INCREASED BASAL PHOSPHORYLATION OF THE CONSTITUTIVELY ACTIVE SEROTONIN 2C RECEPTOR ACCOMPANIES AGONIST-MEDIATED DESENSITIZATION

The 5-hydroxytryptamine (5-HT)(2C) receptor is a G protein-coupled rec eptor that exhibits constitutive receptor activation, defined as agoni st-independent receptor activation of the signal transduction pathway. The present studies were performed to determine whether NIH/3T3 fibro blasts expressing the 5-HT2C receptor exhibited desensitization of ago nist-mediated phosphoinositide hydrolysis. Furthermore, 5-HT2C recepto r-specific antibodies were used to determine whether the 5-HT2C recept or was phosphorylated in the absence of agonist and whether treatment with an agonist or an inverse agonist altered receptor phosphorylation . Time course studies of basal and serotonin-stimulated phosphoinositi de hydrolysis demonstrated that basal values increased in a linear man ner, whereas the response to serotonin plateaued within 60 min. In add ition, pretreatment with serotonin resulted in a rightward shift of th e subsequently determined serotonin dose-response curve. To determine the phosphorylation state of the 5-HT2C receptor, specific antibodies were used to immunoprecipitate the receptor from lysates prepared from P-32-labeled fibroblasts. Phosphorylation of the 5-HT2C receptor was evident under basal conditions, and serotonin treatment increased rece ptor phosphorylation. The inverse agonist mianserin had no detectable effect on 5-HT2C receptor phosphorylation when added alone but blocked the serotonin-stimulated increase in 5-HT2C receptor phosphorylation. The present study is the first to demonstrate that the 5-HT2C recepto r is phosphorylated under basal conditions and phosphorylation is incr eased by agonist treatment conditions that result in desensitization o f receptor signaling. Thus, these studies demonstrate that a cell line exhibiting a high level of constitutive 5-HT2C receptor activity has the ability to undergo agonist-mediated desensitization, consistent wi th current models of G protein-coupled receptor regulation.