Abstract:
The present invention relates to novel engineered Ga proteins and assay methods of using such proteins to advance drug discovery. Engineered Ga proteins described by the invention contain alterations of at least one and preferably two or more amino acid residues that are highly conserved among all four subfamilies of Ga proteins. A preferred engineered protein disclosed here is a double mutant, Gαπ R178M A326S. This specific combination of mutations yields an unexpectedly amplified effect on Ga function both in terms of GTPase activity (GTP hydrolysis) and GDP dissociation. This synergistic effect may have a profound influence on the way GPCR signaling pathways are examined for the development of new pharmacotherapies, particularly in the field of central nervous system disorders such as Parkinson&#39;s disease.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This Application claims priority to U.S. Provisional Application No. 61/093,184, filed Aug. 29, 2008, which is hereby incorporated by reference herein for all purposes. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
       [0002]    This invention was made with United States government support under grant number IR43NS059082-01 awarded by the following government agency: National Institute of Neurological Disorders and Stroke. The United States government has certain rights in this invention. 
     
    
     BACKGROUND OF THE INVENTION 
       [0003]    G-protein-coupled receptors (GPCRs) are by far the most extensively validated class of therapeutic targets, and there remains tremendous potential for targeting new receptors and their downstream effectors [Neubig et al.  Nat Rev Drug Discov,  2002. 1(3): p. 187-97; and Roth et al.  Curr Pharm Des,  2006. 12(14): p. 1785-95]. There are over 900 distinct GPCRs encoded in the human genome, and aside from approximately 300 which are involved in odor and taste reception, it is thought that hundreds represent viable targets for therapeutic intervention. Over half of existing drugs are GPCR ligands, yet the total number of receptors that they target is less than 30 [Esbenshade, T.  G Protein - Coupled Receptors in Drug Discovery,  2005. Taylor and Francis: p. 15-36]. The proven clinical utility of modulating GPCR signal transduction has sustained formidable efforts in the pharmaceutical industry to identify new GPCR-ligand pairs that control clinically relevant signaling pathways [Mertens et al.  Pharmacogenomics,  2004. 5(6): p. 657-72; and Armbruster et al.  J Biol Chem,  2004], as well as equally vigorous efforts to discover further components of the GPCR signaling machinery that have the potential to become therapeutic targets. 
         [0004]    GPCRs are extensively involved in signal transduction in the nervous system, serving as receptors for the major classes of neurotransmitters including GABA, dopamine and serotonin. Most of the drugs used to treat neurological disorders, including pain relievers, antidepressants and anti-psychotics, exert their effects through GPCRs [Esbenshade, T.  G Protein - Coupled Receptors in Drug Discovery,  2005. Taylor and Francis: p. 15-36]. These include dopamine and dopaminergic agents for the treatment of Parkinson&#39;s disease and cholinergic agents for the treatment of Alzheimer&#39;s disease. Of the 2.2 billion prescriptions issued for the top 200 drugs in 2003, 527 million were for drugs targeting GPCRs, and 147 million were for pain medications targeting an opioid receptor alone—more than the total prescriptions for any other single target class [Esbenshade, T.  G Protein - Coupled Receptors in Drug Discovery,  2005. Taylor and Francis: p. 15-36]. Given their extensive role in neurotransmission, GPCR signal transduction pathways clearly represent promising targets for improving the treatment of neurodegenerative diseases. Moreover, development of strategies for modulating these pathways more selectively would expand the potential for more effective treatments. 
         [0005]    The standard model of GPCR signal transduction had long been restricted to a three-component system: receptor, G protein and effector [Gilman, A. G.  Annu Rev Biochem,  1987. 56: p. 615-49]. The receptor, a cell-surface protein that spans the membrane seven times, is coupled to a membrane-associated heterotrimeric complex that comprises a GTP-hydrolyzing Gα subunit and a Gβγ dimeric partner. Agonist-induced conformational changes enhance the guanine-nucleotide-exchange activity of the receptor, leading to the release of GDP (and subsequent binding of GTP) by the Gα subunit depicted in  FIG. 1 . On binding GTP, conformational changes within the three ‘switch’ regions of Gα allow the release of Gβγ. Separated Gα·GTP and GPβγ subunits are thus free to propagate signaling forward via separate (and sometimes converging) interactions with adenylyl cyclases, phospholipase-C (PLC) isoforms, potassium and calcium ion channels, guanine-nucleotide exchange factors for the small GTPase RhoA, and other effector systems ( FIG. 1 ). The intrinsic GTP hydrolysis (GTPase) activity of Gα resets the cycle by forming Gα·GDP, which has low affinity for effectors but high affinity for Gβγ. In this way, the inactive, GDP-bound heterotrimer (Gαβγ) is reformed and capable once again to interact with activated receptor. 
         [0006]    Based on this cycle of GTP exchange and hydrolysis, the duration of heterotrimeric G-protein signaling is thought to be controlled by the lifetime of the Gα subunit in its GTP-bound state. It is precisely this interaction and the lifetime of the Gα-GTP complex which controls the extent and duration of the signal induced. If a pharmaceutical effector is to exert its most favorable response, optimization of the lifetime of signal transduction would be paramount. There is benefit in having the ability to control and possibly extend the lifetime of the Gα-GTP complex, and in doing so, the duration of the signaling response. The invention described here below in the detailed description section of the invention was designed to address this need. 
         [0007]    In 1996, Dr. Siderovski&#39;s group [Siderovski et al.  Curr Biol,  1996. 6(2): p. 211-2], along with other laboratories [Dohlman et al.  Mol Cell Biol,  1996. 16(9): p. 5194-209; and Druey et al.  Nature,  1996. 379(6567): p. 742-6] independently identified a superfamily of RGS (“regulator of G-protein signaling”) proteins that bind Gα subunits via a ˜120 amino-acid RGS domain and dramatically accelerate their GTPase activity (GAP activity) [Hunt et al.  Nature,  1996. 383(6596): p. 175-7; and Watson et al.  Nature,  1996. 383(6596): p. 172-5], thereby attenuating heterotrimer-linked signaling ( FIG. 1 ). The discovery of RGS proteins and their GAP activity towards Gα subunits resolved apparent timing paradoxes between observed rapid physiological responses mediated in vivo by GPCRs and the slow hydrolysis activity of the cognate G-proteins seen in vitro [Arshaysky et al.  Neuron,  1998. 20(1): p. 11-4]. Thus, in this capacity as negative regulators of GPCR signal transduction, the RGS proteins present themselves as excellent potential drug discovery targets [Neubig et al.  Nat Rev Drug Discov,  2002. 1(3): p. 187-97], given that pharmacological inhibition of RGS domain GAP activity should lead to prolonged signaling from G-proteins activated by agonist-bound GPCRs. 
         [0008]    There are at least 37 RGS proteins encoded by the human genome that contain the signature RGS domain (reviewed in [Neubig et al.  Nat Rev Drug Discov,  2002. 1(3): p. 187-97; Siderovski et al.  Crit Rev Biochem Mol Biol,  1999. 34(4): p. 215-51; and Ross et al.  Annu Rev Biochem,  2000. 69: p. 795-827]). The RGS containing proteins are listed in Table 1, along with the sequences of their respective RGS domains. These proteins are grouped according to sequence homology between their RGS domains and fall into subfamilies with similar multi-domain architectures and similar target Gα subunits. For example, the GAP activity of R7 subfamily members is specific to Gα i/o . subunits [Hooks et al.  J Biol Chem,  2003. 278(12): p. 10087-93], whereas that of the GEF subfamily appears specific for Gα 12/13  subunits ([Suzuki et al.  Proc Natl Acad Sci U S A,  2003. 100(2): p. 733-8; and Kozasa et al.  Science,  1998. 280(5372): p. 2109-11]; cf. [Booden et al.  Mol Cell Biol,  2002. 22(12): p. 4053-61]). Based on structural and biochemical studies with RGS4, RGS domains are thought to exert their GAP activity by stabilizing a conformation of Gα that favors the transition state for GTP hydrolysis [Tesmer et al.  Cell,  1997. 89(2): p. 251-61]. Several key questions are currently being addressed in the field to validate RGS proteins as bona fide drug discovery targets, including whether RGS proteins have significant roles in vivo in the physiological timing of GPCR signal transduction. There has been focus on identifying the particular function of RGS proteins in neuronal signaling in the CNS [Neubig et al.  Nat Rev Drug Discov,  2002. 1(3): p. 187-97]. 
         [0000]    
       
         
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 PROTEIN 
                 ACCESSION 
                   
               
               
                 NAME 
                 # 
                 RGS DOMAIN SEQUENCE 
               
               
                   
               
             
             
               
                 RGS1 
                 Q08116 
                 SLEKLLANQTGQNVFGSFLKSEFSEENIEF 
               
               
                   
                   
                 WLACEDYKKTESDLLPCKAEEIYKAFVHSD 
               
               
                   
                   
                 AAKQINIDFRTRESTAKKIKAPTPTCFDEA 
               
               
                   
