Title of Invention

A PROCESS FOR IDENTIFYING A PHARMACEUTICAL

Abstract

ABSTRACT 20/CHENP/2003 A process for identifying a pharmaceutical The present invention relates to a process for identifying a pharmaceutical modifying the action of at least one G-protein-coupled receptor (GPCR) dependent signal transduction pathway of an organism, wherein said process comprises the following steps: a) providing at least one cell which contains at least one GPCR-dependent signal transduction pathway and which produces one or more than one G-protein; b) providing at least one chemical compound to be studied as an agonist or antagonist; c) contact the cell of a) with one or more of the chemical compounds of b); d) determining the quantitative or qualitative effect of the chemical compound or compounds of b) on the signal transduction pathway of the cell of a) by means of a signal transduction pathway-dependent release, wherein the cell provided according to a) produces at least one G-protein hybrid selected from -6qi4myr, -6qs5myr, -6qi4, and -6qs5, whereas the G-proteins -6qi4 and —6qi4myr link the Gs-coupled receptors to the PLCp signal transduction pathway, and the G-proteins -6qs5 and -6qs5myr link the Gi/o-coupled receptors and the Gs-coupled receptors PLCp signal transduction pathway.

Full Text

Description Widely applicable process for identifying modulators of G-protein-coupled receptors The invention relates to a widely applicable process for identifying chemical compounds which modulate G-protein-coupled receptors, by means of novel_hybrid G--proteins_with very broad receptor specificity, and also to chejnicaLCQmpounds which can be identified by such a process. G-protein-coupled receptors (GPCR) play an important role in a multiplicity of physiological processes. They are one of the most important protein families Known to date, and it is assumed that in the human genome about 1000 genes code for this receptor class. GPCRs have a characteristic structure: they are peptide threads which meander in the form of a-helices seven times through the phospholipid bilayer of the cell membrane, arranging themselves in a circle. It is estimated that about 60% of the pharmaceuticals presently available through prescription bind to GPCRs. This underlines the important role of said receptor class for the pharmaceutical research industry. All G-protein-coupled receptors work on a common basic pattern: binding of an extracellular Itgand leads to a conformational change of the receptor protein which can then make contact with a G-protein. The G-proteins located on the cytoplasmic side of the plasma membrane mediate the extracellular signal to the cell interior and can trigger various reactions there. GPCRs are the most important therapeutic target proteins to date. An estimated 40% of the pharmaceuticals prescribed by doctors act as agonists or antagonists of GPCRs. Owing to the size and importance of the protein family and in view of the fact that chemical binding partners are unknown for many GPCRs {orphan GPCRs), it can be assumed that this receptor class will be one of the most important reservoirs for suitable target proteins in the search for novel medicinat substances in the future. GPCRs are integral membrane proteins. They transfer a signal mediated via a mostly hydrophilic signal substance bound to the outer side of the cell into the cell interior via a family of guanine nucleotlde-binding proteins, so-called G-proteins. Depending on the receptor specificity and the G-proteins activated thereby, they trigger-various signal transduction pathways. Depending on the receptor type, various actions are evoked, all of which lead to the formation of second messengers. Thus, activation of a membrane-bound adenylate cyclase may lead to an increase In the intracellular cAMP level, and inhibition may lead to a decrease. Stimulation of a specific phosphodiesterase may lead to a reduction in the cGMP level. Furthermore, the activated G-protein can lead, for example, to an increase of Ca2+ or K+ ions by binding to an ion channel. Furthermore, an activated G-protein can effect activation of a phospholipase and thus formation of inositol 1,4,5-trisphosphate and diacylglycerol. This in turn leads either to a Ga2+ increase or to activation of a protein kinase C, with further effects in both cases. Second messengers are intracellular messenger molecules such as, for example, cAf\1P, cGMP, Ca2+ and others, which trigger reactions in the cell by activating or deactivating intracellular proteins. The heterotrimeric G-proteins are located on the inside of the plasma