G(i) activator region of alpha(2A)-adrenergic receptors: Distinct basic residues mediate G(i) versus G(s) activation

The structural determinants of G protein coupling versus activation by G pr otein-coupled receptors are not well understood. We examine the role of two distinct basic regions in the carboxyl terminal portion of the third intra cellular loop of the alpha(2A)-adrenergic receptor to dissect these aspects of function. Changing three arginines to alanines by mutagenesis and stabl e expression in Chinese hamster ovary-K1 cells impaired the alpha(2)-adrene rgic receptor G(s)-mediated stimulation of cyclic AMP (cAMP) accumulation, whereas G(i)-mediated inhibition was normal. When two (B2) or three (B3) ba sic residues closer to transmembrane span 6 were mutated to alanine, normal ligand binding was observed, but G(i)-mediated inhibition of cAMP accumula tion showed 20-fold and 50-fold decreases in agonist potency for the B2 and B3 mutants, respectively. Surprisingly, a normal G(s) response was seen fo r 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 s howed a 200-fold decrease in agonist potency for G(i)-mediated inhibition o f cAMP accumulation, whereas the G(s) response was nearly completely elimin ated. The three basic residues (which include the BB of the BBXXF motif) pl ay a role as G(i) activators rather than in receptor-G protein coupling, be cause high-affinity agonist binding is intact. Thus, we have identified thr ee basic residues required for activation of G(i) but not required for rece ptor-G protein coupling. Also, distinct basic residues are required for opt imal G(i) and G(s) responses, defining a microspecificity determinant withi n the carboxyl terminal portion of the third intracellular loop of the alph a(2a) adrenergic receptor.