Serotonin-mediated palmitoylation and depalmitoylation of G alpha proteins in rat brain cortical membranes.
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 [3H] palmitate incorporation observed was by G alpha-q (7-fold), followed by G alpha-o (5-fold), 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 [3H] 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.
Bhamre
S
S
Laboratory of Molecular Pharmacology, Department of Pharmacology, MCP-Hahnemann School of Medicine, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania, USA.
Wang
H Y
HY
Friedman
E
E
D000818:Animals; D002540:Cerebral Cortex / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D008297:Male; D019308:Palmitic Acid / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012701:Serotonin / Q000494:pharmacology*
D019308:Palmitic Acid; D012701:Serotonin; D006165:Guanylyl Imidodiphosphate; D019204:GTP-Binding Proteins