                   
                 QKVIYTLMEKDSYPRFLKSDIYLNLL 
               
               
                   
                   
                 (SEQ ID NO: 21) 
               
               
                   
               
               
                 RGS2 
                 P41220 
                 AFDELLASKYGLAAFRAFLKSEFCEENIEF 
               
               
                   
                   
                 WLACEDFKKTKSPQKLSSKARKIYTDFIEK 
               
               
                   
                   
                 EAPKEINIDFQTKTLIAQNIQEATSGCFTT 
               
               
                   
                   
                 AQKRVYSLMENNSYPRFLESEFYQDLC 
               
               
                   
                   
                 (SEQ ID NO: 22) 
               
               
                   
               
               
                 RGS3 
                 P49796 
                 LEKLLVHKYGLAVFQAFLRTEFSEENLEFW 
               
               
                   
                   
                 LACEDFKKVKSQSKMASKAKKIFAEYIAIQ 
               
               
                   
                   
                 ACKEVNLDSYTREHTKDNLQSVTRGCFDLA 
               
               
                   
                   
                 QKRIFGLMEKDSYPRFLRSDLYLDLI 
               
               
                   
                   
                 (SEQ ID NO: 23) 
               
               
                   
               
               
                 RGS4 
                 P49798 
                 SLENLISHECGLAAFKAFLKSEYSEENIDF 
               
               
                   
                   
                 WISCEEYKKIKSPSKLSPKAKKIYNEFISV 
               
               
                   
                   
                 QATKEVNLDSCTREETSRNMLEPTITCFDE 
               
               
                   
                   
                 AQKKIFNLMEKDSYRRFLKSRFYLDLV 
               
               
                   
                   
                 (SEQ ID NO: 24) 
               
               
                   
               
               
                 RGS5 
                 O15539 
                 LDKLLQNNYGLASFKSFLKSEFSEENLEFW 
               
               
                   
                   
                 IACEDYKKIKSPAKMAEKAKQIYEEFIQTE 
               
               
                   
                   
                 APKEVNIDHFTKDITMKNLVEPSLSSFDMA 
               
               
                   
                   
                 QKRIHALMEKDSLPRFVRSEFYQELI 
               
               
                   
                   
                 (SEQ ID NO: 25) 
               
               
                   
               
               
                 RGS6 
                 P49758 
                 SFDEILKDQVGRDQFLRFLESEFSSENLRF 
               
               
                   
                   
                 WLAVQDLKKQPLQDVAKRVEEIWQEFLAPG 
               
               
                   
                   
                 APSAINLDSHSYEITSQNVKDGGRYTFEDA 
               
               
                   
                   
                 QEHIYKLMKSDSYARFLASNAYQDLL 
               
               
                   
                   
                 (SEQ ID NO: 26) 
               
               
                   
               
               
                 RGS7 
                 P49802 
                 GMDEALKDPVGREQFLKFLESEFSSENLRF 
               
               
                   
                   
                 WLAVEDLKKRPIKEVPSRVQEIWQEFLAPG 
               
               
                   
                   
                 APSAINLDSKSYDKTTQNVKEPGRYTFEDA 
               
               
                   
                   
                 QEHIYKLMKSDSYPRFIRSSAYQELL 
               
               
                   
                   
                 (SEQ ID NO: 27) 
               
               
                   
               
               
                 RGS8 
                 P57771 
                 SFDVLLSHKYGVAAFRAFLKTEFSEENLEF 
               
               
                   
                   
                 WLACEEFKKTRSTAKLVSKAHRIFEEFVDV 
               
               
                   
                   
                 QAPREVNIDFQTREATRKNLQEPSLTCFDQ 
               
               
                   
                   
                 AQGKVHSLMEKDSYPRFLRSKMYLDLL 
               
               
                   
                   
                 (SEQ ID NO: 28) 
               
               
                   
               
               
                 RGS9 
                 O75916 
                 NFSELIRDPKGRQSFQYFLKKEFSGENLGF 
               
               
                   
                   
                 WEACEDLKYGDQSKVKEKAEEIYKLFLAPG 
               
               
                   
                   
                 ARRWINIDGKTMDITVKGLKHPHRYVLDAA 
               
               
                   
                   
                 QTHIYMLMKKDSYARYLKSPIYKDML 
               
               
                   
                   
                 (SEQ ID NO: 29) 
               
               
                   
               
               
                 RGS10 
                 O43665 
                 SLENLLEDPEGVKRFREFLKKEFSEENVLF 
               
               
                   
                   
                 WLACEDFKKMQDKTQMQEKAKEIYMTFLSS 
               
               
                   
                   
                 KASSQVNVEGQSRLNEKILEEPHPLMFQKL 
               
               
                   
                   
                 QDQIFNLMKYDSYSRFLKSDLFLKHK 
               
               
                   
                   
                 (SEQ ID NO: 30) 
               
               
                   
               
               
                 RGS11 
                 O94810 
                 SFRELLEDPVGRAHFMDFLGKEFSGENLSF 
               
               
                   
                   
                 WEACEELRYGAQAQVPTLVDAVYEQFLAPG 
               
               
                   
                   
                 AAHWVNIDSRTMEQTLEGLRQPHRYVLDDA 
               
               
                   
                   
                 QLHIYMLMKKDSYPRFLKSDMYKALL 
               
               
                   
                   
                 (SEQ ID NO: 31) 
               
               
                   
               
               
                 RGS12 
                 O14924 
                 SFERLLQDPVGVRYFSDFLRKEFSEENILF 
               
               
                   
                   
                 WQACEYFNHVPAHDKKELSYRAREIFSKFL 
               
               
                   
                   
                 CSKATTPVNIDSQAQLADDVLRAPHPDMFK 
               
               
                   
                   
                 EQQLQIFNLMKFDSYTRFLKSPLYQECI 
               
               
                   
                   
                 (SEQ ID NO: 32) 
               
               
                   
               
               
                 RGS13 
                 O14921 
                 SFENLMATKYGPVVYAAYLKMEHSDENIQF 
               
               
                   
                   
                 WMACETYKKIASRWSRISRAKKLYKIYIQP 
               
               
                   
                   
                 QSPREINIDSSTRETIIRNIQEPTETCFEE 
               
               
                   
                   
                 AQKIVYMHMERDSYPRFLKSEMYQKLL 
               
               
                   
                   
                 (SEQ ID NO: 33) 
               
               
                   
               
               
                 RGS14 
                 O43566 
                 SFERLLQDPLGLAYFTEFLKKEFSAENVTF 
               
               
                   
                   
                 WKACERFQQIPASDTQQLAQEARNIYQEFL 
               
               
                   
                   
                 SSQALSPVNIDRQAWLGEEVLAEPRPDMFR 
               
               
                   
                   
                 AQQLQIFNLMKFDSYARFVKSPLYRECL 
               
               
                   
                   
                 (SEQ ID NO: 34) 
               
               
                   
               
               
                 RGS16 
                 O15492 
                 SFDLLLSSKNGVAAFHAFLKTEFSEENLEF 
               
               
                   
                   
                 WLACEEFKKIRSATKLASRAHQIFEEFICS 
               
               
                   
                   
                 EAPKEVNIDHETHELTRMNLQTATATCFDA 
               
               
                   
                   
                 AQGKTRTLMEKDSYPRFLKSPAYRDLA 
               
               
                   
                   
                 (SEQ ID NO: 35) 
               
               
                   
               
               
                 RGS17 
                 Q9UGC6 
                 NFDKMMKAPAGRNLFREFLRTEYSEENLLF 
               
               
                   
                   
                 WLACEDLKKEQNKKVIEEKARMIYEDYISI 
               
               
                   
                   
                 LSPKEVSLDSRVREVINRNLLDPNPHMYED 
               
               
                   
                   
                 AQLQIYTLMHRDSFPRFLNSQIYKSFV 
               
               
                   
                   
                 (SEQ ID NO: 36) 
               
               
                   
               
               
                 RGS18 
                 Q9NS28 
                 SFDKLLSHRDGLEAFTRFLKTEFSEENIEF 
               
               
                   
                   
                 WIACEDFKKSKGPQQIHLKAKAIYEKFIQT 
               
               
                   
                   
                 DAPKEVNLDFHTKEVITNSITQPTLHSFDA 
               
               
                   
                   
                 AQSRVYQLMEQDSYTRFLKSDIYLDLM 
               
               
                   
                   
                 (SEQ ID NO: 37) 
               
               
                   
               
               
                 RGS19 
                 P49795 
                 SFDKLMHSPAGRSVFRAFLRTEYSEENMLF 
               
               
                   
                   
                 WLACEELKAEANQHVVDEKARLIYEDYVSI 
               
               
                   
                   
                 LSPKEVSLDSRVREGINKKMQEPSAHTFDD 
               
               
                   
                   
                 AQLQIYTLMHRDSYPRFLSSPTYRALL 
               
               
                   
                   
                 (SEQ ID NO: 38) 
               
               
                   
               
               
                 RGS20 
                 O76081 
                 SFDKLMVTPAGRNAFREFLRTEFSEENMLF 
               
               
                   
