter et al. , Science 222:809-814, 1983) and was built in U18. Then plasmid Zm93 were digested with restriction enzyme Sst1 and re-ligated with the formation of promotora.

Proenkephalin CRE was built in the 5'end of the SV40 promoter in Zem106 by splitting first Zem106 restrictase Eco R1 and Sst1 for allocation vector containing fragment. Oligonucleotides Z982 and ZC983 (SEQ 1D 3 and 4, respectively) were designed so that they are coded by hybridization proenkephalin R from nucleotide - 73 to nucleotide - 133 (Come et al., Ntur 323:353-356, 1986), flanked by the 5'-site Eco RI and 3'-site Sst1. Oligonucleotides ZC982 and ZC983 (SEQ ID 3 and 4, respectively) were subjected to the action of kinases, hybridized and ligated with the linearized plasmid Zm106 with the formation of plasmid Zm224.

Plasmid Zem67 was obtained by splitting 119R (Marsh et al., Gene 32: 481-486, 1984) restrictase Sma I and Hind III. Then, a seed crystal (og - district) SV 40 from the position of the card 270 (Pvu II) to position 5171 (ind III) ligated with the linearized pICI9R, receiving plasmid Zm67. Then end Hind III-Bam HI fragment of the gene of resistance to neomycin SV 40 of plasmid pSV 2-peo (ATSS Accession 37149) was built in split ind III-You II plasmid Zem67 getting Zem220.

Expressing unit, SV 40 promoter-gene of resistance to neomycin-SV40 terminator from plasmid Zem220 was allocated in the form of a fragment of Eco RI. plasmid Zem224 were digested Eco RI and treated with alkaline phosphatase calf for preventing the at Zem224 ligated. A plasmid containing SV40 promoter adjacent to the CRE, was named Zem233.

Plasmid Zem233 modified by insertion of additional R sequence, TATA-block and part of the lacZ coding and poly(A) sequences connected directly 3' to proenkephalin sequence R, so that the resulting unit of expression was in the opposite orientation relative units of expression of resistance to neomycin present in Zem233. Plasmid Zem233 was linearizable splitting plasmids SstI and You HI. Oligonucleotides ZC3509 and ZC3510 (SEQ ID 5 and 6, respectively) were designed so that upon hybridization of the received coded duplex-glycoprotein R (Dlgn et al., Mol. ll. il. 7:3994-4002, 1987) with 5' SstI sticky end and a 3' Eco RI adhesive end. The oligonucleotides were hybridized according to standard methods. TATA-block timedancing received in the form of an EcoRI-Pst I fragment from nucleotide -79 to nucleotide +18 gene timedancing (McKnight, Cell 31:355-366, 1982). 3'-the sequence lZ gene and associated poly (A) sequence was obtained in the form of st I-Bam HI fragment from plasmid LF (obtained from Jaques Peschon, Immunex Corp., Seattle, wash.), which contains lZ coding region and murine preteenboy termination sequence, kleinova the TATA-box and Pst I-Bam HI lacZ sequence was enviroware. A plasmid containing the unit of expression in the correct orientation with respect to the unit of expression of the gene of resistance to neomycin Zem233, was named Z5.

Gene luciferase and termination sequence of the human growth hormone (hGH) is used to replace lZ coding and poly (A) sequences present in Z5. Gene luciferase source was obtained from the plasmid-168luc (Dlgn et al., Mol. Cell. il. 7:3994-4002, 1987; de Wet et al. , Mol. Cell. Bil. 7:725-737, 1987) in the form of a Xho I-Xba I fragment of 1.7 KB. The hGH terminator was received in the form of b I-Sal I fragment from Zm219b (deposited in the form of transformant E. Li in ATSC (Rockville, Md.) under Accessi on ATS 68079). Gene is luciferase and hGH terminator was subcloned into the linearized by restrictase Xho I-Sal I plasmid pICI9H (Marsh et al., ibid.) for the convenience. The obtained plasmid Z8 were digested Xho I and Sal I to select sequences of the gene luciferase-terminator hGH. Plasmid KZ5 were digested by Sal I to highlight the vector containing fragment and was treated with alkaline phosphatase calf to prevent re-education of the ring. Xho I - Sal I luciferase-terminator hG fragment ligated with split Sal I plasmid KZ5. A plasmid containing a fragment of the luciferase-terminator hG correctly Orient the bathrooms in ATSS under Accession 10314), using the method of deposition of calcium phosphate, described in Graham and Van dr Eb (Virology 52:456, 1973, incl. here in the form of full references). Transfetsirovannyh cells were grown in the culture medium (modified by way of Dulbecco environment needle (DMEM) containing 10% fetal calf serum, 1x PSN mixture of antibiotics (GIBCO-BRL) and 2.0 mm L-glutamine). After a few days in non-selective medium for the cultivation of this medium was replaced with selective medium G418 (growing medium containing G418 at 500 µg/ml). Then the cells were allowed to grow to confluence, after which they were trypsinization and were sown at limiting dilution in cell tablet with 96 cells. Cells were grown for 1-2 weeks in G418 selective medium. Clones of cells containing single colonies were tested for the ability to respond to Forskolin in the following luciferase test. Forskolin increases the level of cellular camp and, consequently, the associated camp-dependent biological response path-independent receptor. The clone is able to respond to Forskolin, was named VNK/KZ 6-19-46.

