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Method of measuring the activity of g(alpha)i- or g(alpha)o-coupled receptors using ca2+ influx in cellsRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test StripMethod of measuring the activity of g(alpha)i- or g(alpha)o-coupled receptors using ca2+ influx in cells description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20050277110, Method of measuring the activity of g(alpha)i- or g(alpha)o-coupled receptors using ca2+ influx in cells. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of international patent application no. PCT/EP2003/013510, filed Dec. 4, 2003, designating the United States of America, and published in German as WO 2004/051264 on Jun. 17, 2004, the entire disclosure of which is incorporated herein by reference. Priority is claimed based on Federal Republic of Germany patent application no. DE 102 56 947.9, filed Dec. 5, 2002. BACKGROUND OF THE INVENTION [0002] The present invention relates to a method of measuring the activation or deactivation of G(alpha)i-coupled or G(alpha)o-coupled receptors, and to methods of identifying agonists or antagonists of such receptors. [0003] G-protein-coupled receptors (GPCR) are an extensive family of proteins which play an important role in signal transduction in cells. The term "GPCR" is derived from their association with a heterotrimeric complex of G(alpha), G(beta) and G(gamma) subunits. The G(alpha) subunits of the receptors involved in the synaptic transmission of signals can roughly be categorised on the basis of their function and coupling with the GPCRs. The members of the G(alpha)s family stimulate the activity of adenylate cyclases, while those of the G(alpha)i/o family inhibit the activity of adenylate cyclases. The proteins of the G(alpha)q and G(alpha)12/13 family are effective stimulators of the activity of phospholipase C(beta). However, these subtypes do not exhibit any action in respect of adenylate cyclase activity. A given GPCR usually interacts with only a single family of the G(alpha) proteins, although some exceptions to this rule are known. [0004] The name G(alpha) proteins is derived from their ability to bind guanosine di- or tri-phosphate (GDP or GTP), which acts as a switch which regulates the activity and association of the G(alpha) protein with the GPCR and the G(beta)/G(gamma) subunits. GDP binds to G(alpha) proteins in their inactive state, in which they are present in non-covalent association with the G(beta)/G(gamma) subunits and a corresponding GPCR. GPCR activation leads to allosteric conformation changes in the receptor, leading to dissociation of the G-protein heterotrimer from the receptor and to dissociation of the bound GDP from the G(alpha) component. The intracellular concentrations of GTP usually exceed the concentrations of GDP by several orders of magnitude. The dissociation of GDP from the G(alpha) subunit therefore leads to binding of GTP. The binding of GTP to the G(alpha) subunit produces an allosteric conformation change which results in the dissociation of the G(alpha) subunit from the beta and gamma subunits and to activation of the effector functions of the alpha subunit. The beta and gamma constituents remain firmly connected with one another and therefore form a single functional unit. As soon as they are released from the complex with the G(alpha) subunit and from the GPCR, they execute various effector functions independently of the G(alpha) constituent. The described G-protein cycle is completed by hydrolysis of the GTP bound to the G(alpha) subunit by its intrinsic GTPase activity. As a result of this step, the G(alpha) subunit returns to the original state, which leads to reassociation with the beta/gamma subunits and finally to binding of the heterotrimer to the GPCR again. [0005] Measurement of the activity of G(alpha)q-coupled receptors on the basis of measurement of the cytoplasmic Ca.sup.2+ concentration, for example in living cells, with small, cell-membrane-permeable molecules, such as fluorescent dyes which change their fluorescent properties after binding of Ca.sup.2+, is known in the art. These methods are based on the fact that G(alpha)q proteins activate phospholipase C(beta), which catalyses the cleavage of phosphotidylinositol-(4,5)-bisphosphate (PIP2) to inositol triphosphate (IP3) and diacylglycerol (DAG). In contrast to PIP2, which is an integral membrane lipid, IP3 is present in