A cysteine-3 to serine mutation of the G-protein G(i1)alpha abrogates functional activation by the alpha(2A)-adrenoceptor but not interactions with the beta gamma complex

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(i1)alpha were expressed in COS-7 cells along with the porcine alpha(2A)-adrenoceptor. G2A/C3S/C351G G(i1)alpha and G2A/C351G G(i1)alpha were largely cytosolic and ailed to interact with the agonist-occupied alpha(2A)-adrenoceptor in membrane preparations, In contrast, C351G G(i1)alpha was almost entirely particulate and the alpha(2A)-adrenoceptor agonist UK14304 caused a marked stimulation of its GTPase activity and binding of [S-35]GTP gamma S which was not prevented by pertussis toxin treatment of the cells, C3S/C351G G(i1)alpha was present in both the particulate and cytosolic fractions but the GTPase activity of the membrane bound fraction was only slightly activated by the alpha(2A)-adrenoceptor, Coexpression of C3S/C351G G(i1)alpha and the alpha(2A)-adrenoceptor along with beta(1) and gamma(2) subunits increased the P2 membrane complement of the alpha 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 alpha(2A)-adrenoceptor despite the increased proportion of G-protein in the P2 membrane fraction, Despite the low fractional activation of C3S/C351G G(i1)alpha by the alpha(2A)-adrenoceptor compared to C351G G(i1)alpha, 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(i1)alpha and C3S/C351G G(i1)alpha, as both appear to act to sequester beta gamma subunits. By contrast, neither G2A/C351G G(i1)alpha nor G2A/C3S/C351G G(i1)alpha resulted in effective regulation of adenylyl cyclase activity.