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Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, South Carolina
Received January 25, 2006; accepted January 25, 2006
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Abstract |
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fusion proteins were first characterized more than 10 years ago as a strategy for studying receptor-G protein signaling. A large number of studies have used this approach to characterize receptor coupling to members of the Gs, Gi, and Gq families of G subunits, but this strategy has not been widely used to study G12 and G13. As described in the article by Zhang et al. in this issue of Molecular Pharmacology (p. 1433) characterization of the signaling properties of thromboxane A2 receptor (TP) -G12 and -G13 fusion constructs demonstrates the applicability of this strategy to members of this unique family of G subunits, and how this strategy can be used to resolve otherwise difficult problems of receptor pharmacology associated with these proteins. The general strategy of making receptor-G fusion constructs has wide applicability to a number of research problems, but there are perhaps also "hidden messages" in how different receptor-G subunit fusion pairs behave.
12 and G13 are the least understood of the larger family of heterotrimeric G proteins that mediate the effects of a multitude of endogenous and exogenous regulators of cellular function (Riobo and Manning, 2005
). From the beginning, G12 and G13 were pursued by a different tack than their better-characterized cousins that are members of the Gs, Gi, and Gq subfamilies of G subunits. These latter three families were initially described by following the biology of signaling pathways—for example, by looking for the transducers of the regulation of cAMP or phosphatidylinositol turnover. In contrast, G12 and G13 were "discovered" by cloning studies designed to look for homologs of already-identified proteins (Strathmann et al., 1989). Hence, they were accorded numbers instead of the earlier names that were used to denote primary downstream signaling targets, such as "s" for stimulation of adenylyl cyclase, "i" for inhibition of adenylyl cyclase and, whimsically, "q" for stimulation of phospholipase C (p having already been claimed). The G12/G13 proteins, however, segregate into a distinct arm of the G protein subunit family (Strathmann and Simon, 1991) and were, from the beginning, orphan proteins in search of an intracellular function. One such function, at least for G13, turned out to be regulation of a Rho-GEF (Hart et al., 1998) (i.e., a guanine nucleotide exchange factor for a member of the small G protein family of GTP binding proteins). Numerous variants of this protein have been identified as G12/G13 targets, as have several other interacting proteins (Riobo and Manning, 2005).
Several features of the biology of G12 and G13 have made them difficult to study. They have fairly slow nucleotide exchange rates and are hard to express (Singer et al., 1994; Kozasa and Gilman, 1995); they regulate cellular processes that have coincident regulation through multiple other G protein-related processes—as by Gq and G
; and they do not seem to cause generation of a specific small molecule mediator, such as cAMP or inositol 1,4,5-trisphosphate, that would lead to easily assayed downstream effects (Riobo and Manning, 2005). Consequently, it has not been easy to evaluate whether a receptor signals through this family of proteins or to study the pharmacology of their interactions with receptors. To circumvent these limitations, Zhang et al. (2006) report in this issue the characterization of fusion proteins between a thromboxane A2 receptor (TP) and G12 or G13, characterizing direct responses of these constructs expressed in Sf9 cells by [35S]GTPS binding in response to known and suspected TP receptor agonists and antagonists. Using this assay, the authors show that isoprostanes related to 8-iso-prostaglandin F2 (8-iso-PGF2) target G12 and G13 through TP receptor activation. These compounds are generated nonenzymatically from arachidonic acid in response to oxidative stress and may play a role in multiple human diseases (Montuschi et al., 2004). Previous studies have ambiguously associated these compounds with multiple receptors, and uncertainly with TP (for review, see Zhang et al., 2006). Zhang et al. (2006) also characterize the response of TP-G12 and TP-G13 to other agonists (U46619
[GenBank]
and U44069
[GenBank]
) and antagonists [pinane thromboxane A2 (PTA2) and SQ29548] of TP. Their studies indicate that all ligands tested, except SQ29548, have agonist activity for TP-G13, including the purported antagonist PTA2, and that SQ29548 decreases activity of TP-G13, compatible with the idea (but, as admitted by the authors, not definitive proof) that it is an inverse agonist. In contrast to TP-G13, TP-G12 did not respond to PTA2 and had a substantially decreased potency for 8-iso-PGF2 that precluded evaluation of its efficacy. To validate the conclusions from the fusion constructs, Zhang et al. (2006) showed that PTA2 and 8-iso-PGF2 were also agonists for G13 in human embryonic kidney 293 cells through both expressed and endogenous TP receptors that are not fusion constructs. These cells do not express G12, which precluded validation of those results.