                   
                 WMACEELKKEANKNIIEEKARIIYEDYISI 
               
               
                   
                   
                 LSPKEVSLDSRVREVINRNMVEPSQHIFDD 
               
               
                   
                   
                 AQLQIYTLMHRDSYPRFMNSAVYKDLL 
               
               
                   
                   
                 (SEQ ID NO: 39) 
               
               
                   
               
               
                 RGS21 
                 Q2M5E4 
                 NMDTLLANQAGLDAFRIFLKSEFSEENVEF 
               
               
                   
                   
                 WLACEDFKKTKNADKIASKAKMIYSEFIEA 
               
               
                   
                   
                 DAPKEINIDFGTRDLISKNIAEPTLKCFDE 
               
               
                   
                   
                 AQKLIYCLMAKDSFPRFLKSEIYKKLV 
               
               
                   
                   
                 (SEQ ID NO: 40) 
               
               
                   
               
               
                 RGS22 
                 Q9BYZ4 
                 CEHSGNKLWKDSVYFWFDLQAYHQLFYQET 
               
               
                   
                   
                 LQPFKVCKQAQYLFATYVAPSATLDIGLQQ 
               
               
                   
                   
                 EKKKEIYMKIQPPFEDLFDTAEEYILLLLL 
               
               
                   
                   
                 EPWTKMVKSD 
               
               
                   
                   
                 (SEQ ID NO: 41) 
               
               
                   
               
               
                   
                 (2 RGS 
                 KFSDLLNNKLEFEHFRQFLETHSSSRILCA 
               
               
                   
                 domains 
                 DRHWSSSGEITYRDRNQRKAKSIYIKNKYL 
               
               
                   
                 within 
                 NKKYFFGPNSPASLYQQNQVMHLSGGWGKI 
               
               
                   
                 the RGS22 
                 LHEQLDAPVLVEIQKHVQNRLENVWLPLFL 
               
               
                   
                 protein) 
                 ASEQF 
               
               
                   
                   
                 (SEQ ID NO: 42) 
               
               
                   
               
               
                 GRK1 
                 Q15835 
                 EFESVCLEQPIGKKLFQQFLQSAEKHLPAL 
               
               
                   
                   
                 ELWKDIEDYDTADNDLQPQKAQTILAQYLD 
               
               
                   
                   
                 PQAKLFCSFLDEGIVAKFKEGPVEIQDGLF 
               
               
                   
                   
                 QPLLQATLAHLGQAPFQEYLGSLYFLRFL 
               
               
                   
                   
                 (SEQ ID NO: 43) 
               
               
                   
               
               
                 GRK2 
                 P25098 
                 TFEKIFSQKLGYLLFRDFCLNHLEEARPLV 
               
               
                   
                   
                 EFYEEIKKYEKLETEEERVARSREIFDSYI 
               
               
                   
                   
                 MKELLACSHPFSKSATEHVQGHLGKKQVPP 
               
               
                   
                   
                 DLFQPYIEEICQNLRGDVFQKFIESDKFTR 
               
               
                   
                   
                 FC 
               
               
                   
                   
                 (SEQ ID NO: 44) 
               
               
                   
               
               
                 GRK3 
                 P35626 
                 TFDKIFNQKIGFLLFKDFCLNEINEAVPQV 
               
               
                   
                   
                 KFYEEIKEYEKLDNEEDRLCRSRQIYDAYI 
               
               
                   
                   
                 MKELLSCSHPFSKQAVEHVQSHLSKKQVTS 
               
               
                   
                   
                 TLFQPYIEEICESLRGDIFQKFMESDKFTR 
               
               
                   
                   
                 FC 
               
               
                   
                   
                 (SEQ ID NO: 45) 
               
               
                   
               
               
                 GRK4 
                 P32298 
                 DYSSLCDKQPIGRRLFRQFCDTKPTLKRHI 
               
               
                   
                   
                 EFLDAVAEYEVADDEDRSDCGLSILDRFFN 
               
               
                   
                   
                 DKLAAPLPEIPPDVVTECRLGLKEENPSKK 
               
               
                   
                   
                 AFEECTRVAHNYLRGEPFEEYQESSYFSQ 
               
               
                   
                   
                 FL 
               
               
                   
                   
                 (SEQ ID NO: 46) 
               
               
                   
               
               
                 GRK5 
                 P34947 
                 DYCSLCDKQPIGRLLFRQFCETRPGLECYI 
               
               
                   
                   
                 QFLDSVAEYEVTPDEKLGEKGKEIMTKYLT 
               
               
                   
                   
                 PKSPVFIAQVGQDLVSQTEEKLLQKPCKEL 
               
               
                   
                   
                 FSACAQSVHEYLRGEPFHEYLDSMFFDRFL 
               
               
                   
                   
                 (SEQ ID NO: 47) 
               
               
                   
               
               
                 GRK6 
                 P43250 
                 DYHSLCERQPIGRLLFREFCATRPELSRCV 
               
               
                   
                   
                 AFLDGVAEYEVTPDDKRKACGRQLTQNFLS 
               
               
                   
                   
                 HTGPDLIPEVPRQLVTNCTQRLEQGPCKDL 
               
               
                   
                   
                 FQELTRLTHEYLSVAPFADYLDSIYFNRFL 
               
               
                   
                   
                 (SEQ ID NO: 48) 
               
               
                   
               
               
                 GRK7 
                 Q8WTQ7 
                 NEHSLCEQQPIGRRLFRDFLATVPTFRKAA 
               
               
                   
                   
                 TFLEDVQNWELAEEGPTKDSALQGLVATCA 
               
               
                   
                   
                 SAPAPGNPQPFLSQAVATKCQAATTEEERV 
               
               
                   
                   
                 AAVTLAKAEAMAFLQEQPFKDFVTSAFYDK 
               
               
                   
                   
                 FL 
               
               
                   
                   
                 (SEQ ID NO: 49) 
               
               
                   
               
               
                 SNX13 
                 Q9Y5W8 
                 PLDSILVDNVALQFFMDYMQQTGGQAHLFF 
               
               
                   
                   
                 WMTVEGYRVTAQQQLEVLLSRQRDGKHQTN 
               
               
                   
                   
                 QTKGLLRAAAVGIYEQYLSEKASPRVTVDD 
               
               
                   
                   
                 YLVAKLADTLNHEDPTPEIFDDIQRKVYEL 
               
               
                   
                   
                 MLRDERFYPSFRQNALYVRML 
               
               
                   
                   
                 (SEQ ID NO: 50) 
               
               
                   
               
               
                 SNX14 
                 Q9Y5W7 
                 SPLVPFLQKFAEPRNKKPSVLKLELKQIRE 
               
               
                   
                   
                 QQDLLFRFMNFLKQEGAVHVLQFCLTVEEF 
               
               
                   
                   
                 NDRILRPELSNDEMLSLHEELQKIYKTYCL 
               
               
                   
                   
                 DESIDKIRFDPFIVEEIQRIAEGPYIDVVK 
               
               
                   
                   
                 LQTMRCLFEAYEHVLSLLENVFTPMFCHSD 
               
               
                   
                   
                 EYFRQLLRGAESP 
               
               
                   
                   
                 (SEQ ID NO: 51) 
               
               
                   
               
               
                 SNX25 
                 Q9H3E2 
                 QFEDILANTFYREHEGMYMERMDKRALISF 
               
               
                   
                   
                 WESVEHLKNANKNEIPQLVGEIYQNFFVES 
               
               
                   
                   
                 KEISVEKSLYKEIQQCLVGNKGIEVFYKIQ 
               
               
                   
                   
                 EDVYETLKDRYYPSFIVSDLYEKLL 
               
               
                   
                   
                 (SEQ ID NO: 52) 
               
               
                   
               
               
                 Axin 
                 O15169 
                 SLHSLLDDQDGISLFRTFLKQEGCADLLDF 
               
               
                   
                   
                 WFACTGFRKLEPCDSNEEKRLKLARAIYRK 
               
               
                   
                   
                 YILDNNGIVSRQTKPATKSFIKGCIMKQLI 
               
               
                   
                   
                 DPAMFDQAQTEIQATMEENTYPSFLKSDIY 
               
               
                   
                   
                 LEYT 
               
               
                   
                   
                 (SEQ ID NO: 53 
               
               
                   
               
               
                 Axin2 
                 Q9Y2T1 
                 SLHSLLGDQDGAYLFRTFLEREKCVDTLDF 
               
               
                   
                   
                 WFACNGFRQMNLKDTKTLRVAKAIYKRYIE 
               
               
                   
                   
                 NNSIVSKQLKPATKTYIRDGIKKQQIDSIM 
               
               
                   
                   
                 FDQAQTEIQSVMEENAYQMFLTSDIYLEYV 
               
               
                   
                   
                 (SEQ ID NO: 54) 
               
               
                   
               
               
                 D-AKAP2 
                 O43572 
                 TLEQVLHDTIVLPYFIQFMELRRMEHLVKF 
               
               
                   
