Serotonin-Mediated Palmitoylation and Depalmitoylation of G Alpha Proteins in Rat Brain Cortical Membranes1

We investigated serotonin stimulated palmitoylation of G alpha subunits in rat brain cerebrocortical membranes. Serotonin dose dependently stimulated palmitoylation of membrane G alpha proteins. The highest [H] palmitate incorporation observed was by G alpha-q (7-fold), followed by G alpha-o (5fold), G alpha-i (4-fold) and G alpha-s (3-fold) and these increases in palmitoylation were blocked by methiothipin, a serotonin receptor antagonist. Isoproterenol selectively stimulated G alpha-s palmitoylation which was blocked by propranalol. Immunoprecipitates of palmitoylated G alpha subunits yielded single labeled bands on SDS-PAGE. In an attempt to define the sequence of palmitoylation/depalmitoylation that follows receptor stimulation, nonreceptor mediated palmitoylation was carried out in the presence of guanine nucleotides and receptor mediated G alpha depalmitoylation was then monitored. Receptor stimulation did not result in depalmitoylation when membranes were prelabeled with [H] palmitic acid in the presence of the nonhydrolyzable analogue of GTP, Gpp(NH)p. However, serotonin receptor stimulation in the presence of guanine nucleotides, depalmitoylated (90%) membrane G alpha proteins when prelabeled in the presence of GTP. Coimmunoprecipitation experiments revealed decrease in G beta immunoreactivity associated with G alpha immunoprecipitates obtained from membranes prelabeled in presence of GTP prior to reincubation with Gpp(NH)p and serotonin. These observations suggest that receptor occupation results in depalmitoylation of the trimer, followed by guanine nucleotide exchange and dissociation of the alpha subunit from beta-gamma dimer and that the activated alpha subunit is a substrate for repalmitoylation. Heterotrimeric G proteins are central in transducting signals from cell surface receptors to appropiate membrane effectors. G proteins consist of three subunits. To date, ;25 alpha, 5 beta and 11 gamma subunits have been identified (Simon et al., 1991). Activation of G proteins by G protein coupledreceptors results in an exchange of GDP for GTP on the alpha subunit followed by dissociation of the alpha subunit from the beta-gamma dimer. Intrinsic GTPase activity of the alpha subunit subsequently hydrolyzes GTP and the reassociation of alpha-GDP with beta-gamma completes the inactivation of the G protein. The free alpha subunit and beta-gamma complex can interact with target effectors (Birnbaumer, 1992; Neer, 1994, 1995; Sternweis, 1994). The specificity of signal transduction is determined by the association of receptors with different subtypes of G proteins that are an assembly of alpha-, betaand gamma subunits (Birnbaumer and Birnbaumer, 1995; Simon et al., 1991). Homology at the amino acid level has lead to the grouping of the alpha subunits into 4 subfamilies, G alpha-s, G alpha-i, G alpha-q and