The report by Zhang et al. (2006) is the latest to use receptor G protein fusion constructs to study the biology and pharmacology of signaling through specific receptor/G protein interactions (Seifert et al., 1999; Milligan, 2000; Wurch and Pauwels, 2001; Milligan et al., 2004). These constructs express G subunits as a C-terminal extension of the receptor protein so that their expression is linked in a 1:1 stoichiometry, with the expressed proteins in obligatory close proximity. This strategy was first used more than 10 years ago to characterize a 2-adrenergic receptor-Gs fusion construct (2-AR-Gs), showing, first and foremost, that it was expressed as a functional protein and that it had increased sensitivity to agonists (Bertin et al., 1994). Receptor-G protein fusion proteins have now been used extensively to characterize a large number of receptors, notably those coupled to members of the Gs, Gi, and Gq family of proteins (Table 1). (An extended version of Table 1 is available as an online supplement.) Only one previous report has characterized a receptor construct fused to a member of the G12/G13 subunit family (Sugimoto et al., 2003). That study used a similar fusion construct to show that sphingosine-1-phosphate activation of SIP2/Edg5 receptors can use either G12 or G13 to stimulate Rho and inhibit Rac and cell motility, but the authors of that study did not evaluate the utility of the constructs for studies of receptor pharmacology. A primary goal of the work reported here was to establish the G12/G13 family of proteins as targets of this strategy for studying receptor-G protein signaling (Zhang et al., 2006). Thus, this work opens up the possibility of using such constructs both for characterizing responses to additional receptors and, perhaps, for studying the unique signaling properties associated with this particular family of G proteins.
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The future utility of the constructs characterized by Zhang et al. relates in part to the utility of these receptor-fusion constructs in general. The advantages of the 1:1 receptor/G protein stoichiometry of these constructs has led to a large number of studies evaluating specific receptor-G protein pairs (Table 1); there are theoretical reasons for using such constructs to study (GPCR) receptor theory (Colquhoun, 1998). These constructs have been widely used for characterizing mutations and modifications of receptors (Loisel et al., 1999; Ward and Milligan, 1999; Pauwels and Colpaert, 2000; Moon et al., 2001; Stevens et al., 2001; McLean et al., 2002; Ward and Milligan, 2002; Barclay et al., 2005) and G proteins (Wise and Milligan, 1997; Dupuis et al., 1999; Kellett et al., 1999; Loisel et al., 1999; Wang et al., 1999; Moon et al., 2001; Stevens et al., 2001; Liu et al., 2002; Ugur et al., 2003; Barclay et al., 2005). Such constructs are similarly useful for characterizing receptor and G protein polymorphisms (essentially mutant constructs) (Milligan, 2002). Receptor-G protein fusion constructs have also been implemented as a successful means for characterizing orphan receptors, so as to identify exogenous (Takeda et al., 2003), as well as endogenous (Hosoi et al., 2002), regulators of pharmacological significance. They have even been targeted for developing gene therapy reagents (Small et al., 2001). The work of Zhang et al. (2006) indicates the likelihood that members of the G12 family, and possibly all G proteins, will be amenable targets for this research strategy.
Some of the interesting results reported by Zhang et al. (2006) are differences in the responses of the G12 and G13 constructs. TP-G13 responded to PTA2 as a partial agonist with relatively high potency, whereas TP-G12 did not respond to PTA2 and this compound functioned as an antagonist. Nevertheless, both constructs responded to the full agonist U46619
[GenBank]
with similar potency. This may indicate ligand-dependent conformations of TP that differentially interact with G proteins (i.e., some form of agonist-directed trafficking) (Leff et al., 1997; Kenakin, 2003; Perez and Karnik, 2005). There was, however, also a more subtle difference between the two constructs that may or may not relate to the same phenomenon. Zhang et al. (2006) measured activation by agonist-induced GTPS binding. Whereas TP-G12 responded to agonists with slow GTPS binding, the TP-G13 response was rapid and had to be assayed at very short time points to obtain valid estimates of potency. Previous studies of purified proteins do not suggest differences in these two G proteins for GDP/GTPS binding kinetics, and both of them have slow binding kinetics relative to other G proteins (Singer et al., 1994; Kozasa and Gilman, 1995). Thus, results with the receptor fusion contrasts could be due to receptor-G specific interactions indicative of important biological properties or perhaps to differences in the constructs. It is interesting that the recent report of crystals of G12 and G13 as chimeric proteins containing the N-terminal helix of Gi1 found that they crystallized in opposite (active versus inactive, respectively) conformations under otherwise similar conditions in the presence of aluminum fluoride and GDP (Kreutz et al., 2006). Such results argue for G-specific preferences of these proteins that have inversely related interactions with guanine nucleotides, on the one hand, as in the crystallization studies, and with receptors, on the other hand, as in fusion constructs.