the cytosol in dissolved form. Accordingly, IP3 released by the action of phospholipase C(beta) can diffuse to IP3 receptor calcium channels of the endoplasmic reticulum (ER) and effect the release of Ca.sup.2+ from the ER. The resulting increased cytoplasmic Ca.sup.2+ concentration correlates directly with the activation of the GPCR, which is why measurement of the cytoplasmic Ca.sup.2+ permits indirect measurement of receptor activation. Using such methods it is possible, for example, to evaluate potential ligands of the receptor in question with respect to their agonistic or antagonistic properties. [0006] By contrast, measurement of the activity of G(alpha)i- or G(alpha)o-coupled receptors proves to be much more difficult. As discussed above, G(alpha)i and G(alpha)o subunits act on adenylate cyclase. A possible approach therefore consists in measuring the product of this enzyme, cyclic 3'-5'-adenosine monophosphate (cAMP). However, measurement of cAMP is expensive, time-consuming and is limited by a relatively small dynamic range of the test. In a further method, a chimeric G(alpha) protein is introduced into the cells, in which the interaction with the G(alpha)i- or G(alpha)o-coupled GPCR in question is retained, while the downstream effector action of the G(alpha) protein is changed from inhibition of adenylate cyclase to activation of phospholipase C(beta), so that determination of the GPCR activity is again made possible by measuring the cytoplasmic Ca.sup.2+ (see Coward et al. (1999) Anal. Biochem. 270(2): 242-248). However, this technique requires an additional, time-consuming cloning step for the provision of the G(alpha) chimera, especially if stable transfectants are required. Furthermore, owing to the artificial nature of the chimera, artificial results that differ from the actual situation in vivo cannot be ruled out. SUMMARY OF THE INVENTION [0007] The object underlying the present invention is, therefore, to provide an improved method of measuring the activity of G(alpha)i- or G(alpha)o-coupled receptors. [0008] It was also an object of the invention to provide a novel method of measuring the activity of G(alpha)i- or G(alpha)o-coupled receptors which overcomes the disadvantages of methods known in the prior art. [0009] These and other objects have been achieved in accordance with the present invention by providing a [0010] The relates in particular to a method of measuring the activation of a G(alpha)i- and/or G(alpha)o-coupled receptor in cells that express at least one G(alpha)q-coupled receptor, which method comprises: [0011] (a) simultaneously treating the cells with an amount (or concentration) of an agonist of the (at least one, i.e., in the case of a plurality, of any one of the plurality) G(alpha)q-coupled receptor such that a sub-threshold activity is obtained, and with an agonist of the G(alpha)i- and/or G(alpha)o-coupled receptor, and [0012] (b) measuring the cytoplasmic Ca.sup.2+ concentration of the cells. [0013] The measuring method according to the invention permits indirect measurement of the activity of G(alpha)i/o-coupled GPCRs on the basis of a synergistic interaction between activated G(alpha)q-coupled GPCRs and G(alpha)i/o-coupled GPCRs. In particular, phospholipase C(beta) (abbreviated to PLC(beta) hereinbelow) is activated in two separate phases. During the first phase, free G(beta)/G(gamma) subunits bring PLC(beta) to the plasma membrane, as a result of which the enzyme and its substrate (PIP2) are brought close together. During the second phase, G(alpha)q subunits activate the enzymatic activity of PLC(beta). Using an agonist of the G(alpha)q-coupled receptor, for example ATP or UTP, to initiate a specific sub-threshold activation of G(alpha)q receptors, the activity of G(alpha)i/o-coupled receptors can therefore be measured in a simple and effective manner. [0014] In accordance with the invention, the amount or concentration of the agonist of the G(alpha)q-coupled receptor that produces a "sub-threshold" activity is especially that amount or concentration at which the ratio of the result of a measurement of the cytoplasmic Ca.sup.2+ concentration of cells treated with a specific amount of an agonist of the G(alpha)q-coupled receptor to the result of the measurement of the cytoplasmic Ca.sup.2+ concentration of cells treated with the same amount (or concentration) of the above-mentioned agonist of the G(alpha)q-coupled receptor and at