A CYSTEINE-3 TO SERINE MUTATION OF THE G-PROTEIN G(I1)ALPHA ABROGATESFUNCTIONAL ACTIVATION BY THE ALPHA(2A)-ADRENOCEPTOR BUT NOT INTERACTIONS WITH THE BETA-GAMMA COMPLEX

Pertussis toxin-resistant (C351G) and also palmitoylation-negative (C3 S/C351G), myristoylation-negative (G2A/C351G) and combined acylation-n egative (G2A/C3S/C351G) forms of the G-protein G(i1)alpha were express ed in COS-7 cells along with the porcine alpha(2A)-adrenoceptor. G2A/C 3S/C351G G(i1)alpha and G2A/C351G G(i1)alpha were largely cytosolic an d ailed to interact with the agonist-occupied alpha(2A)-adrenoceptor i n membrane preparations, In contrast, C351G G(i1)alpha was almost enti rely 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 ce lls, C3S/C351G G(i1)alpha was present in both the particulate and cyto solic fractions but the GTPase activity of the membrane bound fraction was only slightly activated by the alpha(2A)-adrenoceptor, Coexpressi on of C3S/C351G G(i1)alpha and the alpha(2A)-adrenoceptor along with b eta(1) and gamma(2) subunits increased the P2 membrane complement of t he alpha subunit and increased substantially the ratio of membrane bou nd to cytosolic protein, However, this also failed to allow marked sti mulation of high-affinity GTPase activity by the alpha(2A)-adrenocepto r despite the increased proportion of G-protein in the P2 membrane fra ction, Despite the low fractional activation of C3S/C351G G(i1)alpha b y the alpha(2A)-adrenoceptor compared to C351G G(i1)alpha, the palmito ylation-resistant G-protein caused a marked reduction in pertussis tox in-resistant, agonist (UK14304)-mediated stimulation of adenylyl cycla se activity. UK14304 caused the same degree of effect on adenylyl cycl ase activity in pertussis toxin-treated cells following transfection o f the same amounts of C351G G(i1)alpha and C3S/C351G G(i1)alpha, as bo th appear to act to sequester beta gamma subunits. By contrast, neithe r G2A/C351G G(i1)alpha nor G2A/C3S/C351G G(i1)alpha resulted in effect ive regulation of adenylyl cyclase activity.