In the most general sense, the report of Zhang et al. (2006) focuses attention on the utility of receptor-G fusion constructs for studying GPCR signaling mechanisms. These constructs have been very successful for a number of applications, but as artificial constructs, they have both hidden caveats and potential important surprises for otherwise unapparent biological processes. In general, fusion constructs are perceived to have increased sensitivity to activation, which can include increased constitutive activity. Such observations make sense based upon the proximity and theoretical interactions of the two components of the fusion protein (Seifert et al., 1999; Milligan, 2000; Wurch and Pauwels, 2001; Milligan et al., 2004). These results are not universal, however, and there are substantial differences in the properties of various constructs formed of different receptor-G protein fusion pairs. For example, some constructs, but not all, activate endogenous G proteins as well as their tethered protégé (Burt et al., 1998; Fong and Milligan, 1999; Vorobiov et al., 2000; Massotte et al., 2002; Molinari et al., 2003). Although some of these results may be due to high level overexpression of the fusion constructs (Carrillo et al., 2003), it is not clear that this accounts for all of them or that such signaling is necessarily seen only at high levels of expression. Colquhoun has analyzed the utility of the use of fusion constructs of defined 1:1 stoichiometry for evaluating receptor signaling mechanisms for systems that are otherwise designed to be catalytic with spare receptor and other phenomenon that circumvent an easy analysis of efficacy and potency (Colquhoun, 1998). The activation of endogenous G proteins by fusion constructs seems to minimize these advantages, along with one of the potential uses of the constructs (i.e., evaluation of mechanisms of receptor theory). But is this really true, or are these proteins and this strategy telling us something (important) by disclosing otherwise unsuspected events?
Another emerging concept in GPCR action is the role of receptor dimerization in their synthesis, trafficking and action (Javitch, 2004; Milligan, 2004; Terrillon and Bouvier, 2004). The functional existence of GPCR dimers took years to establish but is now well accepted, particularly for their role in GPCR biosynthesis and maturation (Bulenger et al., 2005) and for the class C receptors (Javitch, 2004; Milligan, 2004; Terrillon and Bouvier, 2004). The general functional role of receptor dimerization in signaling, particularly for the rhodopsin-related class A GPCRs, is still being established (Javitch, 2004; Milligan, 2004; Terrillon and Bouvier, 2004). Is it possible that signaling by fusion constructs is mediated in part through dimer complexes either with themselves or with endogenous proteins? Dimerization of such fusion proteins has in fact been demonstrated, along with the ability of such dimers to cross-regulate one another (Carrillo et al., 2003). Could the dimerization of fusion constructs play a major role in the divergent phenotypes of different receptor-G protein pairs? For example, might the constructs characterized by Zhang et al. have different properties because of differences in their ability to form dimers? Do associated G proteins play a role in receptor dimerization? According to the concepts of agonist-directed trafficking, agonists select different G proteins by inducing agonist-specific conformations of the receptor compatible with that G protein (Leff et al., 1997; Kenakin, 2003; Perez and Karnik, 2005). According to the principle of microscopic reversibility, if agonist induces a conformation of the receptor specific for a G protein, binding of that G protein should also induce a conformation of the receptor specific for that agonist (i.e., G protein-specific receptor conformations). If this interaction is stable, perhaps indicative of precoupling, then some of these G protein-specific receptor conformations may have a tendency to dimerize, whereas others might not. Might such dimerization also explain other observations about fusion constructs that have remained elusive? For example, these constructs often exhibit high- and low-affinity binding states that are not easily explained theoretically or experimentally (Seifert et al., 1999, 2000; Hoare, 2000). Although the functional role of dimers in the actions of these constructs is speculative, access to the full repertoire of G isoforms capable of serving as donors for fusion constructs, in part resulting from the work reported by Zhang et al., provides a mechanism to address this and other questions regarding the signal transduction mechanisms of these proteins and GPCRs in general.
