CHIMERIC MUTAGENESIS OF PUTATIVE G-PROTEIN COUPLING DOMAINS OF THE ALPHA(2A)-ADRENERGIC RECEPTOR - LOCALIZATION OF 2 REDUNDANT AND FULLY COMPETENT G(1) COUPLING DOMAINS

We have investigated potential G(i) and G(s) coupling domains within t he intracellular regions of the alpha(2A)AR subtype using a series of nine chimeric mutations. The second intracellular loop (ICL2, amino ac ids 133-149) and the amino- and carboxyl-terminal regions of the third intracellular loop (ICL3, amino acids 218-235 and 355-371, respective ly) of the cloned human alpha(2A)AR were substituted with the analogou s sequence from either the G(s)-coupled beta(2)AR or the G(i)-coupled serotonin type 1A receptor (5-HT(1A)R). Mutant and wild type alpha(2A) AR were stably expressed in Chinese hamster ovary cells and functional coupling of each receptor to G(i) and G(s) was assessed in membrane a denylyl cyclase assays. Substitution of 5-HT(1A)R sequence into ICL2 a blated coupling to G(s) but not to G(i), whereas substitution of beta( 2)AR sequence significantly depressed coupling to G(i) but not to G(s) . Thus, the ICL2 of the alpha(2A)AR contains elements essential for bo th signaling pathways. Substitution of either the amino- or carboxyl-t erminal. segments of ICL3 with 5-HT(1A)R sequence ablated agonist stim ulation of adenylyl cyclase activity (without affecting inhibition), s uggesting that both domains are necessary for alpha(2A)AR coupling to G(s). In contrast, individual substitution of beta(2)AR sequence into ICL3 amino or carboxyl termini had no appreciable effect on G(i) coupl ing. Concomitant substitution of beta(2)AR sequence into both regions substantially impaired G(i) coupling, implying that each is capable of independently supporting functional coupling. Substitution of 5-HT(1A )R at either locus had no effect on G(i) coupling. Thus, for G(s) coup ling, these two domains within ICL3 are both required for functional c oupling. However, for G(i) coupling, the alpha(2A)AR appears to have t wo distinct regions within ICL3 that are capable of supporting G(i) co upling independently. There has been no previous elucidation of a rece ptor having redundant, fully competent domains for coupling to a singl e class of G-protein. Such duplicity of functional domains within alph a(2)AR may suggest strong evolutionary pressure to maintain G(i) coupl ing.