membrane. They comprise the three subunits a, p and y. An activated receptor makes contact with the G-protein heterotrimer which, as a result, dissociates into an a subunit und the 3Y complex. Both the activated a subunit and the PY complex can influence intracellular effector proteins. The G-protein a subunit family is presently divided into four different classes (Gas, Gai, Gaq and Ga12 classes). GPCRs are classified according to the G-protein involved In the signal transduction. That is, GPCRs of the Gs class mediate adenylate cyclase stimulation via activation of Gets and increase the intracellular cAMP concentration, GPCRs of the Gi class mediate adenylate cyclase inhibition via activation of Gai and decrease intracellular cAMP, GPCRs of the Gq class mediate stimulation of various PLCp isoforms via activation of Gaq and lead to hydrolysis of membrane-bound phosphatidylinositol 4,5-bisphosphate to give diacylglycerol und inositol trisphosphate (IP3). IPS releases Ca2+ from intracellular depots. Most GPCRs can make contact only with one G-protein a subunit family, i.e. they are selective for a particular signal transduction pathway. This narrow specificity is a great hindrance for the purpose of developing a method by which chemical compounds modulating GPCR-dependent signal transduction pathways are to be identified. Moreover, a suitable signal which can be utilized in an assay type with high sample throughput (= high throughput screening = HTS) is obtained only from those signal transduction pathways In which, for example, G-proteIn activation leads to an increase in the intracellular Ca2+ level. Those G-proteins which have altered receptor specifity and are linked to a signal transduction pathway in a different way may be constructed by joining together parts of various G-protelns to give hybrid G-proteins by means of the methods of molecular biology and biochemistry. Hybrid G-proteins are fusion constructs which combine sequences of various Ga subunits within one protein. Thus it is possible, for example by fusion of the Gai receptor recognition region to the Gaq effector activation region, to prepare a Gaq/I hybrid which receives signals from Gi-coupled receptors but switches on the Gaq- PLCP signal transduction pathway. Such a hybrid, in which the C-terminal 5 amino acids of Gaq had been replaced by the corresponding Gai sequence (GaqiS), was first described by Conklin et al., Nature 363, 274 - 276 (1993). This "recoupling" of receptors has the advantage that the assay endpoint (increase in intracellular Ca2+ concentration In comparison with adenylate cyclase inhibition) is more readily accessible through measurement methods and can be used in high throughput screening. However, the disadvantage of said Gaq/Gai fusion constructs Is that they are unable to activate some GPCRs such as, for example, the SSTR1 receptor qi5 (Conklin et al., Mol. Pharmacol. 50, 885 - 890 (1996)). Likewise, fusion constructs between Gaq and Gas have been described. These too have the disadvantage that they cannot link all Gs-coupled receptors to the PLCp signal transduction pathway, like the 32-adrenerglc receptor or the dopamine D1 receptor, for example. Besides C-terminal modifications for altering the linking of receptors to particular signal transduction pathways, an N-terminal modification of Gaq has been described which leads to receptor promiscuity. Receptor promiscuity in this context means the ability of a G-protein to receive and pass on signals from different receptors. In this Gaq protein, the 6 highly conserved N-terminal amino acids were deleted (Kostenis et al., J. Biol. Chem. 272, 19107 -19110 (1997)). This deletion allows the resulting Gq (also called -6q) to receive signals not only from Gq- but also from Gs- or Gl/o- coupled receptors and to pass them on to PLCp. Said Ga subunit now also recognizes receptors such as the SSTR1 somatostatin receptor, the dopamine D1 receptor and the adrenergic p2 receptor. However, even this G-proteIn is unable to recognize the receptor edg5. Moreover, the signal intensity of said G-protein is so weak that it is unusable in practice (Kostenis et al., J. Biol. Chem. 272, 19107 -19110 (1997)). Another known Ga subunit is Gal 6 which links GPCRs from various functional classes to the PLCP-Ca2+ signal transduction pathway. This G-protein is practically nonselective by nature. But even this subunit is not universally applicable, because receptors such as the edgS receptor or the SSTR1 somatostatin receptor couple to Ga16 only weakly, if at all. For this reason, it would be very useful if a G-protein were available