Identification of a Gs coupling domain in the amino terminus of the third intracellular loop of the alpha 2A-adrenergic receptor. Evidence for distinct structural determinants that confer Gs versus Gi coupling.

alpha2-Adrenergic receptors (alpha 2AR) functionally couple not only to Gi but also to Gs. We investigated the amino-terminal portion of the third intracellular loop of the human alpha 2AAR (alpha 2C10) for potential Gs 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 2AAR with the analogous sequence from the 5-HT1A receptor (a Gi-coupled receptor) and the beta 2AR (a Gs-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 2C10-mediated stimulation of adenylyl cyclase activity, while alpha 2C10-mediated inhibition was only moderately affected. This suggested that this region is important for Gs coupling, prompting construction of the chimeric receptor mutants. Substitution of amino acids 218-235 with 5-HT1A receptor sequence entirely ablated agonist-promoted Gs coupling, as compared with a 338 +/- 29% stimulation of adenylyl cyclase activity observed with the wild-type alpha 2C10. In contrast, Gi coupling for this mutant remained fully intact (57 +/- 2% versus 52 +/- 1% inhibition for wild-type alpha 2C10). Similar substitution with beta 2AR sequence had no effect on Gi coupling but did reduce Gs coupling. Two additional mutated alpha 2C10 containing smaller substitutions of the amino-terminal region with 5-HT1A receptor sequence at residues 218-228 or 229-235 were then studied. While Gi coupling remained intact with both mutants, Gs 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 2AR-Gi coupling) and COS-7 cells (which exclusively display alpha 2AR-Gs coupling). Thus, a critical determinant for Gs 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. Taken together, these studies demonstrate the presence of a discrete structural determinant for agonist-promoted alpha 2AR-Gs coupling, which is distinct and separable from the structural requirements for alpha 2AR-Gi coupling.