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Volume 271, Number 22, Issue of May 31, 1996 pp. 12826-12832
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Chimeric Mutagenesis of Putative G-protein Coupling Domains of the _2A-Adrenergic Receptor
LOCALIZATION OF TWO REDUNDANT AND FULLY COMPETENT G

(Received for publication, January 25, 1996, and in revised form, February 28, 1996)

Margaret G. Eason and Stephen B. Liggett ^§¶

From the Departments of  Medicine (Pulmonary), ^§ Molecular Genetics, and ^¶ Pharmacology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

We have investigated potential G_i and G_s coupling domains within the intracellular regions of the _2AAR subtype using a series of nine chimeric mutations. The second intracellular loop (ICL2, amino acids 133-149) and the amino- and carboxyl-terminal regions of the third intracellular loop (ICL3, amino acids 218-235 and 355-371, respectively) of the cloned human _2AAR were substituted with the analogous sequence from either the G_s-coupled ₂AR or the G_i-coupled serotonin type 1A receptor (5-HT_1AR). Mutant and wild type _2AAR were stably expressed in Chinese hamster ovary cells and functional coupling of each receptor to G_i and G_s was assessed in membrane adenylyl cyclase assays. Substitution of 5-HT_1AR sequence into ICL2 ablated coupling to G_s but not to G_i, whereas substitution of ₂AR sequence significantly depressed coupling to G_i but not to G_s. Thus, the ICL2 of the _2AAR contains elements essential for both signaling pathways. Substitution of either the amino- or carboxyl-terminal segments of ICL3 with 5-HT_1AR sequence ablated agonist stimulation of adenylyl cyclase activity (without affecting inhibition), suggesting that both domains are necessary for _2AAR coupling to G_s. In contrast, individual substitution of ₂AR sequence into ICL3 amino or carboxyl termini had no appreciable effect on G_i coupling. Concomitant substitution of ₂AR sequence into both regions substantially impaired G_i coupling, implying that each is capable of independently supporting functional coupling. Substitution of 5-HT_1AR at either locus had no effect on G_i coupling. Thus, for G_s coupling, these two domains within ICL3 are both required for functional coupling. However, for G_i coupling, the _2AAR appears to have two distinct regions within ICL3 that are capable of supporting G_i coupling independently. There has been no previous elucidation of a receptor having redundant, fully competent domains for coupling to a single class of G-protein. Such duplicity of functional domains within ₂AR may suggest strong evolutionary pressure to maintain G_i coupling.

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