which could be activated by other functional GPCR classes and, moreover, would give a sufficiently strong signal In the cell, which could be utilized in an assay, in particular in a high throughput assay, for identifying compounds modulating GPCRs and/or the appropriate dependent signal transduction pathways, for example a signal such as the increase or decrease in the intracellular Ca2+ concentration. The object of the present invention is therefore to provide further hybrid G-proteins for screening processes to Identify chemical compounds, which proteins are characterized by having very broad specificity with respect to the GPCRs which can be recognized, and by the coupling of said G-proteins to a signal pathway leading to an increase in the intracellular Ca2+ concentration. In addition, their expression is at such a high level that signal intensity is improved. The invention relates to a process for identifying a chemical compound modifying the action of at least one G-protein-coupled receptor (GPCR)-dependent signal transduction pathway of an organism, wherein said process comprises the following process steps: a) providing at least one ceil which contains at least one GPCR-dependent signal transduction pathway and which produces one or more than one G-protein; b) providing at least one chemical compound to be studied; c) contacting a cell according to a) with a chemical compound to be studied according fob); d) determining the quantitative or qualitative effect of a chemical compound to be studied from b) on the signal transduction pathway of a cell from a) by means of a signal transduction pathway-dependent measurable signal. The action of at least one G-protein-coupled receptor (GPCR)-dependent signal transduction pathway of an organism can be modified in an inhibiting or stimulating manner. An inhibiting effect of a chemical compound is present if the signal transduction pathway-dependent measurable signal is weaker in the presence of the chemicai compound than in its absence. Compounds evoking such an effect are also called antagonists. On the other hand, a stimulating effect of a chemical compound is present if the signal transduction pathway-dependent measurable signal is stronger than in the absence of the chemical compound. Such compounds are also called agonists. In a preferred embodiment, the process makes use of a cell which produces at least two G-proteins. Said G-proteins may depend on one or on different GPCRs. In principle, all G-proteins are suitable for carrying out the process, regardless of their receptor specificity, their sequence, their structure, the origin with respect to the cell, tissue or organ or with respect to the species for which they are specific. Preference Is given to the cell producing at least one G-protein from among -6qi4myr, -6qs5myr, -6qi4, -6qs5. The G-proteins -6qi4myr, -6qs5myr, -6qi4, -6qs5 are hybrid G-proteins which were assembled from the parts of different G-proteins of the mouse and which, in some cases, contain additional modifications. The G-proteins may be produced by the cell individually or In combination. Apart from the hybrid G-proteins already mentioned, a cell may in particular also produce Galphal 6. Each of the G-proteins may be present in a cell individually or In combination with one or more other G-proteins. Galphal 6 should always be produced in a cell in such a way that it is never produced alone but in combination with another of the G-proteins mentioned above. The amino acid sequences of the preferred G-proteins are disclosed for -6qi4myr In Seq ID No 2, for -6qi5myr in Seq ID No 4, for -6qi4 in Seq ID No 6 , -6qs5 in Seq ID No 8 and for Galphal 6 in Seq ID No 10. The chemical compound Is commonly provided in soluble form. The use of water for dissolving the chemical compound is preferred. Besides the solvent, the solution may contain buffer substances, salts or auxiliaries such as solubilizers, detergents, presen/atives or other substances. Provision of a cell Includes Its production, cultivation and processing. Cell^are. provided, for example, by preparing^u]table„Qgli.o]gte£lal from organs or tissues*^ by propagating suitable cell lineg-eqriicroorganisms. Various suitable culture media can'^'^TJsedforcaltivation. The cetls-^re maintained at the optimum temperature for the organism. Where appropriate, preservatives, antibiotics, pH indicators, blood serum components, blood serum, auxiliaries or other substances are added to the growth medium used In each case. Processes for production, cultivation and further processing are described in standard textbooks (Example; Basic Cell Culture; Ed. J.M. Davis; IRL Press; 1994). ^_^ __ . In preferred embodirnents of the process described above, th^'cel!