the same time with an amount (concentration) of an agonist of a G(alpha)i- and/or G(alpha)o-coupled receptor that is sufficient for the complete activation thereof, is as small as possible, that is to say does not exceed about 1:3. Preferably, this ratio is not more than about 1:10, especially not more than about 1:20, particularly preferably it is minimal for the respective pair of agonists of the receptors. Of course, the above ratio of the measurement results can also be formed the other way round (that is to say, cytoplasmic Ca.sup.2+ concentration after simultaneous treatment with an agonist of a G(alpha)q receptor and an agonist of the G(alpha)i/o-coupled receptor to cytoplasmic Ca.sup.2+ concentration after treatment only with an agonist of a G(alpha)q-coupled receptor). In this case, the amount or concentration of the agonist that initiates a sub-threshold activity of the G(alpha)q-coupled receptor corresponds to the amount or concentration at which the ratio of the measurements of the cytoplasmic Ca.sup.2+ concentrations is as large as possible, that is to say is not less than about 3:1, preferably at least about 10:1, especially at least about 20:1. [0015] This means that the amount (concentration) of the agonist of the G(alpha)q-coupled receptor that produces a sub-threshold activity is the amount (concentration) at which maximum signal amplification possible is detected on measurement of the cytoplasmic Ca.sup.2+ concentration, when the cells are treated simultaneously with the agonist of the G(alpha)q-coupled receptor and with the agonist of the G(alpha)i/o-coupled receptor in question, in comparison with a measurement of the Ca.sup.2+ concentration obtained on treatment of the cells only with the same amount or concentration of the agonist of the G(alpha)q-coupled receptor. Because the cytoplasmic Ca.sup.2+ concentration is a measure of the activity of phospholipase C(beta), the method according to the invention, and the determination of the amount of the agonist of the G(alpha)q-coupled receptor that produces a sub-threshold activity, can of course be carried out with the aid of any other suitable test for determining the activity of phospholipase C(beta). [0016] The measuring method according to the invention is also especially suitable for identifying agonists of a given G(alpha)i- or G(alpha)o-coupled receptor. The present invention accordingly further provides such an identification method, which comprises (i) providing cells that express the G(alpha)i- and/or G(alpha)o-coupled receptor and at least one G(alpha)q-coupled receptor, and (ii) carrying out the above measuring method according to steps (a) and (b). In contrast to the measuring method according to the invention, of course, a (known) agonist of the G(alpha)i- or G(alpha)o-coupled receptor is not used in step (a), but a test substance whose effect on the GPCR in question is to be investigated is employed. [0017] The measurement principle of the present invention can be used not only to measure the activation of G(alpha)i- or G(alpha)o-coupled receptors by corresponding agonists, but likewise to measure deactivation, or prevention of activation, owing to antagonists of these receptors. The present invention therefore provides a method of measuring the deactivation of a G(alpha)i- and/or G(alpha)o-coupled receptor in cells that express at least one G(alpha)q-coupled receptor, which method comprises the steps: [0018] (A) simultaneously treating the cells with an amount (or concentration) of an agonist of the G(alpha)q-coupled receptor such that a sub-threshold activity is obtained, and with an amount (concentration) of an agonist of the G(alpha)i- and/or G(alpha)o-coupled receptor which is just sufficient for complete activation; [0019] (B) measuring the cytoplasmic Ca.sup.2+ concentration of the cells; [0020] (C) simultaneously treating the cells with the same amount (or concentration) as in step (A) of the agonist of the G(alpha)q-coupled receptor, with the same amount (or concentration) as in step (A) of the agonist of the G(alpha)i- and/or G(alpha)o-coupled receptor and with an antagonist of the G(alpha)i- and/or G(alpha)o-coupled receptor; [0021] (D) measuring the cytoplasmic Ca.sup.2+ concentration of the cells; and Continue reading about Method of measuring the activity of g(alpha)i- or g(alpha)o-coupled receptors using ca2+ influx in cells... 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