Method for detecting leptin receptor ligands

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and can be used for screening of compounds with properties of agonists or antagonists of leptin receptor. There is disclosed method for detecting leptin receptor definition that provides contact of a candidate compound and cells expressing after contransfection with two expressing vectors, respectively, two fused proteins, one of which consists of a short isoform of leptin receptor (OBRs) and an energy donor protein (preferentially luciferase), and the second - of OBRs and an energy acceptor protein (preferentially GFP or its mutant form); measurement of energy transfer (BRET) between the fused proteins; comparison of its value to the relevant indicator measured in the same system, however without tested compound, and estimated result where higher energy transfer indicates binding of the candidate compound-candidate with leptin receptor. Prospective application of the invention is related to development of the preparations for prevention or treatment of disease wherein leptin or its receptor are involved.

EFFECT: development of effective method for detecting leptin receptor ligands.

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The present invention relates to a method for determining ligands leptin receptor through the use of energy transfer between the fused protein consisting of leptin receptors and proteins that are donors or acceptors of energy.

It also applies to the fused proteins used for the implementation of this method.

Leptin is a protein with a molecular mass of 16 kDa, which is secreted by adipocytes. This protein is associated with feeling of fullness and plays an important role in controlling weight gain, energy intake, bone formation, angiogenesis, as well as in other physiological processes, such as the beginning of puberty and control over reproduction or regulation of the immune response, regulated by T lymphocytes.

The leptin receptor (OBR) belongs to the family of cytokine receptors. It consists, as shown in figure 1, of a single polypeptide chain consisting of the transmembrane domain (artaglia et al., J.Biol.Chem, 272, 6093-6096, 1995). In the international patent application WO 97/19952 described this receptor.

Also described six different isoforms of the receptor OBR with C-terminal parts of different lengths. All these isoforms derived by alternative splicing from a single gene. There is also a soluble form OBR containing the binding site with leptin, according to the corresponding extracellular domain of the membrane-bound forms. This posttranslational soluble form generated in the plasma membrane as a result of proteolysis of the membrane-bound forms, is in the blood. In very rare cases reveals a different soluble form OBR, which is the result of a mutation that produces a stop codon before the transmembrane domain.

Fused protein consisting of the long form of the leptin receptor (OBRI), merged with the protein EGFP (Enhanced Green Fluorescene Protein), used according to Lundin et al. (Biochemica et Biophysica Acta, 1499, 130-138, 2000) to study the localization of the receptor.

Activation of the receptor OBR flows, apparently, by tetramer complex, consisting of two janus kinase 2 (JAK2) and two OBR. It is assumed that activation of the receptor induced by leptin leads to conformational changes in the receptor OBR, resulting in he activates one kinase JAK2, which in turn transphosphorylate another kinase JAK2, then the receptor OBR.

Activation of the receptor OBR is responsible for all known manifestations of leptin, such as weight loss and all things related to violation of weight.

Recently been discovered inhibiting properties of leptin in relation to the synthesis of bone tissue. Leptin acts as an inhibitor of osteoblast activity,i.e. the population of cells responsible for the formation of bone tissue.

By ISM is in the content of leptin could cure diseases, associated with reduced bone density, such as osteoporosis or, on the contrary, the disease associated with significant calcification.

In 1999, the authors Hee et al (Proc. Natl Acad Si USA A96, 151-156), was described method of determining Libellula interactions in living cells.This method became the object of international patent application WO 99/66324.

This method, called BRET (abbreviation from Bioluminescent Resonance Energy Transfer or energy transfer bioluminescent resonance) based on natural phenomenon, in which marine organisms emit a fluorescent glow. Enzymatic transformation under the action of luciferaseRenilla(Luc), the substrate, which can pass through the membrane, generates bioluminescent energy, which in turn excites the acceptor energy, such as yellow fluorescent protein (YFP). This method corresponds to the LRET (abbreviation Luminscent Resonance eperdu Transfer), described by Wang et al (Mol Gen Genet 264: 578-587(2001)).

The efficiency of energy transfer depends on the physical distance and the relative position of the acceptor and donor energy. It is therefore not enough joint expression of luciferase and YFP to induce energy transfer, because the distance between the two participants of the transfer must be less than 100 Å. To study the interaction between the two potential participants in the interaction between the first protein was fused with luciferase, and the second protein fused with YFP. If two proteins interact, then there may be a transfer of energy.

Since the method is based on the phenomenon BRET, was used to study some receptors, whose structure was significantly different from the structure of the leptin receptor.

Thus, a number of authors describe the application of this method to receptors belonging to the family of receptors associated with G proteins (GPCR), such as beta 2-adrenergic receptors (Angers et al, 2000,Proc. Natl. Acad. Sci. USA10.1073), cholecystokinin type a (SCQ; Cheng et al, 2001.Biol. Chem.276: 48040-48047), and receptors tireotropin-releasing hormone (Kroeger et al, 2001,J.Biol. Chem.276: 12736-12743).

These receptors are of large size have the structure of a complex containing 7 transmembrane domains.

In addition, the authors Boute et al (2001,Mol. Pharmacol.60: 640-645)describe the process of activating the insulin receptor using phenomena BRET.

The insulin receptor consists of covalent dimers, in contrast to non-covalent complexes, as in the leptin receptor. In addition, it has a fairly long intracellular part. In conclusion, the authors showed that the change in BRET induced insulin can be made only with the soluble receptor.