_of^j^rteb_rate' species, an insect specie^, a C. elegansbr a yeast is provided. In particularly preferred embodiments, a HeLa, 293, COS or CHO cell or a Saccharomyces cerevislae cell is provided. In a preferred embodiment, the intracellular Ca2+ concentration will be used for determining the quantitative or qualitative effect of a chemical compound to be studied on a cell signal transduction pathway of a signal transduction pathway-dependent measurable signal. The change in intracellular Ca2+ concentration can be detected, for example, by using aequorin, a dye, or by the FLIPRTM technique from Molecular Devices. in a preferred embodiment, the processes as described above may be used for identifying a pharmaceutical. The invention also relates to at least one chemical compound which modifies the action of at least one G-protein-coupled receptor (GPCR)-dependent signal transduction pathway of an organism, with said chemical compound having been identified by at least one process of this invention. Such chemical compounds coui include, for example, alterations with respect to the chemical structure of hydrophil signal substances which induce GPCRs, such as particular hormones, scents, or particular pharmaceuticals. The invention further relates to a polynucleotide sequence coding for a polypeptide having the property of a G-protein, which comprises selecting the polypeptide from one of the following sequences: a) a polypeptide having an amino acid sequence according to Seq ID No 2, Se ID No 4, Seq ID No 6 or Seq ID No 8; b) a polypeptide according to a) lacking one or more amino acids; c) a polypeptide according to a) having an additional one or more amino acids; d) an allelic variant of the polypeptide according to a). The allelic variants include all polypeptides resulting from the base composition of the characteristic form of the gene at the defined gene locus for the particular partners constituting the hybrid proteins. In addition, the invention relates to a polynucleotide comprising a polynucleotide sequence, wherein the polynucleotide sequence is selected from one of the followii sequences: a) a polynucleotide sequence according to Seq ID No 1, Seq ID No 3, Seq ID ^ 5 or Seq ID No 7 or the corresponding sequence complementary thereto; b) a polynucleotide sequence hybridizing with a polynucleotide sequence according to a) under stringent conditions. The stringency of a solution is determined by the temperature and salt content thereof. Using the stringency, it is possible to adjust the extent of base pairing of two homologous nucleotide sequences. The stringency is dependent on the length and base composition of a polynucleotide. Stringent conditions in accordance with this Invention are present if 95% or more of the polynucleotide sequence and the hybridizing sequence match. A preferred embodiment of a polynucleotide sequence or a polynucleotide as described above is a polynucleotide which is part of a recombinant vector construct. Recombinant vector constructs may be prepared with the help of relevant specialist knowledge as illustrated, for example, in F.M. Ausubel et al., Current Protocols in Molecular Biology, Wiley &Sons, New York. This entails inserting a polynucleotide coding for an amino acid sequence according to the sequence information describee above (Seq ID No 2, 4, 6, 8) or a polynucleotide sequence according to the sequence information described above (Seq ID No 1, 3, 5, 7) into a base vector. A base vector means a vector into which a polynucleotide sequence of a polynucleotide can be inserted by means of the methods of molecular biology and can be cloned in a microorganism, for example a bacterium, fungus or the cell of a cell culture. The base vector may comprise, for example, a plasmid having an antibiotic resistance marker, an origin of replication suitable for propagating the plasmid in bacteria or cell cultures, and also a promoter suitable for expressing a protein. The base vector may also comprise, for example, a phage vector, a phagemid vector, a plasmid vector, a cosmid vector, a viral vector, a YAC vector or other type of vector. Examples of base vectors are pUC 18, pUCi 9, pBluescript, pKS, pSK, etc. The polynucleotide to be inserted is inserted via suitable restriction cleavage sites by means of the appropriate restriction enzymes which are commercially available from companies such as BioLabs, Roche Diagnostics, Stratagene and others. Such restriction cleavage sites may be, for