                   
                 WLEAESFHSTTWSRIRAHSLNTMKQSSLAE 
               
               
                   
                   
                 PVSPSKKHETTASFLTDSLDKRLEDSGSAQ 
               
               
                   
                   
                 LFMTHSEGIDLNNRTNSTQNHLLLSQECDS 
               
               
                   
                   
                 AHSLRLEMARAGTHQVSMETQESSSTLTVA 
               
               
                   
                   
                 SRNSPASPLKELSGKLMKSIEQDAVNTFTK 
               
               
                   
                   
                 YISPDAAKPIPITEAMRNDIIARICGEDGQ 
               
               
                   
                   
                 VDP 
               
               
                   
                   
                 (SEQ ID NO. 55) 
               
               
                   
               
               
                   
                 (2 RGS 
                 YLADILFCESALFYFSEYMEKEDAVNILQF 
               
               
                   
                 domains 
                 WLAADNFQSQLAAKKGQYDGQEAQNDAMIL 
               
               
                   
                 within 
                 YDKYFSLQATHPLGFDDVVRLEIESNICRE 
               
               
                   
                 the D- 
                 GGPLPNCFTTPLRQAWTTMEKVFLPGFLSS 
               
               
                   
                 AKAP2 
                 NLYYKYL 
               
               
                   
                 protein) 
                 (SEQ ID NO: 56) 
               
               
                   
               
               
                 p115 
                 Q92888 
                 NSQFQSLEQVKRRPAHLMALLQHVALQFEP 
               
               
                 RhoGEF 
                   
                 GPLLCCLHADMLGSLGPKEAKKAFLDFYHS 
               
               
                   
                   
                 FLEKTAVLRVPVPPNVAFELDRTRADLISE 
               
               
                   
                   
                 DVQRREVQEVVQSQQVAVGRQLEDFRSKRL 
               
               
                   
                   
                 MGMTPWEQELAQLEAWVGRDRASYEAREHR 
               
               
                   
                   
                 VAERLLMHLEEMQHTISTDEEKSAAVVNAI 
               
               
                   
                   
                 GLYMRHLGVRTKSGDKKSGRNFFRKKVMGN 
               
               
                   
                   
                 (SEQ ID NO: 57) 
               
               
                   
               
               
                 PDZ 
                 O15085 
                 DLEKLKSRPAHLGVFLRYIFSQADPSPLLF 
               
               
                 RhoGEF 
                   
                 YLCAEVYQQASPKDSRSLGKDIWNIFLEKN 
               
               
                   
                   
                 APLRVKIPEMLQAEIDSRLRNSEDARGVLC 
               
               
                   
                   
                 EAQEAAMPEIQEQIHDYRTKRTLGLGSLYG 
               
               
                   
                   
                 (SEQ ID NO: 58) 
               
               
                   
               
               
                 LARG 
                 Q9NZN5 
                 CSCFQSIELLKSRPAHLAVFLHHVVSQFDP 
               
               
                   
                   
                 ATLLCYLYSDLYKHTNSKETRRIFLEFHQF 
               
               
                   
                   
                 FLDRSAHLKVSVPDEMSADLEKRRPELIPE 
               
               
                   
                   
                 DLHRHYIQTMQERVHPEVQRHLEDFRQKRS 
               
               
                   
                   
                 MGLTLAESELTKLDAERDKDRLTLEKERTC 
               
               
                   
                   
                 AEQIVAKIEEVLMTAQAVEEDKSSTMQYVI 
               
               
                   
                   
                 LMYMKHLGVKVKEPRNLEHKRGRIGFLPKI 
               
               
                   
                   
                 (SEQ ID NO: 59) 
               
               
                   
               
             
          
         
       
     
         [0009]    A link between pharmacological modulators of RGS functionality and signal transduction through GPCR activity could result in a drug with important clinical significance, particularly in the field of neurological disorders. It seems likely that pharmacological interventions for many neurological disorders would involve a combination of effects. Such effects may include an agonist to induce a response mediated by a GPCR complex and an inhibitor of RGS activity to prolong the effect seen with the initial agonist. However, efforts to screen compound libraries for inhibitors or activators of RGS proteins have been hampered because the GTPase activity of isolated Gα proteins is limited by GDP dissociation, so steady state GTPase activity cannot be used to measure GAP activity. 
         [0010]    RGS proteins accelerate the rate of Gα-catalyzed GTP hydrolysis by as much as 100-fold, which provides the basis for an in vitro screening assay; moreover both types of proteins are soluble and relatively easy to produce. However, in the absence of GPCR-mediated nucleotide exchange, it is GDP release (rather than GTP hydrolysis) that is the rate-limiting step in the Gα nucleotide cycle. Thus, to examine the effect of an RGS protein in accelerating GTP hydrolysis by an isolated Gα subunit in vitro, a single round of hydrolysis of radiolabelled GTP is usually performed (a.k.a. the “single-turnover GTPase assay”). This standard assay for measuring RGS domain-mediated GAP activity is low-throughput and requires discrete steps of [γ- 32 P]GTP loading onto Gα, purification of the [γ- 32 P]GTP-Gα complex, and its immediate use before significant hydrolysis by intrinsic G α  GTPase can occur. The assay also involves isolation (in discrete time intervals) of released [ 32 P]phosphate with activated charcoal precipitation and centrifugation, and finally scintillation counting. This type of protocol would be very difficult to incorporate into an automated high through put screening (HTS) environment. Moreover, measurement of steady state enzyme activity is the standard approach used for both basic research and HTS; all of the assumptions of Michaelis-Menten kinetic analysis are based on steady state measurements. Use of a single turnover assay thus adds additional complications in data analysis. 
         [0011]    Reliance on reconstituted GPCR/G protein complexes and phosphate detection make steady state Gα GTPase methods unsuitable for HTS. Steady-state GTPase measurements of RGS protein GAP activity are carried out in the presence of an agonist-activated GPCR/heterotrimer complex to effect the exchange of GTP for bound GDP (see  FIG. 2 ). This entails the use of native or heterologously co-expressed GPCRs and Gβγ proteins within membrane preparations from mammalian or Sf9 insect cells, or elaborate reconstitution of purified receptor and heterotrimer in lipid vesicles. [γ- 32 P]GTP radioassays utilizing charcoal to adsorb unhydrolyzed [γ- 32 P]GTP are generally used as a detection method, similar to the single turnover assays. The complexity and expense of using reconstituted GPCRs combined with the regulatory and disposal costs for radioactive waste limits the utility of these assay methods for an industrial HTS environment. Alternatives to radioassays have been developed that rely on colorimetric or fluorescent phosphate detection methods, however the high background levels of phosphate in biological reagents impose stringent requirements on their use. Moreover, the intent in a biochemical HTS assay is to identify inhibitors of a specific molecular target. The difficulty of deconvoluting hits from such a complex assay make it very unattractive; one might as well use a cellular assay, where the potential for interaction with multiple targets—including the GPCR itself—is not generally perceived as a disadvantage. 
         [0012]    The present invention enables the use of biochemical assay methods to screen for modulators of RGS GAP catalytic activity. Altering the relative rates of Gα protein GTPase and GDP dissociation through mutation, so that GDP dissociation is no longer rate limiting, allows the use of steady state enzymatic assays for monitoring changes in Gα GTPase activity. As background, there is literature relating to single amino acid substitutions of important functional residues, which are highly conserved within all Gα proteins subfamilies. These mutations are identified below. 
         [0013]    Single mutation of a conserved arginine: There are two examples of mutant Gα proteins from different subfamilies where GTP hydrolysis has been reduced more than 100-fold without disrupting RGS interactions. Native Gα i1  and Gα q  have similar basal GTP hydrolysis rates (single turnover; Table 3.) of 3.0 min −1  and 0.7 min −1 , respectively [Krumins et al.  Methods Enzymol,  2002. 344: p. 673-85]. Mutation of a highly conserved active site Arg residue in either protein (R178C and R183C, respectively, for Gα i1  and Gα q ) causes an approximate 100-fold reduction in GTPase turnover rate, but it does not abolish their functional interaction with RGS proteins. RGS4 stimulates the GTPase activity of both mutant proteins approximately 100-fold [Berman et al.  Cell,  1996. 86(3): p. 445-5; and Chidiac et al.  J Biol Chem,  1999. 274(28): p. 19639- 43]—a factor equal to or greater than its GAP effect on the wild type protein. In the case of the Gα   q  R183C protein, functional interaction (i.e., stimulation of GTPase) has been demonstrated with several additional RGS proteins including RGS1, RGS2, RGS3, RGS-GAIP and with phospholipase Cβ 1  [Chidiac et al.  Methods Enzymol,  2002. 344: p. 686-702]. Mutation or covalent modification of the cognate Arg in three additional G α  proteins, Gα i2 , Gα s  and Gα t , causes similar losses of GTP hydrolysis activity [Berman et al.  Cell,  1996. 86(3): p. 445-5; Freissmuth et al.  J Biol Chem,  1989. 264(36): p. 21907-14; and Nishina et al.  J Biochem  (Tokyo), 1995. 118(5): p. 1083-9], though their interaction with RGS proteins has not yet been examined. The 20 Gα family members (i.e., reference native Gα proteins) and the locations of the critical Arginine and Alanine amino acids are presented in Table 2. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 SEQ ID 
                 Gα Family 
                 GenBank 
                 Conserved Arg 
                 Conserved Ala 
               
