FUNCTIONAL-ROLE OF AMINO-TERMINAL SERINE(16) AND SERINE(27) OF G-ALPHA(Z) IN RECEPTOR AND EFFECTOR COUPLING

The alpha subunit of G(z) (alpha(z)) harbors two N-terminal serine res idues (at positions 16 and 27) that serve as protein kinase C-mediated phosphorylation sites. The cognate residues in the alpha subunit of G (t1) provide binding surfaces for the beta(1) subunit. We used three s erine-to-alanine mutants of alpha(z) to investigate the functional imp ortance of the two N-terminal serine residues. Wild-type or mutant alp ha(z) was transiently coexpressed with different receptors and adenyly l cyclase isozymes in human embryonic kidney 293 cells, and agonist-de pendent regulation of cyclic AMP accumulation was examined in a settin g where all endogenous alpha subunits of G(i) were inactivated by pert ussis toxin. Replacement of one or both serine residues by alanine did not alter the ability of alpha(z) to interact with delta-opioid, dopa mine D-2, or adenosine A(1) receptors. Its capacity to inhibit endogen ous and type VI adenylyl cyclases was also unaffected. Functional rele ase of beta gamma subunits from the mutant alpha(z) subunits was not i mpaired because they transduced beta gamma-mediated stimulation of typ e II adenylyl cyclase. Constitutively active mutants of all four alpha (z) subunits were constructed by the introduction of a Q205L mutation. The activated mutants showed differential abilities to inhibit human choriogonadotropin-mediated cyclic AMP accumulation in luteinizing hor mone receptor-transfected cells. Loss of both serine residues, but not either one alone, impaired the receptor-independent inhibition of ade nylyl cyclase by the GTPase-deficient mutant. Thus, replacement of the amino-terminal serine residues of alpha(z) has no apparent effect on receptor-mediated responses, but these serine residues may be essentia l for ensuring transition of alpha(z) into the active conformation.