In recent decades, obesity has become a major problem in health care in industrialized countries, in which this paragraph is oblama currently affects 20-30% of the population. These figures appear to be threatening way to grow in subsequent years. Because many causes of obesity, the sources of which are to a greater or lesser extent, on the one hand, environmental factors (the state of food, availability of food, energy costs...) and, on the other hand, numerous genetic factors create a real problem for medicine.

On the other hand, bone diseases such as osteoporosis, also affect increasingly large part of the population.

Thus, the discovery of new molecules, which allows to treat different diseases that involve the leptin receptor, such as obesity and osteoporosis, it would be a big step forward in health.

At the same time, there is no specific way of screening for agonists or antagonists of the receptor for leptin, which would be highly effective.

The applicant has set a task to develop a test for the rapid, specific and effective screening of agonists or antagonists of leptin receptor.

The applicant has unexpectedly found that the change in BRET induced by leptin, may be implemented using one of the isoforms of the leptin receptor, but cannot be implemented, with all the isoforms.

The applicant also determined that the application to BRET d is eToro leptin optimally under certain operating conditions.

Thus, the present invention relates to a method of identifying ligands of the receptor for leptin by using a transfer of resonance energy between the first fused protein consisting of leptin receptor or the main part of the leptin receptor, and protein, which is the donor of energy, or from the main and active part of the protein, which is the donor of energy, and the second fused protein consisting of leptin receptor or the main part of the leptin receptor, and protein, which is the acceptor of energy, or primary and active part of the protein, which is the acceptor of energy.

The invention relates to fused proteins used for the implementation of this method, as well as nucleic acids encoding these proteins.

The invention relates also to a method of treatment or prevention of diseases involving leptin, consisting in the introduction of the ligand, selected according to the above method, the patient who is suffering from the above disease.

Thus, the first object of the present invention is fused protein, characterized in that it consists of leptin receptor or the main part of the leptin receptor, and protein, which is the acceptor or donor energy, or primary and active part of the protein, which is the acceptor or donor of energy.

<> Slit proteins according to the invention consist essentially of the portion that corresponds to the partial or complete sequence of the leptin receptor, and of the part corresponding to the protein - donor or protein-acceptor energy. They may also include other amino acid sequences derived from other proteins, such as signal sequence. Thus, the sequence SEQ ID No. 4 consists of a partial sequence of SEQ ID No. 2 and other sequences, in particular from the signal sequence of the interleukin 3 mouse.

Mainly protein, which is the donor of energy, is to Renilla luciferase. However, this protein may be any protein - donor energy, whose emission spectrum overlaps sufficiently with the excitation spectrum of the acceptor in order to ensure efficient energy transfer between the two partners. Thus, protein - donor of energy can be protein GFP, if the energy transfer is a FRET, or acorin, if the energy transfer is a CRET. Acorin can be obtained and used according to the method described in the patent application EP 0 187 519 or article Inouye et al. (PNAS USA 82: 3154-3158 (1985)).

Fluorescent protein acceptor energy is preferably DsRed, GFP or a mutant, such as YFP, EYFP, GFP wild type, GFPS6T or pz.

It can be represented by any other fluorescent protein acceptor energy, whose excitation spectrum overlap would be sufficient with the emission spectrum of the donor for efficient energy transfer between the two partners.

These proteins are known to the expert, their sequence, he will be able to find in the literature, in particular, the review Blinks et al. (Pharmacol. Rev. 28: 1-93 (1976)). In particular, the protein GFP described by Tsien (Annu. Rev. Biochem. 67: 509-544 (1998)), and its cloning described Prasher et al. (Depe 111: 229-233 (1992)). Cloning DsRed described in Matz et al. (Nat. Biotechnol. 17: 969-973 (1999)). Against Rluc specialist will find the information in the work Blinks et al. (Pharmacol. Rev. 28: 1-93 (1976)) or in the work of Lorenz et al. (PNAS 88: 4438-4442 (1991)).

Mainly isoform of the leptin receptor, which is fully or partially fused protein, is a short isoforms or has a short intracellular domain.

This isoform contains predominantly intracellular domain Box 1, but does not contain intracellular domain Box 3.

Preferably, this isoform is an isoform OBRs, more preferably, isoform OBRs person. This isoform may have a different origin.

It could also be any other isoform, preferably short, and more preferably containing at least the extracellular domain of the receptor OBR, such as the soluble form of the receptor, BR, contains the binding site with leptin, described by the authors Lee et al. Nature 379, 632-635 (1996)), Gavrilova et al. (JBC 272 : 30546-30551 (1997)), Maamr. et al. (Endocrinology 142 : 4389-4393 (2001)) or Clement et al. (Nature 392 : 398-401 (1998)).

According to a particularly preferred variant implementation isoform represents the isoforms OBRs person with the sequence SEQ ID No. 2. It may be a variant of this protein, the sequence of which is identical to the sequence SEQ ID No. 2 of at least 65%, preferably at least 75%, even more preferably at least 85% or 95%.

Fused protein may contain only part of the isoforms OBRs person. Mainly, it contains a part which is between 46 and 866 amino acids in the sequence SEQ ID No. 2.

Thus, the part corresponding to the leptin receptor, may have the sequence SEQ ID No. 4 or a version thereof that is identical to at least 65%, preferably at least 75% and even more preferably at least 85% or 95%.

Particularly preferably fused protein donor has the sequence SEQ ID No6 or a variant of it that is identical to this sequence at least 65%.

