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 receptor that exhibits constitutive receptor activation, defined as agonist-independent receptor activation of the signal transduction pathway. The present studies were performed to determine whether NIH/3T3 fibroblasts expressing the 5-HT2C receptor exhibited desensitization of agonist-mediated phosphoinositide hydrolysis. Furthermore, 5-HT2C receptor-specific antibodies were used to determine whether the 5-HT2C receptor 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 phosphoinositide hydrolysis demonstrated that basal values increased in a linear manner, whereas the response to serotonin plateaued within 60 min. In addition, pretreatment with serotonin resulted in a rightward shift of the 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 32P-labeled fibroblasts. Phosphorylation of the 5-HT2C receptor was evident under basal conditions, and serotonin treatment increased receptor 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 receptor is phosphorylated under basal conditions and phosphorylation is increased by agonist treatment conditions that result in desensitization of 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 with current models of G protein-coupled receptor regulation.
Westphal
R S
RS
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Backstrom
J R
JR
Sanders-Bush
E
E
D016475:3T3 Cells; D000818:Animals; D006868:Hydrolysis; D051379:Mice; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011985:Receptors, Serotonin / Q000187:drug effects* / Q000378:metabolism; D017366:Serotonin Receptor Agonists / Q000494:pharmacology*
D010716:Phosphatidylinositols; D011985:Receptors, Serotonin; D017366:Serotonin Receptor Agonists