               
                 NO: 
                 Member 
                 Accession # 
                 Amino Acid # 
                 Amino Acid # 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 i1 
                 P63096 
                 178 
                 326 
               
               
                 2 
                 i2 
                 NP_002061 
                 179 
                 327 
               
               
                 3 
                 i3 
                 AAM12621 
                 178 
                 326 
               
               
                 4 
                 12 
                 NP_031379 
                 205 
                 353 
               
               
                 5 
                 13 
                 NP_006563 
                 200 
                 349 
               
               
                 6 
                 q 
                 NP_002063 
                 183 
                 331 
               
               
                 7 
                 s 
                 P63092 
                 201 
                 366 
               
               
                 8 
                 z 
                 NP_002064 
                 178 
                 327 
               
               
                 9 
                 i/o 
                 NP_620073 
                 179 
                 326 
               
               
                 10 
                 q11 
                 NP_002058 
                 183 
                 331 
               
               
                 11 
                 q15 
                 NP_002059 
                 186 
                 346 
               
               
                 12 
                 14 
                 AAH27886 
                 179 
                 327 
               
               
                 13 
                 O 
                 NP_066268 
                 179 
                 326 
               
               
                 14 
                 oB 
                 AAM12609 
                 179 
                 326 
               
               
                 15 
                 oA 
                 AAM12608 
                 179 
                 326 
               
               
                 16 
                 olf 
                 AAM12607 
                 188 
                 353 
               
               
                 17 
                 k 
                 AAA35896 
                 178 
                 326 
               
               
                 18 
                 s2 
                 AAA53147 
                 202 
                 367 
               
               
                 19 
                 s3 
                 AAA53148 
                 186 
                 351 
               
               
                 20 
                 s4 
                 AAA53149 
                 187 
                 352 
               
               
                   
               
             
          
         
       
     
         [0014]    The effects of catalytic site Arg mutations on Gα GTPase activity, GDP dissociation and RGS interactions are described in Table 3. 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 Gα 
                 WT 
                 WT 
                 Arg Mutant 
                 Arg Mutant 
                   
               
               
                 Protein 
                 k cat GTPase   
                 k off GDP   
                 k cat GTPase   
                 k off GDP   
                 RGS Interactions 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Gα i1   
                 3.0 
                 0.087 
                 R178C 0.02-0.04 
                 &gt;0.087 
                 RGS4 
               
               
                 Gα i2   
                 4.0 
                 0.02-0.04 
                 R179C 0.01-0.05 
                 0.01-0.04 
                 NA 
               
               
                 Gα q   
                 0.7* 
                 NA 
                 R183C 0.005 
                 NA 
                 RGS1, 2, 3, 4, GAIP, 
               
               
                   
                   
                   
                   
                   
                 PLCβ1 
               
               
                 Gα s4   
                 3.8 
                 0.14  
                 R187A 0.03 
                 0.27 min −1   
                 NA 
               
               
                   
               
               
                 All rates are per minute. All k cat  values were determined using single turnover GTP hydrolysis assays with isolated Gα proteins except WT G αq  kcat, which was determined in reconstituted GPCR/Gβγ system. 
               
               
                 Data from [Posner et al., 1998] and [Coleman et al.  Science , 1994. 265(5177): p. 1405-12] (Gα i1 ), [Nishina et al., 1995] (Gα i2 ), [Chidiac et al., 1999] (Gα q ) and [Freissmuth et al., 1989] (Gα s4 ). 
               
               
                 NA = Not Available. 
               
             
          
         
       
     
         [0015]    Single mutation of a conserved alanine: There are also several examples of Gα mutations that increase GDP dissociation without affecting GTP hydrolysis. The most striking is the A326S mutant of Gα i1 , which exhibits a 25-fold increase in k off (GDP)  relative to wildtype protein and an identical k cat GTP  [Posner et al.  J Biol Chem,  1998. 273(34): p. 21752-8]. Moreover, RGS4 stimulated the steady state GTPase activity of Gα i1  A3265 appreciably, from 1.3 min −1  to 2.2 min −1 . Thus, an additional mutation that caused a relatively small decrease in k cat GTPase  for the Gα i1  A326S mutant would produce a k off (GDP) /k cat (GTPase)  of five or more, enabling detection of RGS GAP activity with good signal-to-noise. 
         [0016]    Efforts to produce mutant Gα proteins have yielded proteins with decreases in their rates of GTP hydrolysis or increases in GDP dissociation from Gα proteins. Neither of these strategies have facilitated a useful system for compound library screening, where the dissociation of GDP is no longer rate limiting. Applicants envision that the ability to possibly achieve such an increase in GDP dissociation relative to GTP hydolysis is highly likely to enable detection of RGS protein GAP activity using steady state GTPase assays. Accordingly, having the tools to examine drug interactions on RGS proteins would result in a significant improvement to the currently existing technology and potentially to important drug discoveries for the treatment of a wide variety of human disorders. 
       BRIEF SUMMARY OF THE INVENTION 
       [0017]    The present invention is summarized as genetically engineered G-alpha proteins. These proteins are components of the G-protein-coupled receptor (GPCR) signal transduction pathway. The engineered protein is a mutant Gα protein, which in some examples contains alterations of at least one, and preferably two or more, highly conserved amino acid residues, which are conserved among all four subfamilies of Gα proteins, and among all specific members of families investigated. The mutations described here yield an unexpectedly high effect on Gα function both in terms of GTPase activity (GTP hydrolysis) and GDP recycling. This effect is more than additive when considered in light of the individual mutations. This unanticipated synergistic effect may have a profound influence on the way GPCR signaling pathways are examined for the development of new pharmacotherapies, particularly in the field of central nervous system disorders such as Parkinson&#39;s disease. 
         [0018]    In one aspect, the invention includes an engineered protein including a Gα protein differing in amino acid sequence from a reference native Gα protein, wherein the difference includes at least two amino acid substitutions, wherein the substitutions have a net effect of an increase in the GDP dissociation rate and a decrease in the GTP hydrolysis rate, so that the rate of GDP dissociation is no longer rate limiting for catalysis relative to a Gα protein without said amino acid substitutions. In a related aspect, the reference Gα protein includes an amino acid sequence from any one of SEQ ID NOs: 1-20. 
         [0019]    In another aspect, the invention includes kit having at least one engineered Gα protein described herein. 
         [0020]    In another aspect, the invention includes an engineered Gα protein described herein, wherein when the engineered Gα protein is in the presence of an RGS protein, the detectable steady state GTPase activity is increased at least two-fold relative to the GTPase activity of Gα protein in the absence of an RGS protein. 
         [0021]    In another aspect, the invention includes an engineered Gα protein differing in amino acid sequence from a reference native Gα protein, wherein the difference consists of two amino acid substitutions, wherein the first substitution is at (i) the conserved Arginine located in the native Gα protein at any one of amino acid positions ranging from about 178 to about 205, and the second substitution is at (ii) the conserved Alanine located in the native Gα protein at any one of amino acid positions ranging from about 326 and about 367, wherein both substitution result in a net effect of an increase in the GDP dissociation rate and a decrease in the GTP hydrolysis rate, so that the rate of GDP dissociation is no longer rate limiting for catalysis relative to a Gα protein without said amino acid substitutions. 
         [0022]    In a related aspect, the invention includes an engineered Gα protein described herein having a protein differing in amino acid sequence from a reference Gα protein of any one of SEQ ID NOs: 1-20, wherein the difference consists of substituting (i) an Arginine, located at positions ranging from about 178 to about 205, to any one of Methionine, Cysteine or Lysine, and (ii) an Alanine, located at positions ranging from about 326 to about 367, to any one of Serine, Threonine or Aspartate. 
         [0023]    In a related aspect, the invention includes the engineered Gα protein described herein having a protein differing in amino acid sequence from a reference Gα protein of any one of SEQ ID NOs: 1-20, wherein the difference consists of an Arginine to a Methionine substitution located at positions ranging from about 178 to about 205, and an Alanine to a Serine substitution located at positions ranging from about 326 to about 367, wherein when the engineered Gα protein is in the presence of an RGS protein, the detectable steady state GTPase activity is increased at least two-fold relative to the GTPase activity of Gα protein in the absence of an RGS protein. 
         [0024]    In another aspect, the invention includes a method of using the engineered Gα protein of Claim  1 , wherein the method includes incubating the engineered Gα protein in the presence or absence of a protein containing an RGS domain. 
         [0025]    In a related aspect, the invention includes a method of using an engineered Gα protein, wherein the method includes incubating the engineered Gα protein in the presence or absence of a protein containing an RGS domain, wherein the engineered Gα protein differs in amino acid sequence from a reference native Gα protein in at least two amino acid substitutions, wherein the substitutions have a net effect of an increase in the GDP dissociation rate and a decrease in the GTP hydrolysis rate, so that the rate of GDP dissociation is no longer rate limiting for catalysis relative to a Gα protein without said amino acid substitutions. 
         [0026]    In a related method, the invention further includes determining GAP activity, wherein when the engineered Gα protein is incubated in the presence of a protein containing an RGS domain, the Gα GTPase activity is stimulated, which is a measure of its GAP activity. 
         [0027]    In a related aspect, the invention includes a method of detecting the enzymatic GAP activity of an RGS protein by using an engineered Gα protein of Claim  1  in the method including the steps of: a) reacting the engineered Gα protein with guanosine triphosphate (GTP) in the presence and absence of another protein containing an RGS domain; b) forming the products guanosine diphosphate (GDP) and phosphate; c) detecting the GDP or phosphate as a measure of Gα GTPase activity; and d) determining the GAP activity by subtracting the GTPase activity in the absence of the protein containing an RGS domain from the GTPase activity in the presence of the protein containing an RGS domain. 
         [0028]    In a related aspect, the invention includes method of detecting the enzymatic GAP activity of an RGS protein by using an engineered Gα protein variant of Claim  1  with the method including the steps of: a) reacting the engineered Gα protein with guanosine triphosphate (GTP) in the presence and absence of another protein containing an RGS domain; b) forming the products guanosine diphosphate (GDP) and phosphate; c) contacting the GDP produced in this reaction with a first complex including an antibody being specific for the GDP and a fluorescent tracer, and capable of producing an observable; d) competitively displacing the tracer of the first complex by the GDP, to generate a second complex GDP-antibody complex and displaced tracer, to directly detect the GDP in the reaction; and e) determining the GAP activity by subtracting the GDP formation in the absence of the protein containing an RGS domain from the GDP formation in the presence of the protein containing an RGS domain. 
         [0029]    Other advantages and a fuller appreciation of specific adaptations, compositional variations, and physical attributes will be gained upon an examination of the following detailed description of the various embodiments, taken in conjunction with the appended claims. 
     
