HUMAN SEROTONIN(1B) RECEPTOR EXPRESSION IN SF9 CELLS - PHOSPHORYLATION, PALMITOYLATION, AND ADENYLYL-CYCLASE INHIBITION

Analysis of the primary protein structure ot the human serotonin1B (5-HT1B) receptor reveals consensus sites for phosphorylation and a putative site for palmitoylation. To investigate these posttranslational modifications, we have expressed a c-myc epitope-tagged 5-HT1B (m5-HT1B) receptor in Sf9 cells. This strategy enabeled receptors to be detected by immunoblot analysis and purified by immunoprecipitation using a monoclonal antibody, 9E10, specific for the c-myc epitope. Agonist radio ligand [H-3]5-HT binding studies showed that the expressed 5-HT1B and m5-HT1B receptors displayed the characteristic pharmacological profile of the neuronal 5-HT1B receptor. The expressed receptors displayed both high- and low-affinity states for [H-3]5-HT, suggesting that the receptors were coupled to endogenous G-proteins. Indeed, agonist binding to the high-affinity receptor state was regulated in the presence of GTPgammaS, Gpp(NH)p, and pertussis toxin. [P-32]ADP-ribosylation experiments identified a major approximately 41-kDa ADP-ribosylated protein present in Sf9 membranes that comigrated with partially purified bovine brain G(ialpha)/G(oalpha) subunits. Measurements of adenylyl cyclase activity in membranes from cells expressing m5-HT1B receptors showed that serotonergic agonists mediated the inhibition of adenylyl cyclase activity with a rank order of potency comparable to their affinity constants. Immunoblot analysis of membranes prepared from cells expressing m5-HT1B receptors and photoaffinity labeling of the immunoprecipitated material revealed photolabeled species at approximately 95 and at approximately 42 kDa. Immunoprecipitated material migrating at approximately 95 and approximately 42 kDa was shown to be posttranslationally modified following whole cell metabolic labeling with [P-32i]phosphate or [H-3]palmitic acid, and this provides the first evidence that the 5HT1B serotonin receptor is phosphorylated and palmitoylated.