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Acknowledgements |
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Footnotes |
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Please see the related article on page 1433.
ABBREVIATIONS: GEF, guanine nucleotide exchange factor; TP, thromboxane A2 receptor ; PTA2, pinane thromboxane A2; U46619
[GenBank]
, 9a,11a-methanoepoxy-15-hydroxyprosta-5,13-dienoic acid; U44069
[GenBank]
, (5Z)-7-((1S,4R,5S,6R)-5-((1E,3S)-3-hydroxy-1-octenyl)-2-oxabicyclo(2.2.1)hept-6-yl)-5-heptenoic acid; SQ29548, 7-[3-[[2-[(phenylamino)carbonyl] hydrazino]methyl]7-oxabicyclo[2.2.1]hept-2-yl]-,[1S-]1,2(Z),3,4]]-]5-heptenoic acid; GPCR, G protein-coupled receptor; GTPS, guanosine 5'-(3-O-thio)triphosphate; 8-iso-PGF2, 8-iso-prostaglandin F2.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. 
Address correspondence to: John D. Hildebrandt, Department of Cell and Molecular Pharmacology, Medical University of South Carolina, 173 Ashley Ave., 303BSB, Charleston, SC 29425. E-mail: hildebjd{at}musc.edu
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References |
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TopAbstractReferences |
|---|
Bertin B, Freissmuth M, Jockers R, Strosberg AD, and Marullo S (1994) Cellular signaling by an agonist-activated receptor/Gs fusion protein. Proc Natl Acad Sci USA 91: 8827-8831.
Bulenger S, Marullo S, and Bouvier M (2005) Emerging role of homo- and heterodimerization in G-protein-coupled receptor biosynthesis and maturation. Trends Pharmacol Sci 26: 131-137.[CrossRef][Medline]
Burt AR, Sautel M, Wilson MA, Rees S, Wise A, and Milligan G (1998) Agonist occupation of an 2A adrenoreceptor-Gi1 fusion protein results in activation of both receptor-linked and endogenous Gi proteins. J Biol Chem 273: 10367-10375.
Carrillo JJ, Pediani J, and Milligan G (2003) Dimers of class A G protein coupled receptors function via agonist-mediated trans-activation of associated G proteins. J Biol Chem 278: 42578-42587.
Colquhoun D (1998) Binding, gating, affinity and efficacy: The interpretation of structure-activity relationships for agonists and of the effects of mutating receptors. Br J Pharmacol 125: 923-947.[CrossRef]
Dupuis DS, Tardif S, Wurch T, Colpaert FC, and Pauwels PJ (1999) Modulation of 5-HT1A receptor signalling by point-mutation of cysteine351 in the rat Go protein. Neuropharmacology 38: 1035-1041.[CrossRef][Medline]
Fong CW and Milligan G (1999) Analysis of agonist funciton at fusion proteins between the IP prostanoid receptor and cognate, unnatural and chimaeric G proteins. Biochem J 342: 457-463.[Medline]
Hart MJ, Jiang X, Kozasa T, Roscoe W, Singer WD, Gilman AG, Sternweis PC, and Bollag G (1998) Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by G alpha-13. Science (Wash DC) 280: 2112-2114.
Hoare SRJ (2000) G protein coupled receptors: what limits high-affinity agonist binding? Trends Pharmacol Sci 21: 82-83.[Medline]
Hosoi T, Koguchi Y, Sugikawa E, Chikada A, Ogawa K, Tsuda N, Suto N, Tsunoda S, Taniguchi T, and Ohnuki T (2002) Identification of a novel human eicosanoid receptor. J Biol Chem 277: 31459-31465.
Javitch JA (2004) The ants go marching two by two: oligomeric structure of G protein coupled receptors. Mol Pharmacol 66: 1077-1082.