example, the recognition sites of the restriction enzymes BamHI, EcoRI, Sail, EcoRV, etc. In a preferred embodiment, the recombinant vector construct comprises an expression vector usable in eukaryotes and/or prokaryotes. An expression vector contains a promoter which can be linked functionally to a polynucleotide sequence so that a protein encoded by said polynucleotide sequence is synthesized In an organism, for example a bacterium, fungus or the cell of a eukaryotic cell line. The promoter may be inducible, by means of tryptophan for example, or may be constitutive. Examples of expression vectors are pUCI 8, pUCI 9, pBluescript, pcDNAS.I etc. Ttie invention further relates to a host cell which may comprise a polynucleotide or a recombinant vector construct as described above. In a preferred embodiment, the host cell comprises a human cell. In further preferred embodiments, the host cell comprises the cell of a vertrebrate species, an insect species, a C. elegans, a bacterium or a yeast. In particularly preferred embodiments, the cell comprises a HeLa, 293, COS or CHO cell, an Escherichia coii cell or a Saccharomyces cerevisiae cell. Moreover, other eukaryotic cells, in particular cell lines, bacteria, in particular Bacillus spec, Streptomyces spec, and fungi, in particular Penicillium spec, Aspergillus spec, may be used. The invention furthermore relates to the production of a host cell as described above by introducing a polynucleotide according to one or more of the polynucleotide sequences as disclosed in Seq ID No (1 - 8) or a recombinant vector construct as characterized above Into a eukaryotic or prokaryotic cell. The polynucleotide sequences may be introduced, for example, by electroporation or by transforming the eukaryotic or prokaryotic cells with the polynucleotide sequence, furthermore by Ca2+ phosphate precipitation of the eukaryotic or prokaryotic cells together with the polynucleotide sequence or by other methods. A host cell of this kind may be used for carrying out an above-described process of this invention. The invention aiso relates to a protein having an amino actd sequence selected from the following sequences: Seq ID No 2, Seq ID No 4, Seq ID No 6, Seq ID No 8. Moreover, the invention relates to a process for preparing a protein comprising an amino acid sequence selected from the following sequences: Seq ID No 2, Seq ID No 4, Seq ID No 6, Seq ID No 8, which process comprises the following process steps: a) producing a host cell containing an appropriate polynucleotide sequence and prepared as described above; b) cultivating said host cell in a growth medium suitable for the host cell and also possibly inducing expression of the protein; c) obtaining the cell material and disrupting the cells; d) removing a protein by means of biochemical methods for protein purification. For preparing and purifying the proteins denoted, known methods, as described in F.M. Ausubel et a!., Current Protocols in Molecular Biology, Wiley SSons, New York, may be used accordingly. A protein having an amino acid sequence according to Seq ID No 2, 4, 6, 8 or prepared according to the process described may be used for producing antibodies. Examples Example 1: Activation of a signal transduction pathway via the Ga-protein mutant -6q by various receptors C0S7 cells were cultured in DMEM {Dulbeccos's modified Eagle's medium) with 10% PCS (ietal calf serum) at 37°C (5% CO2). For transactions, 1 x 10^ cells were seeded in 100-mm plates. About 24 h later, the cells were cotransformed with the expression plasmids aq or-6q (1 /jg DNA/100 mm plate) and, in each case, one of the following receptor constructs (4/yg DNA/100 mm plate): M2 (muscarinic receptor in pCD), D2 (dopamine receptor in pCDNAI), kappa (opioid receptor in pCDNAS), SSTR1 (somatostatin receptor in pCMV), A1 (adenosine receptor in CDM7), D1 (dopamine receptor in pCDNAI), V2 (vasopressin receptor in pCD-ps), P2 (adrenergic receptor in pSVL). About 24 h after transfection, the cells were divided into equal portions in 6-well plates and 3//Ci/ml [3H]myo-inosltol (20 Ci/mmol) in DMEM were added. After incubation for 24 hours, the cells were incubated with HBSS (+ 10 mM LiCI) at room temperature for 20 min. The cells were then stimulated with the appropriate agonists for one hour, and the increase in intracellular inositol monophosphates was determined by anion exchange chromatography. Compared with the wild-type sequence, the Ga-protein mutant -6q lacks the six highly conserved amino acid residues at the amino-terminal end. Constructs of this kind are depicted in fig. 1 with respect to the amino