Particularly preferably fused protein acceptor has the sequence SEQ ID No. 8, or a variant of it that is identical to this sequence at least 65%.

p> Other objects of the invention are nucleic acids encoding these proteins. Such nucleic acid can be complementary or genomic DNA, and RNA. These nucleic acids or polynucleotides can be in the form of a single chain or duplex.

Especially preferred complementary DNA.

Preferably the invention relates to nucleic acid, the degree of identity which the nucleic acid sequence SEQ ID No. 5 or SEQ ID No. 7 to nucleotide is at least 65%, preferably at least 75%, even more preferably at least 85% or 95%.

According to another aspect of the invention relates to nucleic acid hybridization in the harsh conditions of hybridization with nucleic acid,such as described above, more specifically with a nucleic acid having the nucleotide sequence of SEQ ID No. 5 and SEQ ID No. 7, or a nucleic acid having a complementary sequence.

“The degree of identity between two nucleotide sequences or amino acid can be determined according to the invention when comparing two optimally aligned sequences in the comparison window.

So, a part of the nucleotide or polypeptide sequence in the comparison window may contain insertions or deletions (for example the EP “gaps”) in comparison with the reference sequence (which does not contain these inserts or deletions) for in order to achieve optimal alignment of two sequences.

Percentage calculated by determining the number of positions that are identical to the base of the nucleic acid or the identical amino acid residue detected for the two compared sequences (nucleic acid or peptide), then divide the number of positions in which there is identity between the two bases or amino acid residues, the total number of positions in the comparison window, then multiply the result by 100 to obtain the degree of sequence identity in percent.

Optimal alignment of the compared sequences can be implemented on computer systems using known algorithms contained in the software package company WISCONSIN GENETICS SOFTWARE PACKAGE, GENETICS CONPUTER GROUP (GCG), 575 Science Doctor, Madison, WISCONSIN.

For example, the degree of sequence identity can be calculated using the software BLAST (BLAST version 1.4.9 March 1996, BLAST 2.0.4 February 1998 and BLAST 2.0.6 September 1998), using only the default settings (S.F.Altschul et al., J.Mol.Biol. 1990, 215: 403-410, S.F.Altschul et al, Nucleic Acids Res. 1997, 25: 3389-3402). BLAST search of sequences similar or homologous to the sequence as a reference, is performed using the algorithm of Altschul et al. The corresponding reference sequence is alnost and set the Foundation can be a peptide or nucleic acid sequences or to represent any combination of them.

By “stringent hybridization conditions“ is understood in this description and the following conditions:

1.Competitive binding to membranes and prehybridization:

- Mixing 40 µl of DNA salmon sperm (10 mg/ml) and 40 μl of the DNA of the human placenta (10 mg/ml),

- Denaturation for 5 minutes at 96°C, followed by immersing the mixture in ice

- Remove 2X SSC and the infusion of 4 ml formamide mixture in the test tube for hybridization containing membrane

Add a mixture of two denaturirovannykh DNA

- Incubation at 42°C for 5-6 hours with stirring in a rotary mixer.

2.Competitive binding of labeled probe:

Add to the labeled and purified probe 10 to 50 ál DNA Cot I depending on the number of repetitions,

- 7-10 min denaturation at 95°C,

- Incubation at 65°C for 2-5 hours.

3.Hybridization:

- Remove pre-hybridizing mixture,

- Mixing 40 µl of DNA salmon sperm and 40 µl of the DNA of the human placenta and denaturation for 5 minutes at 96°C, followed by immersion in ice

Add in a test tube for hybridization 4 ml formamide mixture, a mixture of two DNA and the labeled probe is denatured DNA Cot I,

- Incubation for 15-20 hours at 42aboutWith mixing in a rotary mixer.

4.Washing:

- Washing at room temperature in 2X SSC for washing,

- PR is mivka 2 times for 5 minutes at room temperature using 2X SSC and SDS to 0.1% at 65°C,

- Washing 2 times for 15 minutes at 65aboutWith 1X SSC at 65aboutWith and SDS to 0.1% at 65°C.

Wrap the membrane in Saran and leave them for storage.

The hybridization conditions described above are suitable for hybridizing in stringent conditions to nucleic acid molecules with a chain length in the range from 20 nucleotides to several hundred nucleotides.

Of course, the above conditions can be appropriately adjusted according to the knowledge of a specialist, depending on the length of the nucleic acid, which is then subjected to hybridization, or in accordance with the selected type of marking.

Appropriate hybridization conditions can be selected, for example, in accordance with information contained in the work HAMES et HIGGINS (1985, "Nucleic acid hydridization: a prtical approach", Hames and Higgins Ed., IRL Press, Oxford) or work F.AUSUBEL et al. (1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.).

The proteins according to the invention can be obtained in accordance with methods known to the expert. But mostly they are obtained by expression of the nucleic acids described above, encoding these proteins, possibly built into the expression vectors, respectively, selected cells, followed, if necessary, extraction and purification, which may be full or partial.

The invention relates to a recombinant vector, sod is rasamu nucleic acid according to the invention.

