IDENTIFICATION OF A G(S) COUPLING DOMAIN IN THE AMINO-TERMINUS OF THE 3RD INTRACELLULAR LOOP OF THE ALPHA(2A)-ADRENERGIC RECEPTOR - EVIDENCE FOR DISTINCT STRUCTURAL DETERMINANTS THAT CONFER G(S) VERSUS G(I) COUPLING

alpha(2)-Adrenergic receptors (alpha(2)AR) functionally couple not only to Gi but also to G(5). We investigated the aminoterminal portion of the third intracellular loop of the human alpha(2A)R ((alpha 2)C10) for potential G(s) coupling domains using site directed mutagenesis and recombinant expression in several different cell types. A deletion mutant and four chimeric receptors consisting of the alpha(2A)R with the analogous sequence from the 5-HT1A receptor (a G(i)-coupled receptor) and the beta(2)AR (a G(s)-coupled receptor) were expressed in Chinese hamster ovary cells, Chinese hamster fibroblasts, or COS-7 cells and examined for their ability to mediate stimulation or inhibition of membrane adenylyl cyclase activity or whole cell cAMP accumulation. In stably expressing Chinese hamster ovary cells, deletion of amino acids 221-231, which are in close proximity to the fifth transmembrane domain, eliminated alpha(2)C10-mediated stimulation of adenylyl cyclase activity, while alpha(2)C10-mediated inhibition was only moderately affected. This suggested that this region is important for G(s) coupling, prompting construction of the chimeric receptor mutants. Substitution of amino acids 218-235 with 5-HT1A receptor sequence entirely ablated agonist-promoted G(s) coupling, as compared with a 338 +/- 29% stimulation of adenylyl cyclase activity observed with the wild-type alpha(2)C10. In contrast, G(i) coupling for this mutant remained fully intact (57 +/- 2% versus 52 +/- 1% inhibition for wild-type alpha(2)C10). Similar substitution with beta(2)AR sequence had no effect on G(i) coupling but did reduce G(s) coupling. Two additional mutated alpha(2)C10 containing smaller substitutions of the amino-terminal region with 5-HT1A receptor sequence at residues 218-228 or 229-235 were then studied. While G(i) coupling remained intact with both mutants, G(s) coupling was ablated in the former but not the latter mutant receptor. Similar results were obtained using transfected Chinese hamster fibroblasts (which exclusively display alpha(2)AR-G(i) coupling) and COS-7 cells (which exclusively display alpha(2)AR-G(s) coupling). Thus, a critical determinant for G(s) coupling is contained within 11 amino acids (218-228) of the amino-terminal region of the third intracellular loop localized directly adjacent to the fifth transmembrane domain. `