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
         [0030]      FIG. 1  illustrates a model of the G-protein signaling complex, which is described in this invention. Agonist activation of the GPCR results in conformational changes transmitted to the Gαβγ heterotrimer resulting in the release of GDP from Gα and subsequent exchange for GTP (thus serving as guanine nucleotide exchange factors or GEFs), release of Gβγ, and signal commencement [Hamm, H. E.  Proc Natl Acad Sci U S A,  2001. 98(9): p. 4819-21]. Activated Gα·GTP and liberated Gβγ modulate the activity of several downstream effectors responsible for cellular responses to extracellular ligands. RGS proteins facilitate signal termination by increasing the intrinsic GTP hydrolysis rate of Ga·GTP, thus serving as GTPase-accelerating proteins (GAPs). 
           [0031]      FIG. 2  shows the effect of RGS4 on steady state GTPase activity of wild type (WT) and mutated Giα1 proteins shown as: (A) change in polarization, and (B) GDP produced. Dashed lines are in the absence and solid lines are in the presence of 250 nM RGS4. 
           [0032]      FIG. 3  provides a sequence alignment for some members of the human Gα protein family, including all 4 subfamilies; Gα i  (members 1, 2, 3, z, and i/o; SEQ ID NOs: 1, 2, 3, 8, and 9 respectively), Gα q  (members q and 11; SEQ ID NOs: 6 and 10 respectively), Gα 12 &amp; 13  (SEQ ID NOs: 4 and 5 respectively), and Gα s  (SEQ ID NO: 7). Also shown is the described invention, a Gα i1  double mutant (SEQ ID NO: 60), which demonstrates the changes conferred upon the conserved residues of interest (R178 and A326). 
           [0033]      FIG. 4  shows the polynucleotide sequence (SEQ ID NO: 61) and the amino acid sequence (SEQ ID NO: 1) for the human guanine nucleotide binding protein alpha i1 (GNAI1). All mutants described in  FIG. 4  were derived from this reference sequence, GenBank Accession # AF493905 and P63096. 
       
    
    
       [0034]    Before the various embodiments of the invention are explained in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description. The invention is capable of being practiced or being carried out in a variety of ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting in any way. 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0035]    The present invention is directed to novel and non-obvious genetically engineered G-alpha proteins. These proteins have been engineered in a novel way to contain at least one or more mutations, which result in a greater than additive effect than the single mutations alone. This synergistic effect was greater than that expected from previous work. 
         [0036]    The k off (GDP) /k cat (GTPase)  for wildtype Gα i1  is 0.03. By introducing mutations that affect k off (GDP)  and k cat (GTPase) , inventors hoped to achieve a ratio of at least five, a 150-fold increase. This would allow detection of a five-fold enhancement of steady state GTPase activity by RGS proteins, which inventors believe provides adequate signal-to-noise ratio for an HTS assay. (Note that it is not necessary to detect the full potential GAP activity of an RGS protein in an assay for identifying inhibitors.) 
         [0037]    This approach was acknowledged to be of high risk because it required Gαproteins with GDP dissociation rates that are not just equal, but significantly greater than their GTP hydrolysis rates—a reversal of the natural situation. Mutations of Gα i1  or other closely related Gα proteins that affect either (but not both) k off (GDP)  and D cat (GTPase)  without affecting functional interaction with RGS proteins have been previously identified and are set forth in Table 4 below. The most striking of these were mutations of a highly conserved active site Arg R178C, which causes an approximate 100-fold reduction in Gα i1  GTPase turnover rate, and A326S which results in a 25-fold increase in k off (GDP)  relative to wildtype protein and an identical k cat GTP  [Posner et al.  J Biol Chem,  1998. 273(34): p. 21752-8]. The novel approach described in this invention was to combine mutations to yield a synergistic effect of these separate inventions to yield a mutant Gα protein with a profoundly lower k cat (GTPase)  and a profoundly higher k off(GDP) . 
         [0038]    Previously reported mutations that were used to develop strategy for altering Gα i1  GTP hydrolysis and GDP dissociation rates are identified below in Table 4. 
         [0000]                                        TABLE 4                   Gα protein   Decrease in   Increase   Equivalent   RGS Interactions       Mutation   mutated   k cat (GTPase)     in k off (GDP)     Site in Gα i1     (with mutant)                   A326S   Gα i1     none   25x   A326   RGS4       [Posner et al., 1998]       D55G/G56S   Gα t     none   10x   A50/G60   NA       [Mello et al.,  J Protein           Chem , 2002. 21(1): p.       29-34]       R144A   Gα i1     ND    5x   R144   NA       [Remmers et al.,         Biochemistry , 1999.       38(42): p. 13795-800]       R178C   Gα i1     &gt;100x   Reported, not   R178   RGS4       [Coleman et al., 1994]           quantified       T182A   Gα i2     &gt;100x    2x   T181   NA       [Nishina et al., 1995]                    
Accordingly, the present invention will now be described in detail with respect to such endeavors; however, those skilled in the art will appreciate that such a description of the invention is meant to be exemplary only and should not be viewed as being limiting on the full scope thereof.
 
