A Cysteine-3 to Serine Mutation of the G-Protein Gi1α Abrogates Functional Activation by the α2A-Adrenoceptor but Not Interactions with the βγ Complex

Abstract

Pertussis toxin-resistant (C351G) and also palmitoylation-negative (C3S/C351G), myristoylation-negative (G2A/C351G) and combined acylation-negative (G2A/C3S/C351G) forms of the G-protein G_i1α were expressed in COS-7 cells along with the porcine α_2A-adrenoceptor. G2A/C3S/C351G G_i1α and G2A/C351G G_i1α were largely cytosolic and failed to interact with the agonist-occupied α_2A-adrenoceptor in membrane preparations. In contrast, C351G G_i1α was almost entirely particulate and the α₂-adrenoceptor agonist UK14304 caused a marked stimulation of its GTPase activity and binding of [³⁵S]GTPγS which was not prevented by pertussis toxin treatment of the cells. C3S/C351G G_i1α was present in both the particulate and cytosolic fractions but the GTPase activity of the membrane bound fraction was only slightly activated by the α_2A-adrenoceptor. Coexpression of C3S/C351G G_i1α and the α_2A-adrenoceptor along with β₁ and γ₂ subunits increased the P2 membrane complement of the α subunit and increased substantially the ratio of membrane bound to cytosolic protein. However, this also failed to allow marked stimulation of high-affinity GTPase activity by the α_2A-adrenoceptor despite the increased proportion of G-protein in the P2 membrane fraction. Despite the low fractional activation of C3S/C351G G_i1α by the α_2A-adrenoceptor compared to C351G G_i1α, the palmitoylation-resistant G-protein caused a marked reduction in pertussis toxin-resistant, agonist (UK14304)-mediated stimulation of adenylyl cyclase activity. UK14304 caused the same degree of effect on adenylyl cyclase activity in pertussis toxin-treated cells following transfection of the same amounts of C351G G_i1α and C3S/C351G G_i1α, as both appear to act to sequester βγ subunits. By contrast, neither G2A/C351G G_i1α nor G2A/C3S/C351G G_i1α resulted in effective regulation of adenylyl cyclase activity.