terminus. The wild-type sequence is denoted aq (WTq). The results which follow were obtained with the Ga-protein construct-6q. Moreover, fig. 1 presents additional sequence examples. Mutants of this kind or the receptor constructs used were prepared with the aid of standard molecular biological methods, as described in detail, for example in F.M. Ausubel et al., Current Protocols in Molecular Biology, Wiley & Sons, New York. C0S7 cells expressing WTq or -6q and various Gi/o-coupled receptors (A) or Gs-coupled receptors (B) were incubated (37°C) In the presence and absence of the appropriate agonists (see hereinbelow) for 1 h. The increase in intracellular IP1 concentration was measured as in the attached protocol 1. The data represent averages'+ S.E. of 3-7 independent experiments, with each determination performed in triplicate. The following ligands were used: Fig. 2 A: m2 (muscarinic receptor): carbachol (100 ;/M); D2 (dopamine receptor): (-)-quinpirole (10//M); kappa (opioid receptor): (-)-U50488 {^0 fjM); SSTR1 (somatostatin receptor): somatostatin14 (1 //M); B, A1 (adenosine receptor); R(-)- PlA(10mM); Fig. 2 B: D1 (dopamine receptor): dopamine (1 mM); V2 (vasopressin receptor): AVP (1 nM); 32 (adrenergic receptor): (-)-isoproterenol (200 /JM). The numbers below the figures indicate the extent of the particular PLC stimulation as relative increase In PLC stimulation from -6q to WTq. Fig. 2 shows that the Ga-protein mutant -6q stimulates IP1 formation depending on different receptor classes. IP1 is a signal molecule, which is generated in the PLC-P-signal transduction pathway and leads in the further course of the signal transduction to an increase in intracellular Ca2+ concentration. The experimental results for -6q in fig. 2 are compared with stimulation by means of the wild-type construct (WTq) and of a further control with the vector construct without any Ga insert (vector). IP1 release by means of the -6q construct succeeds both with Gi/o-coupled (fig. 2 A: m2, D2, k-OR, SSTR1, A1) and with Gs-coupled (fig 2 B: D1, V2, p2) receptors. Example 2: Preparation of highly expressed mutants of Ga proteins with broad receptor specificity Initially, hybrid G-protein a subunits, which lack the six highly conserved amino acids of the amino terminus and which simultaneously have either an ai or as sequence at the C terminus were constructed. They are denoted -6qj4 or -6qs5, corresponding to the contained ai sequence or as sequence. The construct -6qi4 links the Gs-coupled receptors and also some of the Gi/o-coupled receptors to the PLCp signal transduction pathway. Said receptors also include the SSTR1 receptor or the edgS receptor. Gal 6 cannot link the edgS receptor to the PLCp signal transduction pathway. Gal 6 is a G-protein with broad receptor specificity and has been disclosed in WO 97/48820 (title: Promiscuous G-protein compositions and their use). The construct -6qs5 links the Gi/o-coupled receptors and additionally also the Gs-coupled receptors to the PLCP signal transduction pathway and now also recognizes receptors such as the dopamine D1 receptor or the adrenergic P2 receptor. A combination of said two G-protein a subunits in one cell line thus recognizes a wider range of GPGRs than each subunit separately or than Ga16. The applicability in technical screening procedures could be further improved if expression of said Ga subunits (-6qi4; -6qs5) were increased, because this would also result in a stronger signal. For this reason, additional myristoylation/palmitoylation recognition sequences were inserted into the amino-tenninal region of the Ga subunits. This led to the construction of the G proteins -6qi4myr and -6qs5myr. The protein sequence of -6qi4myr and -6qs5myr with respect to the amino terminus is MGGC, in contrast to MAGC in the original sequence of the -6q variants. The novel constructs, -6qi4myr and -6qs5myr, now contain a consensus sequence for myristoylation/palmitoylation. -6qi4myr was prepared from -6qi4 and -6qs5myr from -6qs5. It is known that removing myristyl or palmltyl residues from G-proteins leads to a redistribution in the cell: loss of palmitate or myristate residues influences the expression pattern of the G-proteins In such a way that removing the fatty acid residues leads to mainly cytosolic localization. G-protein a subunits are found both in the cell membrane and in the cytosol. However, only the membrane-bound G-proteins can pass the signals from GPCRs on to intracellular effectors. Only the consequences of removing a consensus sequence for palmitoylation/myrlstoylation by mutation have been known. It