Reciprocal modulation of alpha(2A)-adrenoceptor and G(alpha o) protein states as determined by carboxy-terminal mutagenesis of a G(alpha o) protein

The C-terminal portion of G(alpha) proteins plays a key role in their selec tive activation by cognate receptors. alpha (2A)-Adrenoceptors (alpha (2A)- ARs) can differentially inhibit or stimulate adenylyl cyclases by the activ ation of distinct G(i/o) and G(s) protein families. The implication of the C-terminal portion of G(alphao) and G(alphas) proteins in their activation by alpha (2A)-ARs was analyzed by constructing mutant G(alphao) proteins in which each of the last five amino acid positions were exchanged for those corresponding to a G(alphas) protein. Agonist-dependent, pertussis toxin-re sistant binding of guanosine 5'-O-(3-[S-35]thio)triphosphate (S-35]GTP gamm aS) revealed that the degree of positive efficacy of clonidine was highly d ependent on the presence of a G(alphao) protein-derived Gly amino acid as t he -3 residue at the C-terminal portion of the protein. In contrast, antago nist properties for clonidine were observed for those mutants carrying a G( alphas) protein-derived Glu residue at this position. (-)-Epinephrine yield ed almost similar maximal [S-35]GTP gammaS binding responses, but its poten cy was decreased 22- to 150-fold at the -3 Glu containing mutant G(alphao) proteins compared with those mutants containing a Gly. A 9- to 39-fold incr ease in the alpha (2A)-AR agonist equilibrium dissociation constants furthe r reflected changes in the G(alpha) protein-induced alpha (2A)-AR state med iated by the specific Gly to Glu mutation in the C-terminal portion of the G(alpha) protein. The present data emphasize the unique role of the -3 posi tion at the G(alpha) protein C-terminal portion, independent of its surroun ding peptidic environment, in constraining a structure favorable for activa ted receptor interaction and transmission of the mutation-induced conformat ional change from the G(alphao) protein to the alpha (2A)-AR.