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The actions of the neurotransmitter 5-hydroxytryptamine (5-HT) (serotonin) are mediated by multiple receptor subtypes. One of the prominent serotonin receptors in the brain is the 5-HT2C receptor (5-HT2C-R). We report the occurrence of a second 5-HT2C-R transcript, first identified using S1 nuclease protection of total RNA isolated from the choroid plexus. Analyses of the distribution of these two RNAs revealed that the short form is expressed in the same structures as the 5-HT2C-R mRNA, including choroid plexus, striatum, hippocampus, hypothalamus, olfactory tubercles, and spinal cord. Cloning and sequence analyses revealed a second cDNA with a 95-nt deletion in the region coding for the putative second intracellular loop and the fourth transmembrane domain of the 5-HT2C-R. This deletion leads to a frameshift in the coding sequence and the introduction of a premature stop codon. The predicted truncated protein (5-HT2C-tr) contains 172 amino acids, with 153 residues at the amino terminus, identical to the 5-HT2C-R, and 19 carboxyl-terminal amino acids that are unique. Although antibodies specific to the 5-HT2C-tr protein showed that the truncated form is expressed in a transfected fibroblast cell model system, there was no serotonergic ligand binding activity or phosphoinositide hydrolysis. Analyses of the 5-HT2C-R gene revealed that the two transcripts arise from a single gene by differential splicing using alternative donor sites and a common 3'-splice acceptor. Polymerase chain reaction amplification of mouse and human brain cDNAs demonstrated the occurrence of the same splicing patterns in these species. Although this study demonstrates tissue-specific expression of two 5-HT2C mRNA splice variants in rat, mouse, and human, the significance of the truncated form in these three species remains to be established.

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.