Mainly, this recombinant vector containing the nucleic acid, selected from the following nucleic acids:

a) a nucleic acid that encodes a protein, the identity of which, according to the amino acid sequence SEQ ID No. 6 or SEQ ID No. 8, or a peptide fragment or variant of this sequence is at least 65%,

b) a nucleic acid that contains polynucleotide with the sequence SEQ ID No. 5 or SEQ ID No. 7, or a fragment or variant,

C) nucleic acid identity which, nucleotides, nucleic acid sequence SEQ ID No. 5 or SEQ ID No. 7 or a fragment or variant of the latter is at least 65%,

g) nucleic acid hybridization under conditions of stringent hybridization with nucleic acid having the sequence SEQ ID No. 5 or SEQ ID No. 7 or a fragment or variant of the latter.

By “vector“ is understood in the present invention are circular or linear DNA or RNA, which independently may be in the form of a single chain and double chain.

According to a variant implementation, the expression vector comprises in addition to the nucleic acid according to the invention, regulatory sequences directing transcription and/or translation.

According to a preferred variant, recombinant vector according to the invention including the AET, in particular, the following elements:

(1) the elements regulating the expression of embedded nucleic acid, such as promoters and enhancers;

(2) the coding sequence included in a nucleic acid according to the invention, to be embedded into the specified vector, and specified the coding sequence is localized regulatory signals listed in (1) and

(3) the corresponding sequence of initiation and stop of transcription.

In addition, the recombinant vectors according to the invention can include one or multiple sites of initiation of replication in the cells of the host,in which amplification or expression is required, and markers or markers selection.

For example, promoters for eukaryotic cells include the promoter timedancing virus HSV or promoter of L-metallothionine mouse.

Usually to select the appropriate promoter specialist refers to the work of SAMBROOK et al. (1989, “olecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.) or to the methods described by FULLER et al. (1996,Immunology in Current Protocols in Molecular Biology,Ausubel et al).

Preferred vectors according to the invention are plasmids, such as, for example, vectors pCDNA3 (Invitrogen), pQE70, pQE60, pQE9 (Qiagen), psiX174. pBluescript SA, pNH8A, pNH16A, pNH18A, pNH46A, pWLNE0, pSV2CAT, pOG44, pXTI, pSG(Stratagene).

Suitable vectors of typebaculovirus,such as the vector pVL1392/1393 (Pharmingen), used for transfection of the cell line Sf9(ATCC N CRL 1711), obtained fromSpodoptera frugiperda.

Can be also suitable adenoviral vectors, such as human adenovirus type 2 or 5.

Recombinant vector according to the invention can also be retroviral vector or adeno-associated vector (AAV). Such adeno-associated vectors are described, for example FLOTTE et al. (1992,Am.J.Resir.Cell. Mol.Biol.,7: 349-356.

The present invention also relates to cells containing the protein, nucleic acid or vector as described above, or fragments of these cells, lysates of these cells, or membranes of these cells.

Such cells may represent cells isolated from the body and grown in appropriate culture medium. However, such cells are preferably cell lines. Particularly preferred cell line HEK 293, COS (ATCC N CRL 1650), COS-M6 and HeLa(ATCC N CCL2) or Cv I (ATCC N CL 70), Sf-9 (ATCC N CRL 1711), CHO(ATCC N CCL-61) or 3T3 (ATCC N CRL-6361).

The membranes of these cells can be obtained by any known specialist way.

Preferably, the membrane get by mechanical grinding of the cells followed by centrifugation of the obtained emulsions, as shown in the following examples.

The present invention relates also to compositions containing cells that described above, and saponin.

Present from Britanie relates also to a method of determining the binding of the compounds with the leptin receptor, which includes stages, namely, that:

enter into contact with the specified connection with the merged protein, which is the donor of energy, such as described above, and with the merged protein, which is the acceptor of energy, such as described above, or cells, or fragments, or with lysates, or cell membranes containing such protein, and with the appropriate enzyme substrate, and

- measure the energy transfer.

Preferably, the method is carried out using cells treated with saponin.

Fused proteins, which is the donor of energy, and fused proteins, which are acceptors of energy is chosen so that the energy released from the activation of the donor, could most effectively be transmitted to the acceptor.

In an advantageous embodiment of the above method fused protein, which is a donor of energy, is a protein obtained as a result of the merger of the leptin receptor or the main part of the leptin receptor, with luciferase or the main part luciferase, and in this case, the substrate is mainly coelenterazine.

In a preferred embodiment, the method fused protein, which is an acceptor of energy, is a protein obtained as a result of the merger of the leptin receptor or main frequent the leptin receptor with YFP or the main part of YFP.

According to a preferential variant of the method, the transfer of energy, measured in the presence of the compounds, compared with the energy transfer, measured in the absence of the compounds.

Preferably, the method is carried out on the membranes of cells, which are described above.

Preferably, proteins, donors and acceptors of energy, according to the invention, is chosen so that energy transfer occurred through resonance BRET or LRET. However, this energy transfer can be carried out through resonance FRET (Fluorescent Resonance Energy Transfer or energy transfer fluorescence resonance) or RET (Chemioluminescent Resonance Energy Transfer or energy transfer chemiluminescence resonance).

Regardless of the type of energy transfer protein pairs, consisting of the fused protein-donor/fused protein acceptor, is chosen in such a way as to ensure such transfer.

CRET-transfer is the transfer of energy between aquarium, which represents the luciferase and GFP.

FRET-transfer is the transfer of energy between two proteins a family of GFP with different spectra.

For these transfers, the technician can use the work Baubet et al. (PNAS USA 97: 7260-7265 (2000)) in the case of a transfer type CRET, work Matyus (J.Photochem. Photobiol. 12: 323-337 (1992)) and work Pollok et Heim (Trends Cell. Biol. In: 57-60 (1999)) in case the e transfer type FRET.