         [0039]    All technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the relevant art. 
       DEFINITIONS 
       [0040]    “Amino acid” embraces all compounds (natural and synthetic) including both amino functionality and acid functionality, including amino acid analogs and derivatives. The instant amino acids may be those naturally occurring amino acids found in proteins, or the naturally occurring anabolic or catabolic products of such amino acids which contain amino and carboxyl groups. Particularly suitable amino acid side chains include side chains selected from those of the following amino acids: glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan. “Amino acid sequence” and terms such as “polypeptide” or “protein” are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule. 
         [0041]    “G-alpha protein” or “Gα protein” refers to the first described of three subunits which together comprise the receptor component of GPCR (G-Protein Coupled Receptor) essential to this important signal transduction system (see  FIG. 1 ). The G-alpha subunit is responsible for nucleotide binding. An agonist induces a conformation changes in the GPCR complex which enhances the nucleotide-exchange activity of the receptor, which leads to the release of GDP and subsequent binding of GTP. Throughout this application inventors use Gα to refer in general to the alpha subunit of the Gαβγ heterotrimer. There are twenty Gα proteins classified in four subfamilies, which inventors denote by Gs, Gi, G 12 , and Gq. The individual isoforms of the Gi subfamily are Gα i1 , Gα i2 , Gα i3 , and Gα i/o . These proteins have been isolated, sequenced and characterized. The twenty known Gα protein family members are homologues and possess substantial sequence similarity. The G alpha proteins are listed with GenBank Accession numbers in Table 2. 
         [0042]    “Engineered G-alpha protein” includes at least one or a limited number of amino acid substitutions (e.g., conservative or non-conservative substitutions), additions, or deletions (e.g., truncations) compared to wild type protein. The present invention further encompasses engineered G-alpha variants that have at least two amino acid substitutions at positions that are functionally equivalent to positions 178 and 326 of the human G alpha i1 protein (SEQ ID NO: 1) and which result in the desired function. In one aspect, such substitutions have a net effect of an increase in the GDP dissociation rate and a decrease in the GTP hydrolysis rate, such that the rate of GDP dissociation is no longer rate limiting for catalysis relative to a Gα protein without the amino acid substitutions. Suitable regions for amino acid substitutions include an Arginine to a Methionine, Cysteine or Lysine substitution from positions 178 to 205, and an Alanine to a Serine, Threonine or Aspartate substitution from positions 326 and 367 of native Gα proteins described here in Table 2. 
         [0043]    “RGS protein” refers to multifunctional GTPase-accelerating proteins that contain a ˜120 amino acid RGS sequence domain and inactivate G-protein signaling pathways. GTPase-accelerating protein activity is a general feature of RGS proteins and serves to facilitate the inactivation of the G protein rather than the receptor. Thus, agents that bind and inhibit RGS proteins could modulate endogenous neurotransmitter and hormone signaling in a manner analogous to neurotransmitter uptake inhibitors. The thirty-seven known RGS proteins are listed with GenBank Accession numbers and RGS domain sequences in Table 1. Functionally equivalent fragments of RGS proteins and fragments thereof are also included in the methods of this invention. 
         [0044]    “GPCR” refers to G-Protein Coupled Receptor signal transduction three component systems. GPCRs are composed of a receptor, a G protein and an effector component. 
         [0045]    “G-protein inhibitors” refers to any agent or set of agents that interferes with G-protein function either directly or indirectly. These inhibitors may act competitively or in an allosteric fashion and may act directly through RGS binding. Such an inhibitor may affect the GTPase activity, the nucleotide exchange activity of the G-protein or both. 
         [0046]    “Allosteric” means regulation of G-protein activity by binding of an effector molecule at a site other than the GTP binding site or the site of interaction with an RGS protein. 
         [0047]    “Homologous,” ‘homolog” or “homologue” means amino acid sequences that share at least 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90% or 95+% homology and a common functional activity. 
         [0048]    “Conservative amino acid substitutions” means substitutions predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain. The invention encompasses substitution of amino acids based upon the probability of an amino acid substitution resulting in conservation of function. 
         [0049]    “Conserved sequence” means similar or identical sequences of nucleic acids or amino acids to multiple other species of organisms, or to different molecules produced by the same organism. 
         [0050]    “Substantial sequence similarity” in the amino acid sequence comparison context means either that the segments (or, their complementary strands) when compared, are identical when optimally aligned with appropriate amino acid insertions, deletions or substitutions in at least about 50% of the amino acids, at least 56%, at least 59%, at least 62%, at least 65%, at least 68%, at least 71%, at least 74%, at least 77%, at least 80%, at least about 85%, at least about 90%, at least about 95 to 98%, or, as high at about 99% or more of the amino acids. 
         [0051]    “Wild-type protein” may be produced by synthetic methods. Wild-type proteins include, but are not limited to, forms that include post-translational modifications such as glycosylation as well as any preprocessed forms. In contrast, the terms “modified”, “mutant”, and “variant” refer to a gene or gene product that displays modifications in sequence when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered nucleic acid or polypeptide sequence when compared to the wild-type gene or gene product. This is in contrast to synthetic mutants that are changes made in a sequence through human (or machine) intervention. 
         [0052]    “Fragment” as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion as compared to the native protein, but where the remaining amino acid sequence is identical to the corresponding positions in the amino acid sequence deduced from a full-length cDNA sequence. Fragments typically are at least 4 amino acids long, preferably at least 20 amino acids long, usually at least 50 amino acids long or longer, and span the portion of the polypeptide required for intermolecular binding of the compositions with its various ligands and/or substrates. In some embodiments, fragments (e.g., G-alpha protein or RGS protein) possess an activity of the native protein or a functionally equivalent activity of the native protein. 
         [0053]    “Kit” refers to any delivery system for delivering materials. In the context of reaction materials (e.g., compositions comprising at least one G-alpha protein described herein). Such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents and/or supporting materials (e.g., written instructions for using the materials, etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials. Also encompassed here is a “fragmented kit” which refers to delivery systems comprising two or more separate containers that each contain a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain a composition comprising at least one G-alpha protein described herein for a particular use, while a second container contains a second agent. Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all of the components of a reaction materials needed for a particular use in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits. 
       METHODS AND MATERIALS 
       [0054]    Molecular Biology and Protein Production Methods. 
         [0055]    Full length human Gα i1  and mutants were made from a pProEX HTb-Gα i1  vector using a Stratagene QuikChange® II Site-Directed Mutagenesis Kit. Mutagenesis primers were designed using Stratagene&#39;s QuikChange® primer design program and were synthesized and PAGE purified by Sigma-Genosys. All sequencing primers were purchased from Sigma-Genosys. All constructs were sequence verified at Functional Biosciences LLC, Madison Wis. His-tagged wild type and mutant Gα i1  proteins and human His-tagged RGS4 (23-298) were expressed in Rosetta2 (DES) cells. 250 ml cultures were grown at 37° C. to an A 600  of approximately 0.8 and then induced with 1 mM isopropyl-β-D-thiogalactopyranoside for 4 h at 25° C. All proteins were purified using a Qiagen QIAexpress® Ni-NTA Fast Start Kit following the protocol for purification of 6×His-tagged proteins under native conditions. Purified proteins were analyzed on SDS-PAGE and determined to be &gt;95% purity. Protein concentrations were determined using a standard Bradford protein determination assay with BSA as standard. Purified proteins were stored at −20° C. in 20 mM Tris pH 7.5, 200 mM NaCl, 1 mM DTT, 5% Glycerol and 1 uM GDP for Gα i1  proteins, and the same storage buffer without GDP for RGS4. 
         [0056]    Inventors chose Gα i1  as the native Gα protein background for this invention because: (a) the previous single mutations of R178C and A326S had been shown to exhibit significantly decreased k cat (GTPase)  and increased k off (GDP) , respectively, and to functionally interact with RGS proteins, (b) the wildtype protein interacts with a variety of RGS proteins [Krumins et al.  Methods Enzymol,  2002. 344: p. 673-85], and (c) the wild type protein and the R178C and A326S mutants are easily expressed in  E. coli,  and the purified proteins are stable. 
         [0057]    Combining Active Site and GDP Dissociation Mutations Enables Detection of RGS GAP Activity. 
         [0058]    Based on the previous single mutation studies, inventors constructed 18 variants of Gα i1  with the mutations shown in Table 5. Note that multiple substitutions were made at most sites, including amino acids that were intended to be more or less disruptive than the original reported mutation. For instance, R178K was tested as a conservative substitution at the catalytic arginine, and R178M was intermediate relative to the original R178C variant; it was thought that either of these alternative substitutions might result in a smaller decrease in k cat (GTPase)  than R178C. 
         [0000]    
       
         
               
             
               
               
             
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Gαi1 variants 
               
             
          
           
               
                   
                 Protein 
               
               
                   
                   
               
             
          
           
               
                   
                 Gαi1 wild type 
               
               
                 1 
                 Gαi1 A326D 
               
               
                 2 
                 Gαi1 A326T 
               
               
                 3 
                 Gαi1 A326S 
               
               
                 4 
                 Gαi1 R178C 
               
               
                 5 
                 Gαi1 T181V 
               
               
                 6 
                 Gαi1 T181S 
               
               
                 7 
                 Gαi1 T181A 
               
               
                 8 
                 Gαi1 R178M 
               
               
                 9 
                 Gαi1 R178K 
               
               
                 10 
                 Gαi1 F336A 
               
               
                 11 
                 Gαi1 K192A 
               
               
                 12 
                 Gαi1 K192A F336A 
               
               
                 13 
                 Gαi1 R178C A326S 
               
               
                 14 
                 Gαi1 T181A A326S 
               
               
                 15 
                 Gαi1 A326S D26G G27S 
               
               
                 16 
                 Gαi1 R178M/A326S 
               
               
                 17 
                 Gαi1 R178M/A326T 
               
               
                 18 
                 Gαi1 R178C/A326S 
               
               
                   
               
             
          
         
       
     
