EXPRESSION OF A MUTANT G(I2) ALPHA-SUBUNIT INHIBITS ATP AND THROMBIN STIMULATION OF CYTOPLASMIC PHOSPHOLIPASE-A(2)-MEDIATED ARACHIDONIC-ACID RELEASE INDEPENDENT OF CA2-ACTIVATED PROTEIN-KINASE REGULATION( AND MITOGEN)

The 85-kDa cytoplasmic phospholipase A2 (cPLA2) is the major hormone a nd growth factor-regulated enzyme that catalyzes release of arachidoni c acid in mammalian cells. Activation of cPLA2 requires elevation of i ntracellular Ca2+ and the phosphorylation of the cPLA2 enzyme by mitog en-activated protein (MAP) kinase. Down-regulation of protein kinase C by phorbol esters or pertussis toxin catalyzed ADP-ribosylation of G( i) proteins inhibits thrombin and ATP receptor-stimulated MAP kinase a nd arachidonic acid release, indicating that functional protein kinase C and G(i) proteins are required for G protein regulation of arachido nic acid release. A mutant Galpha(i2) subunit having Gly203 mutated to Thr (alpha(i2)G203T) inhibited thrombin and ATP receptor stimulation of arachidonic acid release independent of adenylyl cyclase inhibition , Ca2+ mobilization, and MAP kinase activation. Overexpression of the wild-type alpha(i2) polypeptide or the inactive mutant alpha(i2)G204A (Gly204 mutated to Ala) polypeptide had no effect on thrombin or ATP r eceptor stimulation of arachidonic acid release. The phenotype observe d with expression of the mutant alpha(i2)G203T polypeptide defines a r ole for G(i2) in the control of cPLA2 activity and subsequent arachido nic acid release in addition to the regulation of intracellular Ca2+ l evels and MAP kinase activity.