has not been known, however, that introducing the additional consensus site for myristoylation/palmitoylation into the Ga deletion mutants leads to an increase In expression. It was possible to show that introducing additional palmitoytatlon/myristoylation sites increases the amount of Ga subunits expressed in the cell membrane (fig. 3, fig. 4). The SDS-PAGE Western blot (sodium dodecyl sulfate polyacrylamide gel electrophoresis Western blot) in fig. 3 shows distinctly increased expression of -6qi4myr compared to -6qi4. Fig. 4 depicts an SDS-PAGE Western blot of a fractionation into particle fraction (p; membrane-containing) and soluble fraction {s; sc) of qwt and -6qi4myr. The variant with a higher degree of myristoylation/palmitoylation, -6qi4myr, is present only in the particle fraction. For this purpose, 20 ^g of membrane proteins were prepared from transfected C0S7 cells, fractionated by SDS-PAGE gel electrophoresis (10%) and analyzed by means of Western blot analysis using the 12CA5 monoclonal antibody (Roche Biosclences). All G-protein a subunits were detected by the 12CA5 monoclonal antibody (coupled to horseradish peroxidase), which is directed against the HA epitope tag. The HA tag is contained in all G-protein constructs. In qwt und ql5, it replaces amino acids 125-130, in the N-terminaily deleted G-proteins (-6q, -6qi4, -6qi4myr) amino adds 119-124. 20/yg of membrane protein, prepared from transfected C0S7 cells, were in each case fractionated by means of SDS PAGE gel electrophoresis and blotted onto nitrocellulose, and the G-protein a subunits were detected by the 12CA5 antibody. Immunoreactive G-proteins were visualized using a chemiluminescence system (Amersham). Example 3: Stimulation of various highly expressed Ga-proteins with broad receptor specificity by different receptors Stimulation of the highly expressed Ga variants, -6qs5myr and -6qi4myr, by different receptors is depicted in fig. 5 and fig. 6. Fig. 5 shows that -6qi4myr (=A6qi4myr) is connected by Gi/o-coupled receptors (for example dopamine D2, edg5, CCR5, SSTR1, KOR) to the PLCp signal transduction pathway and leads to a strong signal which is proportional to Ca2+ release. The controls used were a vector construct and the Gal 6 protein (+16). Fig. 6 shows that Gs-coupled receptors are linked to the PLCp signal transduction pathway by -6qs5myr (=A6qs5myr). The G-protein Ga16 acts as reference here too. To experimentally determine the released Ca2+ by means of the aequorin system, CHO ceils were cotransfected with the apoaequorin expression plasmid cytAEQ/pCDNAI, the receptor DNA mentioned (SSTR1, KOR, D2, D1, beta2) and the G-protein a subunits G16 and -6qi4myr with the use of lipofectamine. After incubation in OPTIMEM medium for 10 hours, the cells were washed once with RPMl 1640 medium and incubated with 5/jMcoelenterazinef in RPMl 1640at37°C for 2 h. The cells were then washed twice with PBS and stimulated using the appropriate receptor agonists: somatostatin 14 for the SSTR1 receptor, U50488 for the kappa opioid receptor, (-)-quinpirole for the dopamine D2 receptor, dopamine for the dopamine D1 receptor und isoproterenol for the beta2 receptor. Agonist stimulation of Gi/o-coupled receptors (SSTR1, KOR, D2) and Gs-coupled receptors (D1, beta2) leads to activation of the G-proteins G16 and -6qi4myr followed by stimulation of PLCp and intracellular Ga release. Ga binding to the apoaequorin-coelenterazine complex leads to light emission which was measured using a luminometer (TOPCount, Hewlett Packard). Description of the figures: Fig. 1 represents an alignment of the amino-terminal regions of various Ga proteins. Fig. 2 shows a stimulation of the PLCp signal transduction pathway by means of the -6q-Ga protein variation by Gi/o-coupled (A) and Gs-coup!ed (B) receptors using the maximum concentration of the relevant agonist. Fig. 3 shows an SDS-PAGE Western blot with increased expression of -6qi4myr in comparison with -6qi4. in addition, the expression of further Ga proteins is shown. Fig. 4 depicts an SDS-PAGE Western blot showing fractionation into a particle fraction (p; membrane-containing) and a soluble fraction (s; sc) of qwt and -6qi4myr. The G-protein subunits were detected by the 12CA5 monoclonal antibody resulting in protein bands of ~ 42 KD. Fig. 5 shows the linking of various Gi/o-coupled receptors to the PLCp signal transduction pathway by -6qi4myr (=A6qi4myr}. D2, kappa and SSTR1 are Gi/o-coupled receptors. The controls used were a vector construct and the Gal 6 protein (+16). Fig. 6 shows that Gs-coupled receptors are linked to the PLCp signal