Functional role of amino-terminal serine16 and serine27 of Gα(z) in receptor and effector coupling

Abstract

The α subunit of G(z) (α(z)) harbors two N-terminal serine residues (at positions 16 and 27) that serve as protein kinase C-mediated phosphorylation sites. The cognate residues in the α subunit of G11 provide binding surfaces for the β1 subunit. We used three serine-to- alanine mutants of α(z) to investigate the functional importance of the two N-terminal serine residues. Wild-type or mutant α(z) was transiently coexpressed with different receptors and adenylyl cyclase isozymes in human embryonic kidney 293 cells, and agonist-dependent regulation of cyclic AMP accumulation was examined in a setting where all endogenous a subunits of G1 were inactivated by pertussis toxin. Replacement of one or both serine residues by alanine did not alter the ability of α(z) to interact with δ- opioid, dopamine D2, or adenosine A1 receptors. Its capacity to inhibit endogenous and type VI adenylyl cyclases was also unaffected. Functional release of βγ subunits from the mutant α(z) subunits was not impaired because they transduced βγ-mediated stimulation of type II adenylyl cyclase. Constitutively active mutants of all four α(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 hormone receptor-transfected cells. Loss of both serine residues, but not either one alone, impaired the receptor-independent inhibition of adenylyl cyclase by the GTPase-deficient mutant. Thus, replacement of the amino-terminal serine residues of α(z) has no apparent effect on receptor-mediated responses, but these serine residues may be essential for ensuring transition of α(z) into the active conformation.