Kellett E, Carr C, and Milligan G (1999) Regulation of G protein activation and effector modulation by fusion proteins between the human 5-hydroxytryptamine1A receptor and the subunit of Gi1: Differences in receptor-constitutive activity imparted by single amino acid substitutions in Gi1. Mol Pharmacol 56: 684-692.
Kenakin T (2003) Ligand-selective receptor comformations revisited: the promise and the problem. Trends Pharmacol Sci 24: 346-354.[CrossRef][Medline]
Kozasa T and Gilman AG (1995) Purification of recombinant G proteins from Sf9 cells by hexahistidine tagging of associated subunits. J Biol Chem 270: 1734-1741.
Kreutz B, Yau DM, Nance MR, Tanabe S, Tesmer JJG, and Kozasa T (2006) A new approach to producing functional G alpha subunits yields the activated and deactivated structures of G alpha 12/13 proteins. Biochemistry 45: 167-174.[CrossRef][Medline]
Leff P, Scaramellini C, Law C, and McKechnie K (1997) A three-state receptor model of agonist action. Trends Pharmacol Sci 18: 355-362.[Medline]
Liu S, Carrillo JJ, Pediani JD, and Milligan G (2002) Effective information transfer from the 1b-adrenoceptor to G11 requires both interactions and an aromatic group four amino acids from the C terminus of the G protein. J Biol Chem 277: 25707-25714.
Loisel TP, Ansanay H, Adams L, Marullo S, Seifert R, Lagace M, and Bouvier M (1999) Activation of the 2-adrenergic receptor-Gs complex leads to rapid depalmitoylation and inhibition of repalmitoylation of both the receptor and Gs. J Biol Chem 274: 31014-31019.
Massotte D, Brillet K, Kieffer BL, and Milligan G (2002) Agonists activate Gil alpha or Gi2 alpha fused to the human mu opioid receptor differently. J Neurochem 81: 1372-1382.[CrossRef][Medline]
McLean AJ, Zeng F-Y, Behan D, Chalmers D, and Milligan G (2002) Generation and analysis of constitutively active and physically destabilized mutants of the human 1 adrenoceptor. Mol Pharmacol 62: 747-755.
Milligan G (2000) Insights into ligand pharmacology using receptor-G-protein fusion proteins. Trends Pharmacol Sci 21: 24-28.[CrossRef][Medline]
Milligan G (2002) The use of receptor G protein fusion proteins for he study of ligand activity. Recept Channels 8: 309-317.[CrossRef][Medline]
Milligan G (2004) G protein-coupled receptor dimerization: function and ligand pharmacology. Mol Pharmacol 66: 1-7.
Milligan G, Feng G-J, Ward RJ, Sartania N, Ramsay D, McLean AJ, and Carrillo JJ (2004) G protein-coupled receptor fusion proteins in drug discovery. Curr Pharm Des 10: 1989-2001.[CrossRef][Medline]
Molinari P, Ambrosio C, Riitano D, Sbraccia M, Gro MC, and Costa T (2003) Promiscuous coupling at receptor-G fusion proteins. J Biol Chem 278: 15778-15788.
Montuschi P, Barnes PJ, and Roberts LJ (2004) Isoprostanes: markers and mediators of oxidative stress. FASEB J 18: 1791-1800.
Moon H-E, Bahia DS, Cavalli A, Hoffmann M, and Milligan G (2001) Control of the efficiency of agonist-induced information transfer and stability of the ternary complex containing the delta opioid receptor and the alpha subunit of the Gi1 by mutation of a receptor/G protein contact interface. Neuropharmacology 41: 321-330.[CrossRef][Medline]
Pauwels PJ and Colpaert FC (2000) Disparate ligand-mediated Ca2+ responses by wild-type, mutant Ser200Ala and Ser204Ala alpha-2A-adrenoceptor: G15 fusion proteins: evidence for multiple ligand-activation binding sites. Br J Pharmacol 130: 1505-15112.[CrossRef][Medline]
Perez DM and Karnik SS (2005) Multiple signaling states of G protein-coupled receptors. Pharmacol Rev 57: 147-161.