Another object of the invention is a method of screening for or identifying compounds intended for the prevention and/or treatment of pathologies involving leptin, which includes stages, namely, that:

- carry out the contact specified connection with the merged protein-donor energy described above, and with the merged protein-acceptor energy described above, or with cells in the presence of saponin or without it, or with fragments, or with lysates, or cell membranes containing such proteins, and possibly with an appropriate enzyme substrate, and

- measure the energy transfer.

This method can be used for screening of agonists or antagonists of leptin receptor.

The method according to the invention is suitable for 96-bit or 384 well plates, which are typically used. This method does not require the use of radioactive molecules, it is sensitive, reproducible, fast and the result is easy to read. Thus, the method provides a satisfactory signal/background noise and low cross-reactivity with other ligands that are not leptin. This is due, at least partially, by the fact that activation of the receptor OBR is detected directly at the level of the receptor, which eliminates possible sources of cross-reactivity of the and other levels of signaling pathways, as you can observe in case analysis based on reporter genes or growth of cells.

Furthermore, the method is not limited to one by transduction with a specific signal, on the contrary, it is able to detect all molecules that interact with receptors OBR.

This ability is especially important for large-scale screening, as an increasing number of ligands to membrane receptors can activate certain way and no other.

The present invention relates, furthermore, to the use of compounds selected according to the method, consisting in the fact that

- enter in contact specified connection with the merged protein-donor energy, and with the merged protein-acceptor energy described above, or cells, or fragments, or with lysates, or cell membranes comprising such a protein, and possibly with an appropriate enzyme substrate, and

- measure the transfer of energy,

to obtain drugs intended for the treatment or prevention of diseases involving leptin or its receptor.

Finally, the invention relates to a method for the treatment or prevention of diseases associated with leptin or its receptor, which includes stages:

- selecting the connection method consisting in:

+ kontaktierung the specified connection with the merged protein donor and with the merged protein the acceptor energy, or cells, or fragments, or with lysates, or cell membranes containing such protein, and with the appropriate enzyme substrate, and

+ in the measurement of energy transfer, and

- the introduction of the specified connection to a patient suffering from a specified disease.

Such diseases can be disease associated with decrease in bone density, such as osteoporosis or, on the contrary, the disease associated with significant calcification.

Such diseases can be also diseases that affect weight, such as obesity, diabetes or anorexia.

Compounds according to the invention can be included in pharmaceutical compositions for introducing them to topical, oral, parenteral, nasal, intravenous, intramuscular, subcutaneous, intraocular, etc. by. Preferably, the pharmaceutical composition for injectable formulations contain pharmaceutically acceptable carriers. Refer below them, in particular, sterile, isotonic saline solutions (odnonatrieva phosphate, disodium phosphate, sodium chloride, potassium, calcium or magnesium, etc. or mixtures of such salts), or dry compositions, in particular, liofilizovannye that, after addition, depending on the necessity, of sterilized water or physiological serum, create injecor the administered solutions.

Finally, the method according to the invention also enables the screening of serum from obese patients to determine the presence or absence of functional leptin or screening of molecules that affect the dimerization OBR.

Figure 1 schematically presents the slit proteins. Box 1 indicates the JAK2 binding site; Box 3 indicates the binding site STAT protein; TM denotes the TRANS-membrane domain.

In Fig. 2A and 2B illustrated expression constructs OBR in COS cells, experimentally implemented using RFID tags using the125I-leptin as a radio-ligand. On figa and 2B are indicated respectively the total content in the cells of the OBR and the number of binding sites with the surface of the cells.

On figs shows localization in cells expression constructs OBRI-YFP and OBRs-YFP in the presence and in the absence of leptin.

In Fig. 2D illustrates the activation of JAK2 different designs OBR.

On file illustrates the effect of stimulation by leptin cells, coexpression reporter gene for STAT3 and various designs OBR.

In Fig. 3 shows constitutive dimerization of OBR. The HEK 293 cells expressing these different designs OBR, incubated in the presence coelenterazine. Energy transfer measured using a luminometer.

In Fig. 4A and 4B illustrates the effect of the binding of leptin to design itatively BRET receptors ORB.

On fega: cells HeL expressing these different designs OBR, incubated in the presence of leptin before initiating the reaction of luciferase. Energy transfer measured using a luminometer.

On figw: effect of leptin compared with whole-cell coexpressing OBRs-Luc OBR-YFP in the presence or in the absence of saponin in the total lysates and membrane preparations.

In Fig. 5A-5E shows the optimization and characterization of changes BRET induced by leptin, using receptors OBRs. Membranes obtained from cells HeLa or COS, coexpression OBRs-Luc and OBRs-YFP, preincubation with leptin or without it before initiating the reaction with luciferase.

On fega: Optimization of the relative and absolute levels expresii OBRs-Luc and OBRs-YFP.

On figw: BRET signal Change induced by leptin, depending on time.

On figs: Curves dose-response and BRET/concentration of leptin on the membrane and in intact cells in the presence of saponin (0.05 per cent).

On fig.5D: Competitive binding125I-leptin increase when increasing concentrations of leptin.