VNK/KZ 6-19-46 cells were cotranslationally with pLJ4 and pLJ1 or ZP and LJ1 (Z the transfectants were used as negative controls) as described above using mediawindow calcium phosphate TRANS is in triplicate in inducing R-luciferase response selected agonists. Tested 6 randomly selected LJ4 of transfectants and arbitrary ZP the transfectants (negative controls). Microtiter tablets were prepared so that each cell contained 2104cells in 100 μl of selective among, and cells were grown over night. Agonists were prepared in selective medium at 2-final concentration

1 μm - glucagon

200 nm glucagon-like peptide (GL)

20 nm - vasoactive intestinal peptide (VIP)

100 nm calcitonin (CT)

20 µm Forskolin (CalBiochem, Sn Dig, lif.)

Induction was initiated by adding 100 µl of each of 2 solutions in a cell with three replications. Neindutsirovannom levels were determined in three cells, to which was added 100 μl of DMEM containing 10% fetal calf serum. The plates were incubated for 4 hours at 37oC and 5% CO2to induce the formation of luciferase.

After induction, the medium was removed and cells were washed 1 time with 200 ál/cell PBS. After washing each cell was added 25 μl of 1x reagent for lysis of cell cultures (Luciferase Assay System, Promega Corp., Madison, Wis.) and the plates were incubated for 15 minutes at room temperature. Then the tablet was transferred into a Labsystems Luminoskan microtiter luminometer (Labsystems Inc. , Morton Grove, Ill.), which is Kunda and integrated luciferase signal for 2 seconds per cell. The induction of some number of times luciferase for each agonist was calculated as follows:

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One clone pLJ4 of transfectant, Z6/rGR-DHEFR-2, discovered the induction of 6-10 times luciferase glucagon, but he was not induced GLP, VIP or ARTICLE.

camp-reply clone transfectant KZ6/rGR-DHFr-2, glucagon and Forskolin were tested by radioimmunoassay using camp [125I] scintillation test systems proximity (Amersham) according to the manufacturer's instructions. Briefly, 100 ál 2105cells per ml KZ6/rGR-DHFR-2 cells were sown in aceki cultural tablet with many cells and were grown overnight in selective medium. Glucagon and Forskolin were prepared in DMEM, 10% fetal calf serum, 10 μm VMH when of 0.0001-1000 nm and 26 μm, respectively.

Environment for the cultivation was replaced by 50 μl of the cell agonist (either glucagon or Forskolin). Cells were incubated with agonists for 10 minutes at 37oC, 5% CO2. After incubation the cells were literally by adding 200 μl of boiling water in each cell. After 25 minutes was collected supernatant and diluted 1: 5 or 1:40 in acetate buffer (camp [125I] Scintillation Proximity Assay System (Amersham)). Samples were azetilirovanie using triethylamine and acetic ang is oiginal with 75 ál125I-camp, 75 ál artsyfartsy against succinyl-camp and 75 μl of SPA beads connected with donkey anti-rabbit IgG (all test solutions are provided in the camp [125I] Sintilltin Primity Assay System (Amersham)) in cell LKB T tablet. The tablets were closed tightly and incubated overnight with continuous swing on a rotary shaker at 200 rpm Samples considered for radioactivity in a liquid scintillation counter 1205 BETAPLATE (Pharmacia LKB Instruments Inc., Gaithersburg, Md.). Also received a standard curve of 2-128 Foley acetylated camp. Was defined as the total associated125I-camp and nonspecific binding. Z6/rGR-DFR-2 detected an induction of 140 times the levels of camp in saturating glucagon (10-100 nm) and ED50of 0.25 nm.

