Receptor-G Protein γ Specificity: γ11 Shows Unique Potency for A 1 Adenosine and 5-HT 1A Receptors † William K. Lim, ‡ Chang-Seon Myung, §,| James C. Garrison, § and Richard R. Neubig* ,‡,⊥ Departments of Pharmacology and Internal Medicine/Hypertension, The UniVersity of Michigan, Ann Arbor, Michigan 48109-0632, and Department of Pharmacology, UniVersity of Virginia Health Sciences Center, CharlottesVille, Virginia 22908 ReceiVed May 9, 2001; ReVised Manuscript ReceiVed June 22, 2001 ABSTRACT: G protein coupled receptors activate signal transducing guanine nucleotide-binding proteins (G proteins), which consist of an R subunit and a γ dimer. Whole cell studies have reported that receptors signal through specific γ subtypes. Membrane reconstitution studies with the adenosine A 1 and R 2A adrenergic receptors have reached a similar conclusion. We aimed to test the generality of this finding by comparing the γ subtype specificity for four G i -coupled receptors: R 2A adrenergic; A1 adenosine (A 1 -R); 5-hydroxytryptamine 1A (5-HT 1A -R); mu opioid. Membranes were reconstituted with GR i1 and five γ subtypes (dimerized to 1). Using a sensitive R-γ binding assay, we show that all recombinant γ (except 1γ1) had comparable affinity for R i1 . Using high affinity agonist binding as a measure of receptor-G protein coupling, γ-containing γ11 was the most potent for A 1 -R and 5-HT 1A -R (p < 0.05, one way ANOVA) while γ7 was most potent for the other two receptors. γ11 was 3-8-fold more potent for the A 1 -R than were the other γ subtypes. Also, γ11 was 2-8-fold more potent for A 1 -R than at the other receptors, suggesting a unique coupling specificity of the A 1 -R for γ11. In contrast, the discrimination by receptors for the other γ subtypes (1 and γ1, γ2, γ7, and γ10) was limited (2-3-fold). Thus the exquisite γ specificity of individual receptors reported in whole cell studies may depend on in vivo mechanisms beyond direct receptor recognition of γ subtypes. Transmembrane G protein coupled receptors convey signals from the extracellular milieu into the interior of the cell (1). 1 They activate signal transducing G proteins, which consist of the R subunit and a γ dimer (2). To date, more than 17 R (3), 5  (4), and 12 γ (5) isoforms have been identified. The large number of members in the G protein coupled receptor family compared to the more limited repertoire of G proteins suggests that each receptor signals through specific G protein partners. While receptors have selectivity for one or more of the four R subunit families, it is less clear if there is a specific γ partner for each receptor. There is 80-90% amino acid identity between the 1-4 isoforms which bind γ. We (6) and others (7, 8) have provided evidence that the  subunit directly contacts the receptor, and there is emerging evidence for receptor- coupling specificity (9-11). There is substantially less homology among γ isoforms (only 40-60%), and they have been classified into four classes by sequence homology (12). Hence the γ subunit may be more significant as a determinant for γ signaling diversity. Data from certain whole cell studies have provided evidence that each receptor signals via a specific G protein heterotrimer (13). Using an antisense approach, it was reported that the M4 muscarinic receptor inhibits voltage- sensitive Ca 2+ channels through a G protein heterotrimer composed of R 01 /3/γ4, while the somatostatin receptor was coupled through R 02 /1/γ3(14). This approach has been extended to other receptors (M1 muscarinic, angiotensin AT 1A , and galannin) and effector systems (15-18). A ribozyme approach has identified 1γ7 to be involved in  adrenergic receptor stimulation of adenylyl cyclase (19). However, neither of these approaches clearly distinguishes between γ specificity for receptor or effector or possibly both. Although -subtype specificity has been reported for modulation of adenylyl cyclase (10), several effector systems showed no specificity for γ dimers formed from various  and γ combinations (20, 21) (except for a lower potency of † Supported by NIH Grants HL-46417 (R.R.N.) and DK-19952 (J.C.G.), a postdoctoral fellowship from the AHA (C.-S.M.), and a training grant from the University of Malaysia at Sarawak (W.K.L.). * To whom correspondence should be addressed. Phone: 734-763- 3650. Fax: 734-763-4450. E-mail: Rneubig@umich.edu. ‡ Department of Pharmacology, University of Michigan. § Department of Pharmacology, University of Virginia Health Sciences Center. | Current address: Department of Pharmacology, Kwandong Uni- versity College of Medicine, Kangwon-do 210-701, Korea. ⊥ Department of Internal Medicine/Hypertension, University of Michigan. 1 Abbreviations: G proteins, heterotrimeric guanine nucleotide- binding regulatory proteins; Sf9, Spodoptera frugiperda; PIC, p-iodo- clonidine; YOH, yohimbine; CCPA, 2-chloro-N 6 -cyclopentyladenosine; CPX, 8-cyclopentyl-1,3-dipropylxanthine; ABA, aminobenzyladenosine; 8-OH-DPAT, 8-hydroxy-2-(di-n-propylamino)tetralin; MPPF, 4-(2′- methoxy)phenyl-1-[2′-(N-2′′-pyridinyl)-p-fluorobenzamido]thylpipera- zine; NLX, naloxone; R-PIA, R-phenylisopropyladenosine; GTPγS, guanosine 5′-(3-o-thiotriphosphate); CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; PMSF, phenylmethanesulfonyl fluoride; DTT, dithiothreitol; buffer A, 50 mM Tris-HCl, pH 7.6, 5 mM MgCl 2, 1 mM EDTA; E. coli, Escherichia coli; FITC, F-R, fluorescein isothiocynate-labeled Ri1; b-γ, biotinylated bovine brain γ; Gpp- (NH)p, guanyl-5′-yl imidodiphosphate; ANOVA, analysis of variance; PDZ, PSD-95, Dlg, ZO-1 homology. 10532 Biochemistry 2001, 40, 10532-10541 10.1021/bi010950c CCC: $20.00 © 2001 American Chemical Society Published on Web 08/07/2001