On file: Specificity of changes BRET induced by leptin. Membrane preincubation in the presence of saturated concentrations of erythropoietin (EPO, 10U/ml), thrombopoietin (TRO, 10 nm), colony stimulating factor, granulocyte/macrophage (GM-CSF, 250 ng/ml), in erlykin 3 (IL3, 280 ng/ml), interleukin 6 (IL6, 100 ng/ml), prolactin (PRL, 200 ng/ml), factor α stem cells (SCFα, 250 ng/ml), epidermal growth factor (EGF, 100 ng/ml), insulin (Ins, 100 nm), lipopolysaccharide (LPS, 100 ng/ml) and factor α tumor necrosis (TNFα, 50 ng/ml).

Figure 6: Cotransfected COS cells by a constant amount OBRs-Luc (50 ng) and increasing the number of OBRs-YFP: ο,200 ng; ●,400 ng; Δ,800; ♦1600; ◊,3200. Measurement BRET made on cells in the presence of saponin (0,015%), incubated or not incubated in the presence of increasing doses of leptin, and expressed in BRET.

The present invention is illustrated in the following examples, without limiting it.

Materials and methods used in the examples

Construction of plasmids, transfection and culturing of cells

Slit proteins OBR-YFP and OBR-Luc constructed by stitching YFP and Luc-end receptors classical methods of molecular biology. Coded areas YFP obtained from the vector pGFPtpz-N1 CytogemR- Topaze (Packard, Meriden, CT) were introduced into the EcoRV site of the vector pcDNA3/CMV (Invitrogen, Groningen, Netherlands), which contains a modified polylinker website. Coded plot ofRenillaluciferase derived from pRL-CV (Promega, Madison, WI) and is built into the EcoRV site of the modified vector pcDNA3. Coded areas OBRI and OBRs (Dr.Gainsford, Royal Melbourne Hospital, Victoria, Australia) is built in two vectors described above, respectively with whom ity cloning EcoR1/BamH1 and Nhel. Stop codons were demetroulis by site-directed mutagenesis, and set phase fused protein.

Cell line HEK 293, COS-M6 and HeLa were cultured in DMEM, supplemented with the following components: 10% (V/V) FBS, 4.5 g/l glucose, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 1 mm glutamine (Life Technologies, Gaithersburg, MD).

Transit tranfection was performed using the transfection reagent FuGene 6 (Roche, Bale, Sweden).

Fluorescence microscopy

After two days of transfection constructs YFP cells were incubated in the presence of 100 nm leptin for 60 min and 0.01 mm bis-benzamidine for 15 min, then washed using PBS and fixed for 20 minutes at room temperature in 4%cold solution of paraformaldehyde in PBS. Fractions were examined by fluorescence microscopy using FITC filters and DAPI.

Preparation of membranes and solubilization.

The cells were placed on ice, washed twice in PBS at ice temperature and mechanically detached in buffer 1 (5 mm Tris, 2 mm EDTA, pH 7.4, 5 mg/l of trypsin inhibitor soybean, 5 mg/l leupeptin and 10 mg/l of benzamidine) at ice temperature.

The cell suspension homogenized three times for 5 sec on the homogenizer transmitter station (Janke and unkel Ultra-Turrax T25). The lysate was centrifuged at 450 x g for 5 min at 4aboutC and the supernatant was centrifuged at 4000 x g for 30 minutes at 4°C. the Final precipitate was washed twice with buffer 1 and re-suspended in solution (75 mm Tris (pH of 7.4), 12.5 mm MgCl2, 5 mm EDTA with protease inhibitors as described above) and immediately used in the experiments on the binding of the radioactive ligand or in BRET experiments.

Immunoprecipitate JAK2.

HeLa cells were cotransfection plasmids expressing labeled JAK2 via HA2 (Dr.Wojchowsky, Pensylvania State University, Pensylvania, USA), and plasmids containing different designs OBR.

The obtained cells were lysed in lyse buffer (10 mm Tris, 150 mm NaCl, 5 mm EDTA, 5% glycerol, 0.02% of NaN3, NP40 0.1%), orthovanadate 1 mm, 5 mg/l of trypsin inhibitor soybean and 10 mg/l of benzamidine), then centrifuged for 15 min at 13,000 rpm Soluble fraction was immunoprecipitated within 2 hours polyclonal antibody to JAK2 (HR-758) (1 μg/ml) (Santa-Cruz Biotechnology, Santa Cruz, CA).

Immunoabsorption LTO-EF on PAG

Immunoprecipitate JAK2 was denaturiruet in solution (62.5 mm Tris/HCl (pH 6,8), 5% SDS, 10% glycerol and 0.05% blue Bromphenol) at 100aboutC for 10 min, the Proteins were separated using LTO-EF on SDS page in 7%polyacrylamide and transferred to nitrocellulose. Immunodetection carried out antiphosphotyrosine antibody 4G10 (2 μg/ml) (Upstate Biotechnology, Lake Placid, NY). Immunoreactivity was detected using the appropriate secondary antibody linked to the CSO with horseradish peroxidase and chemiluminescent reagent ECL (Amersham, Aylesbury, UK).

Test bind125I-leptin.

Transfetsirovannyh cells worsened serum in DMEM (1% BSA) for 24 h before experiments on binding. To measure the binding of leptin to the surface of the cells, the cells were washed twice using PBS at the temperature of ice and incubated in binding buffer (DMEM, 25 mm Hepes, pH 7.4, 1% BSA)containing 100000 cpm/well125I-leptin (PerkinElmer life sciences, Paris, France) in the absence or in the presence of 200 nm nonradioactive leptin (recombinant human leptin (PeproTech Inc, USA)for 4 h at 4°C. Cells are washed twice using PBS when the temperature of the ice was literally in 1N NaOH and radioactivity was counted on the counter with gamma radiation. To measure the total quantity of leptin associated in the extract, the cells were suspensively in 1.5 ml of binding buffer containing 0.15% of digitonin, for 2 h at 4°C. the Extracts were centrifuged for 30 min in an Eppendorf centrifuge maximum speed at 4°C. the Supernatant (0.2 ml) were incubated125I-leptin concentration 100000 CPM in the total volume of 0.25 ml in the absence or in the presence of 200 nm leptin under stirring over night at s.