E. determining the concentration of intracellular calcium

The responses of intracellular calcium pLJ4 of transfectants to glucagon was tested using the method described by Grynkiewicz et al. (J. Biol. Chem. 260: 3440-3450, 1985, incl. here in the form of full references). Plasmid pLJ4 VNK the transfectants were sown in 2-hole camera cover glass (NUNC) at 5104the cells on the camera. Cells were grown 1-3 days under normal culture in selective medium with methotrexate. The medium was removed by suction and the cells were rinsed twice in 1 m is matney temperature with a solution of Fura-2 AM (table 3). After incubation the solution of Fura-2 AM was removed and cells were washed with 1 ml Imaging Buffer three times. After final rinsing with 0.5 ml of buffer was left in each cell. The cells were kept in the dark at room temperature for 30-120 minutes.

The image acquisition was performed in inverted fluorescent microscope Nicon Diaphot, equipped with a lamp with mercury arc and 10x and 40x Nicon Fluor dry objective lenses. The experiments were controlled and analyzed using SUN SPR II Work Station and software Inovision (Rsrh Triangle Park, N. C.) RATIOTOOL. Alternating excitatory wavelengths were controlled by the software via an automated filter disk (the disk with filters) containing filters, permeable strip them 340 and 380 nm. Emission images were sent dichroic mirror (shut-380 nm) to the camera Dage-MTI 72 CD, amplified image Genesis II, and recorded in digital form by the software.

Intracellular calcium concentration was controlled by calculating the relationship of the intensities of emission at each of the two excitatory wavelengths (340/380) for each picture element (pixel) digital microscopic image field (512480 pixels). Grynkiewicz et al. (ibid.) showed h is that absorb and deesterification acetoxypiperidine (fura-2 AM) used to load cells. Software RATIOTOOL depict this information in the form of a false color image, which can be calibrated to the calcium concentration. Were obtained image and the calculation was performed with an interval of 5 seconds during each experiment.

Cells were monitored for at least 60 seconds (12 images) to establish the reference level before stimulation. Stimulation was performed by adding 0.5 ml buffer for images containing 200 nm glucagon, 0.5 ml of this buffer in the camera, which gave a final concentration of 100 nm. spent monitoring cells and images were recorded for at least 3 minutes after stimulation.

The relationship of the images corresponding to the number of cells in each field was drastically changed soon after the addition of glucagon. This was expressed in quantitative form using RIOL software to calculate an average value for the specific areas of the relationship of the image corresponding to each of the responding cells. Cells with an average fixed ratio of approximately 1.4 to detect the rapid growth of this ratio to a value of 3-4. They remained at this kalibrovochnye experiments testify, it reflects changes in the intracellular calcium concentration from an initial value of approximately 150 nm to a peak of approximately 400 nm.

F. Definition of insiststhat

KSS 570 cells expressing the receptor for glucagon from pLJ4, or false transfetsirovannyh KSS 570 cells were sown in 24-hole culture plates to tissue culture at 200,000 cells per well. After 24 hours the cells in each well were labeled by incubation in 0.5 ml of selective medium with methotrexate (MTX), containing a 2.0 µci myo-(2-3H)Inositol (specific activity 20 CI/mmol; Amersham). At the end of the 24-hour incubation, the cells were washed in 1 ml pre-warmed DMEM (modified by way of Dulbecco medium Needle; JR Biosciences, Lenexa, Kan.), which included a buffer of 20 mm HEPES, pH 7.0 (Sigma Chemical Co.), containing 10 mm LiCl. Leaching medium was removed by suction and replaced with 900 μl of fresh buffered environment. Cells were incubated 5 minutes at 37oC. After incubation, each agonist or antagonist was added to three wells (three frequency), and incubated in accordance with the amounts and conditions set forth in table 8.

The reaction was stopped by placing the cells on ice. After sucking environments cells literately by adding 1 ml DM and 1 ml Leda the La 500 μl of 10 mm EDTA, pH 7.0. Samples were neutralized by adding 900 ál of 1.5 M KOH in 60 mm HEPES-buffer and added (dropwise) a solution of KOH-HEPES up until not reached a pH between 7 and 7.5. Neutralized samples were frozen at -20oWith during the night. Frozen samples were thawed and precipitation was allowed to settle. Supernatant inflicted on microcolony APREP (Amicon), successively washed with 5 ml methanol and 5 ml of 1 M KHCO3with subsequent washing with 15 ml of water. After application of the samples collected passing through the column a solution. The column was washed with 1 ml water 4 times and after each wash was collected fractions of 1 ml of Insiststhat was suirable from column four sequential application of 1 ml of 0.25 M knso3that gave a 1 ml sample after each application of knso3. 10 ml of OPTIFLUOR (packard Instrument Co., Menden, Conn. ) was added to each sample and samples were considered to radioactively. Stimulation inozitfosfornah path was confirmed by increased levels of labeled InsideOut. None of the sample did not observe any increase in the education of insiststhat.