Riobo NA and Manning DR (2005) Receptors coupled to heterotrimeric G proteins of the G12 family. Trends Pharmacol Sci 26: 146-154.[CrossRef][Medline]
Seifert R, Wenzel-Seifert K, and Kobilka B (1999) GPCR-G alpha fusion proteins: molecular analysis of receptor-G protein coupling. Trends Pharmacol Sci 20: 383-389.[CrossRef][Medline]
Seifert R, Wenzel-Seifert K, and Kobilka BK (2000) Reply to G protein coupled receptors: what limits high-affinity agonist binding? Trends Pharmacol Sci 21: 83-84.[Medline]
Singer WD, Miller RT, and Sternweis PC (1994) Purification and characterization of the subunit of G13. J Biol Chem 269: 19796-19802.
Small KM, Brown KM, Forbes SL, and Liggett SB (2001) Modification of the 2 adrenergic receptor to engineer a receptor effector complex for gene therapy. J Biol Chem 276: 31596-31601.
Stevens PA, Pediani JD, Carrillo JJ, and Milligan G (2001) Coordinated agonist regulation of receptor and G protein palmitoylation and functional rescue of palmitoylation-deficient mutants of the G protein G11 following fusion to the 1b-adrenoceptor: palmitoylation of G11 is not required for interaction with complex. J Biol Chem 276: 35883-35890.
Strathmann M and Simon MI (1991) G alpha-12 and G alpha-13 subunits define a fourth class of G protein alpha subunits. Proc Natl Acad Sci USA 86: 7407-7409.
Strathmann M, Wilkie TM, and Simon MI (1989) Diversity of the G protein family: Sequences from five additional alpha subunits in the mouse. Proc Natl Acad Sci USA 86: 7404-7409.
Sugimoto N, Takuwa N, Okamoto H, Sakurada S, and Takuwa Y (2003) Inhibitory and stimulatory regulation of Rac and cell motility by the G12/13-Ro and Gi pathway integrated downstream of a single G protein-coupled sphingosine-1-phosphate receptor isoform. Mol Cell Biol 23: 1534-1545.
Takeda S, Yamamoto A, Okada T, Matsumura E, Nose E, Kogure K, Kojima S, and Haga T (2003) Identification of surrogate ligands for orphan G protein-coupled receptors. Life Sci 74: 367-377.[CrossRef][Medline]
Terrillon S and Bouvier M (2004) Roles of G-protein-coupled receptor dimerization. From ontogeny to signalling regulation. EMBO (Eur Mol Biol Organ) Rep 5: 30-34.
Ugur O, Onaran HO, and Jones TLZ (2003) Partial rescue of functional interactions of a nonpalmitoylated mutant of the G protein G alpha-s by fusion to the beta-adrenergic receptor. Biochemistry 42: 2607-2615.[CrossRef][Medline]
Vorobiov D, Bera AK, Keren-Raifman T, Barzilai R, and Dascal N (2000) Coupling of the muscarinic m2 receptor to G protein-activated K+ channels via Gz and a receptor-Gz fusion protein. J Biol Chem 275: 4166-4170.
Wang Y, Windh RT, Chen CA, and Manning DR (1999) N-Myristoylation and play roles beyond anchorage in the parlitoylation of the G protein o subunit. J Biol Chem 274: 37435-37442.
Ward RJ and Milligan G (1999) An Asp79Asn mutation of the alpha-2A adrenoceptor interferes equally with agonist activation of individual Fa alpha-family G protein subtypes. FEBS Lett 462: 459-463.[CrossRef][Medline]
Ward RJ and Milligan G (2002) Reciprocal mutations of highly conserved residues in transmembrane helices 2 and 7 of the alpha-2A adrenoceptor restore agonist activation of Gi1 alpha. Cell Signal 14: 139-144.[CrossRef][Medline]
Wise A and Milligan G (1997) Rescue of functional interactions between the 2A-adrenoceptor and acylation-resistant forms of Gi1 by expressing the proteins from chimeric open reading frames. J Biol Chem 272: 24673-24678.
Wurch T and Pauwels PJ (2001) Analyatical pharmacology of G protein-coupoled receptors by stoichiometric expression of the receptor and G alpha protein subunits. J Pharmacol Toxicol Methods 45: 3-16.[CrossRef][Medline]
Zhang L, Dilizio C, Kim D, Smyth E, and Manning DR (2006) The G12 family of G proteins as a reporter of thromboxane A2 receptor activity. Mol Pharmacol 69: 1433-1440.
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