Added 0.5 ml of γ-globulin (1.25 mg/ml) and 0.5 ml of polyethylene glycol 6000 (25% weight/vol.) for the deposition of complexes of receptor-ligand formed during centrifugation (17000 x g for 3 min). Sediment p is washed with 1 ml of polyethylene glycol 6000 (12% weight/vol.) and counted.

Activation of the reporter gene.

HeLa cells co-transfusional with the help of 2.6 µg plasmid carrying reporter gene STAT3 (from Dr. Levy, New York University, New York, USA), 200 PG pcDNA3 containing coded plot of Renilla luciferase (used as internal control) and with the help of 1.4 µg different designs OBR or only a carrier. After 48 h after transfection, cells worsened by DMEM (1% BSA) overnight, then stimulated with 1 nm leptin for 6-8 hours. Then cells were washed and lysed in passive lyse buffer (Promega Corporation, Madison, WI) for 15 minutes at room temperature. Total lysates were centrifuged for 2 minutes at 15,000 rpm and the supernatant was used for the quantitative analysis of Luciferase ((Promega Corporation, Madison, WI) using a Berthold luminometer (Lumat LB 9507).

The results are expressed as the ratio of activity of Firefly luciferase activity luciferaseRenilla.

Measurement BRET

After 48 h after transfection of HeLa cells, COS cells and HEK 293 expressing OBR, were detached and washed using PBS. Cells in the amount of 1-2x105distributed optical 96-hole plates (Packard Instrument Company, Meriden, CT) in the absence or in the presence of the ligand at 25aboutC. Membranes obtained from cells expressively OBR, were used in the test for measuring range is of BRET. The substrate coelenterazine h (Molecular Probes, Eugene, OR) was injected to a final concentration of 5 μm and was carried out by reading on fluro/luminometer FusionTM(Packard Instrument Company, Meriden, CT), which provides a consistent integration of fluorescent signals captured using two filters (filter Luc: 485 +/- 10 nm; YFP filter: 530+/-12,5 nm).Attitude BRET describes as the difference between the emission at 530 nm/485 nm karanfilovski fused proteins Luc and YFP and emission at 530 nm/485 nm only one fused protein Luc. The results are expressed in units BRET (BU), 1 unit BRET corresponds to the value of the relationship BRET, multiplied by 1000. The following ligands were used to determine specificity of the quantitative analysis: recombinant human erythropoietin (EPO), insulin (Ins), lipopolysaccharide (LPS, Sigma Aldrich, St.Luis, USA), recombinant thrombopoietin person (TPO), GM-CSF, interleukin 3 (IL3), interleukin 6 (IL6), prolactin (PRL), SCF, EGF and TNFα.

Example 1. Functional expression of the fused protein OBR

Long form (OBRI) and short form (OBRs) receptors OBR were merged With their ends with YFP or Luc (Figure 1). The expression of these fused proteins is confirmed in COS cells, transfected in experiments on binding125I-leptin (Figa). Similar results were obtained in transfected HeLa cells.

Expression on the surface is rnost cells fused proteins and wild-type receptors, expressed in COS cells, varies in the range from 5 to 10%, which is consistent with the already described values. Similar results were obtained with HEK 293 cells expressing endogenous OBR (14+/-3%).

Localization merged OBR protein in HeLa cells was studied by the method of fluorescence microscopy for protein, fused to YFP. Fluorescence caused OBR1-YFP, a point is distributed in the cells, while the fluorescence caused OBRs-YFP localized to the leafs. The stimulation caused by leptin, localizes OBR1-YFP in large intracellular leafs, relevant, apparently, endosomal the compartment. Localization OBRs-YFP does not introduce any significant changes. The results obtained by fluorescence microscopy confirmed the localization of the OBR mainly in the intracellular compartment and are consistent with the described localization of the fused protein OBR1-GFP in COS cells.

Functional expression of the fused protein is assessed by the magnitude of the activation pathway JAK-STAT. Kinase JAK2 is associated with the intracellular domains OBRs and OBR1. Ligand binding induces transphosphorylation JAK2 and phosphorylation OBR1, but not OBRs. Fosforilirovanii OBR1 creates a site zakurivaniya for STAT proteins, which are activated by phosphorylation of tyrosine after binding to the receptor. Activated STAT proteins then dimerize is raised and transferred to the nucleus, where they stimulate the transcription of genes via elements, responsive to STAT, as described Tartaglia (1997, J.Biol.Chem. 272, 6093-6096).

As shown in Fig. 2C, all designs OBRs induce phosphorylation of JAK2, which indicates the activation of JAK2. Activation of reporter gene STAT3 increases 2-4 times using OBR1-wt and fused protein OBR1, while the short isoform does not affect the activity of the reporter gene. These results show that in HeLa cells expressed functional fused proteins OBR.

Example 2. Constitutive dimerization of OBR in living cells.