       EXAMPLES 
       [0059]    Inventors have developed a biochemical assay to screen for modulators of RGS GAP catalytic activity. There are two key components to our approach: (1) altering the relative rates of Gα GTPase and GDP dissociation so that GDP dissociation is no longer rate limiting will allow the use of steady state enzymatic assays for monitoring changes in Gα GTPase activity, and (2) selective immunodetection of GDP will enable homogenous, fluorescence-based detection of Gα GTPase activity in a multiwell format. In combination, these developments will enable direct detection of RGS-catalyzed stimulation of Gα GTP hydrolysis in a robust HTS format. 
         [0060]    Inventors have produced a novel double mutant of the Gα i1  protein to overcome the disparity between GDP dissociation and GTPase activity. Both parameters can be significantly altered by mutation without affecting functional interaction of Gα i1  with RGS proteins [Berman et al.  Cell,  1996. 86(3): p. 445-52; and Posner et al.  J Biol Chem,  1998. 273(34): p. 21752-8]. Moreover, other Gα proteins have been shown to be similarly affected by mutation of cognate amino acids [Chidiac et al.  J Biol Chem,  1999. 274(28): p. 19639-43; and Iiri et al.  Nature,  1994. 371(6493): p. 164-8], so the use of mutant Gα proteins for steady state GTPase assays is potentially a generic approach. 
       Example 1 
     Testing of the Novel Gαi1 Mutants for GDP Dissociation and GTP Hydrolysis Rates 
       [0061]    The effect of RGS4 on GTP hydrolysis by WT and mutated Gα i1  proteins is illustrated in  FIG. 2 . In this experiment, Gα i1  proteins were incubated with and without RGS4 in the presence of GDP assay reagents, and plates were read at intervals starting at 15 minutes. The polarization data is shown in  FIG. 2A , and in  FIG. 2B  a subset of the data in the linear region has been converted to GDP formation using a standard curve and normalized to the amount of Gα i1  protein present.  FIG. 2A  is complex, and the main observations to point out are as follows: a) The variants with mutations at the catalytic arginine only, R178C and R178M, had lower activity than wild type Gα i1  and, like wild type, were unaffected by RGS4. These results are expected because the observed GTPase rate is limited by the slow dissociation of GDP from enzyme following the hydrolysis reaction for all of these proteins. b) The A326S variant exhibits a much higher GTPase rate, as would be expected from its higher reported k off GDP  and is also unaffected by RGS4, presumably because a further increase in GTPase is limited by k off GDP . c) Most importantly, the two double mutants, R178M/A326S and R178C/A326S had very low basal GTPase activity and much higher activity in the presence of RGS4; the GAP effect on R178M/A326S was greater than with R178C/A326S. The effects of the R178M and A326S mutations, alone and in combination, are shown in more detail in  FIG. 2B . The maximum polarization shift resulting from RGS stimulation was 73 mP for R178M/A326S (at 120 min); this is an adequate window for an HTS assay. 
         [0062]    GTPase rates for Gα i1  proteins in the presence and absence of RGS4 are shown in the Table 6. 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 6 
               
             
             
               
                   
                   
               
               
                   
                   
                 GTPase rate 
                   
               
               
                   
                   
                 Rates are in min −1   
                 GAP 
               
             
          
           
               
                   
                 No RGS4 
                 +RGS4 
                 Factor 
               
               
                   
                   
               
             
          
           
               
                   
                 WT 
                 0.016 
                 0.013 
                 0.81 
               
               
                   
                 R178M 
                 0.0059 
                 0.007 
                 1.19 
               
               
                   
                 R178C 
                 0.00038 
                 0.00035 
                 0.92 
               
               
                   
                 A326S 
                 0.10 
                 0.11 
                 1.10 
               
               
                   
                 R178C/A326S 
                 0.019 
                 0.069 
                 3.63 
               
               
                   
                 R178M/A326S 
                 0.015 
                 0.097 
                 6.47 
               
               
                   
                   
               
             
          
         
       
     
         [0063]    The rates of GTP hydrolysis calculated from the data in  FIG. 2A , are shown in Table 6. Note that the observed rates may still be limited by GDP dissociation. However, our hypothesis was that inventors would be able to increase k off (GDP) /k cat (GTPase) —which is 0.03 for WT Gα i1 —only enough to detect 4-fold stimulation by RGS using a steady state GTPase assay. 
       Example 2 
     The Specific Combination of R178M and A326S Accelerates GDP Dissociation More than Expected 
       [0064]    To gain an accurate understanding of how catalysis was affected in mutated Gα i1  proteins, classic radioassay methods were employed to directly measure GDP dissociation and GTP hydrolysis rates. Single turnover GTP hydrolysis assays, which are not rate-limited by GDP dissociation [Ross, E. M.  Methods Enzymol,  2002. 344: p. 601-1], were used to measure the intrinsic k cat , and GTPγS binding assays were used to measure GDP dissociation. The single turnover assay measures  32 P i  released from enzyme-bound γ- 32 P-GTP; reactions are terminated before a stoichiometric amount of phosphate is formed. Binding of the non-hydrolyzable GTP analog, GTPγ- 35 S, to Gα i1  which had been preloaded with GDP was used as a measure of the rate of GDP dissociation; the assumption is that k on  for GTPγ- 35 S is much more rapid than k off  for GDP. 
         [0065]    The results from single turnover GTP hydrolysis assays indicated that, as expected, all of the Gα i1  variants with a mutation in the catalytic arginine have very low or undetectable levels of GTP hydrolysis, whereas the variant with a single mutation that only affects GDP dissociation, A326S, has a rate similar to wild type Gα i1  (Table 5). The GTPγ- 35 S binding assays showed that wild type Gα i1  and the two variants with mutations only at the catalytic site, R178M and R178C, had similar rates of GDP dissociation; whereas introduction of the A326S mutation, either alone or in combination with R178C, caused a three-fold acceleration in GDP dissociation less than would be expected from previous studies. However, when A326 was combined with the methionine substitution at R178 instead of cysteine, the GDP dissociation rate increased more than ten-fold from 0.008 min −1  to 0.130 min −1  (Table 7). Inventors do not know why the particular combination of R178M and A326S resulted in more rapid GDP release than A326 alone; it is not an additive effect since the singly-mutated R178M variant exhibits wild type GDP dissociation. However, the data is consistent with our steady state GAP assays (Table 5), in which inventors observed a 6.5-fold RGS4 GAP effect with R178M/A326S. 
         [0066]    The rates of GTP binding (GDP dissociation) and hydrolysis determined by radioassays are shown in Table 7. 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                 TABLE 7 
               
               
                   
                   
               
               
                   
                   
                 GTP 
                 GTP 
               
               
                   
                   
                 binding 
                 hydrolysis 
               
               
                   
                 Gαi1 
                 (min −1 ) 
                 (min −1 ) 
               
               
                   
                   
               
             
             
               
                   
                 WT 
                 0.009 
                 1.718 
               
               
                   
                 R178M 
                 0.008 
                 0.001 
               
               
                   
                 R178C 
                 0.009 
                 0.008 
               
               
                   
                 A326S 
                 0.027 
                 1.097 
               
               
                   
                 R178M/ 
                 0.130 
                 0.000 
               
               
                   
                 A326S 
               
               
                   
                 R178C/ 
                 0.025 
                 0.000 
               
               
                   
                 A326S 
               
               
                   
                   
               
             
          
         
       
     
         [0067]    Inventors also used radiometric GTPase assay methods to confirm the RGS4 GAP effect with Gα i1  R178M/A326S. In this case, steady state assays were performed since there is no need for the single turnover approach. RGS4 caused a very significant enhancement of GTPase activity (Table 7) for R178M/A326S. Thus, inventors have used two independent methods—radioactive phosphate detection and GDP immunodetection to show that inventors can detect a significant GAP effect for RGS4 using the R178M/A326S variant of Gα i1 . 
       Example 3 
     The Gαi1 R178M/A326S Double Mutant is Specifically Recognized by RGS Proteins 
       [0068]    A concern about the use of mutated Gα proteins for RGS GAP assays is that the mutations could disrupt the normal specificity that RGS proteins show for the various Gα substrates. To test for this possibility with Gα i1  R178M/A326S, inventors measured the GAP effects of three additional RGS domains on the R178M/A326S. Inventors used RGS2, which is not expected to have a functional interaction with wild type Gα i1  in vitro based on multiple previous studies [Heximer et al.  J Biol Chem,  1999. 274(48): p. 34253-9; and Heximer et al.  Proc Natl Acad Sci U S A,  1997. 94(26): p. 14389-93], and RGS21, the newest member of the R4 subfamily, which has been shown to bind Gα i1  [von Buchholtz et al.  Eur J Neurosci,  2004. 19(6): p. 1535-44] and have a GAP effect on Gα i1  in single turnover radio assays. In addition, inventors included an RGS21 variant in which a conserved arginine (R126) at the interface of the complex with Gα proteins has been substituted with glutamate. This mutation has been shown to disrupt the functional interaction of RGS21 with Gα i1 . The selectivity of these RGS domains for Gα i1  is not affected by the R178M/A326S double mutation. RGS4 and wild type RGS21 caused a stimulation of Gα i1  R178M/A326 GTPase activity of 6.6- and 8.8-fold, respectively, whereas the Gαq-selective RGS2 and the mutated RGS21 had no effect. Though not a comprehensive analysis, these results suggest that the R178M/A326S double mutation to Gα subunits will serve as useful reagents for identifying RGS selective inhibitors and lead to potentially powerful mediators of disease pathways. 
         [0069]    All publications cited herein are hereby incorporated by reference in their entirety. In the case of conflict between the present disclosure and the incorporated publications, the present disclosure should control. 
         [0070]    While the present invention has now been described and exemplified with some specificity, those skilled in the art will appreciate the various modifications, including variations, additions, and omissions that may be made in what has been described. Accordingly, it is intended that these modifications also be encompassed by the present invention and that the scope of the present invention be limited solely by the broadest interpretation that lawfully can be accorded the appended claims.