Gi Activator Region of α2A-Adrenergic Receptors: Distinct Basic Residues Mediate Gi versus Gs Activation
- Susan M. Wade1,
- William K. Lim1,
- Keng-Li Lan1,
- Duane A. Chung1,3,
- Masakatsu Nanamori1 and
- Richard R. Neubig1,2,3
- Departments of 1Pharmacology (S.M.W, W.K.L, K.-L.L, D.A.C, M.N., R.R.N.) and 2Internal Medicine/Hypertension (R.R.N.), 3Biophysics Research Division (D.A.C, R.R.N.), The University of Michigan, Ann Arbor, Michigan
Abstract
The structural determinants of G protein coupling versus activation by G protein-coupled receptors are not well understood. We examine the role of two distinct basic regions in the carboxyl terminal portion of the third intracellular loop of the α2A-adrenergic receptor to dissect these aspects of function. Changing three arginines to alanines by mutagenesis and stable expression in Chinese hamster ovary-K1 cells impaired the α2-adrenergic receptor Gs-mediated stimulation of cyclic AMP (cAMP) accumulation, whereas Gi-mediated inhibition was normal. When two (B2) or three (B3) basic residues closer to transmembrane span 6 were mutated to alanine, normal ligand binding was observed, but Gi-mediated inhibition of cAMP accumulation showed 20-fold and 50-fold decreases in agonist potency for the B2 and B3 mutants, respectively. Surprisingly, a normal Gs response was seen for the B2 mutant, and the B3 mutant showed only a 6-fold decrease in agonist potency. Mutation of both the three alanines and B3 residues to alanines showed a 200-fold decrease in agonist potency for Gi-mediated inhibition of cAMP accumulation, whereas the Gs response was nearly completely eliminated. The three basic residues (which include the BB of the BBXXF motif) play a role as Gi activators rather than in receptor-G protein coupling, because high-affinity agonist binding is intact. Thus, we have identified three basic residues required for activation of Gi but not required for receptor-G protein coupling. Also, distinct basic residues are required for optimal Gi and Gs responses, defining a microspecificity determinant within the carboxyl terminal portion of the third intracellular loop of the α2a adrenergic receptor.
Footnotes
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Send reprint requests to: Richard R. Neubig, M.D., Ph.D., Department of Pharmacology, 1301 MSRB III, 1150 W. Medical Center Dr, Ann Arbor, MI 48109-0632. E-mail:rneubig{at}umich.edu
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↵1 The endogenous Gi-family proteins present in CHO cells are Gi2 and Gi3 (Gerhardt and Neubig, 1991). Both contribute to high affinity binding of and adenylyl cyclase inhibition by PIC and UK 14,304, although Gi2 appears to play the larger role (Gerhardt and Neubig, 1991).
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↵2 Attempts to reconstitute high affinity binding with bacterially expressed αs (gift of Dr. Ron Taussig) plus brain βγ were unsuccessful. Negative data are difficult to interpret but this may be due to the relatively inefficient coupling of the α2-AR with Gs versus Gi. Evaluation of receptor reserve for Gi versus Gsindicates that Gi couples to receptor 100 times better than Gs (Brink et al., 1999). Thus our conclusions about physical RG coupling apply to R-Gi coupling and we can not make any conclusions about physicalR-Gs coupling, only functional coupling.
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This work was supported by National Institutes of Health Grant HL46417, the University of Michigan Multipurpose Arthritis Center (AR20557), and Natural Sciences and Engineering Research Council of Canada APP 207830-1998 (D.A.C.).
- Abbreviations:
- GPCR
- G protein-coupled receptor
- AR
- adrenergic receptor
- i3n
- amino-terminal end of the third intracellular loop
- i3c
- carboxyl-terminal end of the third intracellular loop
- IBMX
- isobutyl-1-methylxanthine
- GppNHp
- 5′-guanylyimidodiphosphate
- R3
- mutation of the RWRGR to AWAGA at residues 361 to 365
- R3B3
- clone in which receptors with both the R3 and B3 mutations were expressed
- B2
- mutation of 2 basic residues
- B3
- mutation of 3 basic residues
- BBXXB
- structural motif including basic (B) and non-basic (X) residues
- CHO
- Chinese hamster ovary
- PIC
- p-iodoclonidine
- PTX
- pertussis toxin
- myr-αi1
- myristoylated recombinant αi1 subunit
- WT
- wild-type
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- Received April 20, 1999.
- Accepted July 30, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