Dimerization of OBR-Luc and OBR-YFP was investigated in living cells. The most notable energy transfer is observed between OBRs-Luc and OBRs-YFP, as well as between OBR1-Luc and OBR1-YFP expressed in equimolar amounts, which indicates that the constitutive homodimer characteristic of the two receptors (Figa,B). The existence of heterodimers OBRs/OBR1 in living cells is confirmed by the fact that the detected BRET-transfer between OBRs-Luc and OBR1-YFP, as well as between OBR1-Luc and OBRs-YFP. The specificity of these interactions says no significant energy transfer between OBRs-Luc and OBR1-Luc and the fusion protein YFP and insulin receptor described above (BOUTE et al., 2001, cited above).

These results demonstrate that the short and long isoforms are included in hetero - and holocomplex living cells.

Note the p 3. The influence of the binding of leptin on constitutive BRET receptors OBR.

In order to assess the effect of agonists on constitutive BRET, cells were preincubate in the presence of leptin, then initiated the reaction of luciferase with its substrate.

Found no changes in constitutive BRET in the case of homodimers OBR1 and two combinations of heterodimers OBRs/OBR1, while BRET was increased in the case of homodimers OBRs (Figure 4 A).

Changes BRET in the case of homodimers OBRs, induced by leptin was studied in different cell preparations.

Mechanical destruction of cells in hypotonic buffer significantly increases BRET caused by leptin, whereas the initial BRET remains unchanged.

Similar results were obtained with the membrane fraction after separation of cytosol. Since all pairs of OBRs-Luc/OBRs-YFP involved in the formation of the initial signal BRET, only the receptors present on the surface of cells (5-10%)can be stimulated by leptin, which is unable to pass through the membrane of intact cells. The disintegration of cell membranes increases the fraction OBR, permeable to leptin, and thereby increases BRET, induced by leptin.

Similar results were obtained in cells treated with saponin. This component makes holes in the membranes and both the accounts for the permeability of proteins, as, for example, leptin, in intracellular compartments, where the majority of receptors OBRs.

In similar experiments carried out using preparations derived from cells expressively homodimer OBR1 or heterodimer OBRs/OBR1, there were no changes BRET induced by leptin.

Were then determined the number and ratio of OBRs-Luc and OBRs-YFP to optimize BRET induced by leptin (Figa). The best results were obtained when used 500 ng of DNA that encodes OBRs-Luc and 250 ng DNA, coding OBRs-YFP.

Under these optimal conditions, the saturated concentration of leptin increases in 2-2,5 times the initial BRET signal in cells incubated in the presence of saponin, or in membranes derived from cells expressing homodimer OBRs. This increase is a function of time. Maximum values are reached after 20 min after incubation with 1 nm leptin at room temperature (Pigv). At high concentrations of leptin maximum values are reached after 5 minutes after incubation at room temperature.

The effect of leptin is dose-dependent when the value IS about 100 PM (Figs), which is consistent with the values of Ki obtained with fused proteins OBRs-Luc (116 PM) and OBRs-YFP (35 PM) (Fig.5D). The specificity of the test is confirmed by the absence of RET, inetsirovany ligand with intense concentrations of several cytokines and other ligands of membrane receptors, such as erythropoietin, thrombopoietin, GM-CSF, IL3, IL6, PRL, SCFα, EGF, insulin, and LPS and TNFα.

The distribution of receptors in the form of dimers occurs according to the statistical distribution law and the numerical ratio of receptors 1/1 you may get the following distribution of receptors, if they are all in dimeric form: 1/4 Luc/Luc, 1/4 YFP/YFP and 1/2 receptors can run the BRET signal (1/4 Luc/YFP, 1/4 YFP/Luc). However, when measuring BRET a collection of molecules, merged with Luc, gives the luminescence signal and, therefore, at a ratio of 1/1 is observed that only half of the entire population of receptors capable of running the BRET signal. Therefore, for signal amplification BRET was conducted an experiment with the saturation of the molecules Luc-YFP molecules so that the set of molecules Luc was in the form of dimers with YFP molecules (capable to run the BRET signal). The results presented in Fig.6 show that the initial BRET signal is amplified at saturation, and that the stimulation of leptin is proportional to the initial signal and increases in 2-2,5 times the initial signal BRET. At saturation receive the best resolution of the original and induced BRET signal, which allows for easier selection of the desired molecules.

1. The method of determining svyazyvanie the connection with the leptin receptor, which includes stages, namely, that
a) receive cells, together transfetsirovannyh two vectors, one of which provides the expression of the first fused protein, characterized in that it consists of short isoforms of the leptin receptor (OBRs) and luciferase, and the second vector provides for expression of a second fused protein, characterized in that it consists of OBRs and fluorescent protein, presents GFP or one of its mutant forms, and the fact that it is characterized by an excitation spectrum that overlaps the emission spectrum of luciferase, which is part of the first fused protein;
b) shall contact the specified connection with the cells obtained in stage a), and measure the energy transfer;
c) compare the energy transfer measured in the presence of the compounds, with the transfer of energy, measured in the absence of the compounds, and
d) analyze the results obtained in stage C), where the increase in energy transfer correlates with the binding connection with the leptin receptor.

2. The method according to claim 1, characterized in that it is carried out with cells treated with saponin.

3. The method according to claim 1, characterized in that the substrate is coelenterazine.

4. The method according to claim 1, characterized in that the leptin receptor represents the isoforms OBRs person with the sequence SEQ ID NO2.



 

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