transduction pathway by-6qs5myr (=A6qs5myr). pi, 32 and D1 are Gs-coupled receptors. A vector construct and the G-protein Gal 6 (+16) serve as reference. Fig. 7 shows the linking of the Gi/o-coupled dopamine D2 receptor to the PLCp-Ga signal transduction pathway in the presence of the low-sensitivity a subunit GIB, in the presence of the very sensitive Ga subunit -6qi4myr and for a combination of G16 and -6qi4myr. It is evident that the potential activation of calcium release by -6qi4myr is not adversely affected by the presence of G16. WE CLAIM: 1. A process for identifying a pharmaceutical modifying the action of at least one G protein-coupled receptor (GPCR)-dependent signal transduction pathway of an organism, wherein said process comprises the following steps: a) providing at least one cell which contains at least one GPCR-dependent signal transduction pathway and which produces one or more than one G-protein; b) providing at least one chemical compound to be studied as an agonist or antagonist; c) contacting the cell of a with one or more of the chemical compounds of b); d) determining the quantitative or qualitative effect of the chemical compound or compounds of b) on the signal transduction pathway of the cell of a) by means of a signal transduction pathway-dependent Ca release, wherein the cell provided according to a) produces at least one G-protein hybrid selected from -6qi4myr having SEQ ID N0:2, -6qs5myr having SEQ ID N0:4, -6qi4 having SEQ ID NO:6, and -6qs5 having SEQ ID N0:8, whereas the G-proteins -6qi4 and -6qi4myr link the Gs-coupled receptors to the PLCp signal transduction pathway, and the G-proteins -6qs5 and -6qs5myr link the Gi/o-coupled receptors and the Gs-coupled receptors PLCp signal transduction pathway, whereas the Ca released is detected by using the dye aquaria or the FLIPR technique and measuring the light emission in a luminometer. 2. The process as claimed in claim 1, wherein the cell provided according to a) produces at least two G-protein hybrids. 3. The process as claimed in claim 1 or 2, wherein the cell provided according to a) produces at least two G-protein hybrids selected from - SEQ ID N0:2, -6qs5myr having SEQ ID N0:4, -6qi4 having SEQ ID N0:6, -6qs5 having SEQ ID N0:8, or Ga]phal6 having SEQ ID NO: 10. 4. The process as claimed in claim 1 or 2, wherein the cell provided according to a) produces at least two G-protein hybrids selected from -6qi4myr, -6qs5myr, -6qi4 or -6qs5. 5. The process as claimed in one or more of claims 1 to 4, wherein the cell according to a) is the cell of a vertebrate species, an msec species, a yeast species, or a C elegans. 6. The process as claimed in claim 5, wherein the cell is a Heal, 293, COS or CHO cell or a cell of Saccharomyces cerevistae. 7. A polynucleotide sequence coding for a polypeptide having the property of a G-protein hybrid, which comprises selecting the polypeptide from one of the following sequences: a) SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8. 8. A polynucleotide comprising a polynucleotide sequence coding for a G-protein hybrid wherein the polynucleotide sequence is selected from one of the following sequences: SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7 or the corresponding sequence complementary thereto. 9. The polynucleotide as claimed in one or both of claims 7 and 8, wherein the polynucleotide is part of a recombinant vector construct. 10. The polynucleotide as claimed in claim 9, wherein the recombinant vector construct is an expression vector usable in eukaryotes and/or prokaryotes. 11. The polynucleotide as claimed in claim 10, wherein the expression vector contains a constitutive and/or inducible promoter, 12. A host cell comprising a polynucleotide as claimed in one of claims 9 to 11, wherein the host cell is a bacterium or a yeast. 13. The host cell as claimed in claim 12, wherein the cell is an Escherichia coli cell or Saccharomyces cerevisiae cell. 14. The production of a host cell as claimed in claims 12 and 13 by introducing a polynucleotide as claimed in one or more of claims 7 to 11 into a prokaryotic cell.

Documents:

20-chenp-2003 abstract.pdf

20-chenp-2003 claims duplicate.pdf

20-chenp-2003 claims.pdf

20-chenp-2003 correspondence others.pdf

20-chenp-2003 correspondence po.pdf

20-chenp-2003 description (complete) duplicate.pdf

20-chenp-2003 description (complete).pdf

20-chenp-2003 drawings.pdf

20-chenp-2003 form-1.pdf

20-chenp-2003 form-13.pdf

20-chenp-2003 form-18.pdf

20-chenp-2003 form-26.pdf

20-chenp-2003 form-3.pdf

20-chenp-2003 form-5.pdf

20-chenp-2003 pct.pdf

20-chenp-2003 petition.pdf