Abstract

The C-terminal portion of G_α proteins plays a key role in their selective activation by cognate receptors. α_2A-Adrenoceptors (α_2A-ARs) can differentially inhibit or stimulate adenylyl cyclases by the activation of distinct G_i/o and G_s protein families. The implication of the C-terminal portion of G_αo and G_αs proteins in their activation by α_2A-ARs was analyzed by constructing mutant G_αo proteins in which each of the last five amino acid positions were exchanged for those corresponding to a G_αs protein. Agonist-dependent, pertussis toxin-resistant binding of guanosine 5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) revealed that the degree of positive efficacy of clonidine was highly dependent on the presence of a G_αo protein-derived Gly amino acid as the −3 residue at the C-terminal portion of the protein. In contrast, antagonist properties for clonidine were observed for those mutants carrying a G_αs protein-derived Glu residue at this position. (−)-Epinephrine yielded almost similar maximal [³⁵S]GTPγS binding responses, but its potency was decreased 22- to 150-fold at the −3 Glu containing mutant G_αo proteins compared with those mutants containing a Gly. A 9- to 39-fold increase in the α_2A-AR agonist equilibrium dissociation constants further reflected changes in the G_α protein-induced α_2A-AR state mediated by the specific Gly to Glu mutation in the C-terminal portion of the G_αo protein. The present data emphasize the unique role of the −3 position at the G_α protein C-terminal portion, independent of its surrounding peptidic environment, in constraining a structure favorable for activated receptor interaction and transmission of the mutation-induced conformational change from the G_αo protein to the α_2A-AR.