G. Expression of the human glucagon receptor in COS - 7 cells

Plasmid pLJ6' was transfusional in COS 7 cells by the method with DEAE-dextran, as described above. Cells were grown on slides the application of125I-glucagon followed by the emulsion radioautography performed as described in example 3. More than 50% of cells transfected with plasmid pLJ6', specifically bound glucagon.

N. Expression of the human glucagon receptor in UNK cells

Cells BHK570 (deposited in ATSS under Accession 10314) were transliterowany the plasmid pLJ6' using the method of transfection with calcium phosphate (example 8). Transfetsirovannyh cells were selected in the presence of G418 until it became visible a separate colony. Colonies were cloned using cloning cylinders. It was shown that these clones bind glucagon. Linking in situ with itinerancy glucagon were performed as described above.

Transfetsirovannyh the plasmid pLJ6' cells are tested for the accumulation of cAMP as described in example 8C. The transfectants were tested after stimulation by glucagon, secretion, ETC GL-I. Tests showed that transfetsirovannyh pLJ6' cells accumulated higher concentrations of cAMP after stimulation by glucagon, compared with control cells. Stimulation of transfectants by secretion, VIP or GLP-I found no increased accumulation of camp. The response of intracellular calcium was determined basically as described in primarlly, as shown by the rapid increase in fluorescence of the calcium indicator Fura-2. These results show that pLJ6' encodes a functional human glucagon receptor, capable of binding to the glucagon and facilitate the transaction signal.

Based on the above it will be understood that although specific variants of the present invention described herein for purposes of illustration, may be made of numerous modifications without departing from the essence and scope of this invention. Therefore, the invention is not limited to the described, with the exception of the attached claims.

1. An isolated DNA molecule encoding a glucagon receptor, containing the nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 14 from nucleotide 145-nucleotide 1599 and nucleotide sequence of SEQ ID NO: 24 from nucleotide 53 to nucleotide 1486, or encoding a peptide glucagon receptor containing the nucleotide sequence of SEQ ID NO: 14 from nucleotide 226 to nucleotide 570.

2. The DNA molecule under item 1, where the glucagon receptor or peptide glucagon receptor selected from the group consisting of rat and human glucagon receptor and the rat and human inoculate sequence, presented in SEQ ID NO: 15 and selected from the group of amino acids 1 to 485, from amino acid 28 to 142, from amino acid 1 to 477 or peptide glucagon receptor containing the amino acid sequence represented in SEQ ID NO: 15 from amino acid 28 to 150.

4. DNA design, which is a DNA molecule according to any one of paragraphs. 1-3, containing the first segment of DNA encoding a glucagon receptor or peptide receptor glucagon, operatively associated with an additional segment of DNA that is required for expression of the first DNA segment.

5. Line COS cells containing a DNA construct under item 4.

6. Cell line KSS containing a DNA construct under item 4.

7. The method of producing glucagon receptor, wherein implementing the cultivation of a host cell transformed with a DNA construct under item 4.

8. An isolated peptide glucagon receptor containing the amino acid sequence from amino acid Gln at position 28 to amino acids Tyr at position 150.

9. An isolated antibody that is specific associated with glucagon receptor encoded by the DNA molecule described in paragraph 1 or 4, where the aforementioned antibody is a monoclonal the introduction of animal immunogen effective amount of the specified receptor and the subsequent allocation of the specified antibodies.

10. A probe comprising at least 12 nucleotides capable of gibridizatsiya with the DNA molecule characterized in any one of paragraphs. 1-3.

11. The method of detecting the presence of antagonists of glucagon, characterized in that carry out the cultivation of host cells containing the construct DNA under item 4, encoding a glucagon receptor, contacting the antagonist in the presence of agonist with recombinant glucagon receptor glucagon, paired with by the reaction, under conditions and for a time sufficient to allow binding of the antagonist to the receptor and associated with this binding response at the specified path and the detection of reducing the stimulation of the response pathway the cells caused by binding of the antagonist to the receptor glucagon, compared with the stimulation of the response, the way one agonist of glucagon and determining from the received data presence of the antagonist of glucagon.

12. The method according to p. 11, characterized in that by means of the reaction is the adenylyl cyclase pathway.

13. The method according to p. 11, characterized in that the way the response includes a luciferase reporter system.

Priority points and features:

28.08.1992 on PP. 1, 3, 8, PP.R>
01.07.1993 on PP. 2, 4-7, 9-13, which directly or indirectly refer to p. 1 regarding the receptor of the human glucagon (SEQ ID 24).

 

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