Phosphorylation-deficient serotonin 5-HT 2C receptors were generated to determine whether phosphorylation promotes desensitization of receptor responses. Phosphorylation of mutant 5-HT 2C receptors that lack the carboxyl-terminal PDZ recognition motif (Ser 458 -SerVal-COOH; ⌬PDZ) was not detectable based on a bandshift phosphorylation assay and incorporation of 32 Serotonin 5-HT 2C (formerly 5-HT 1C ) receptors exists as several isoforms throughout the brain, due to RNA editing and alternative splicing, and function to stimulate phospholipase C through activation of the G protein G q (1). Whereas RNA editing generates isoforms in the second intracellular loop that modify receptor signaling (2-4), alternative splicing creates truncated nonfunctional receptors (5, 6). In addition, agonists promote phosphorylation of nonedited 5-HT 2C receptors (7), raising the possibility that 5-HT 2C receptors are also regulated dynamically.Phosphorylation of G protein-coupled receptors regulates signaling through multiple mechanisms including receptor desensitization, which attenuates second messenger responses. In the case of phospholipase C-linked receptors, phosphorylation has been demonstrated to promote desensitization based on observations that receptors lacking the corresponding phosphorylation sites display sustained phosphoinositide hydrolysis responses relative to wild-type receptors (8 -13). Furthermore, a second application of agonist produces amplified phosphoinositide hydrolysis (14) and calcium release (13, 15, 16) by phosphorylation-deficient mutants relative to wild-type receptors, illustrating that mutation of phosphorylation sites involved in desensitization enhances both initial and secondary responses.Although desensitization of 5-HT 2C receptor-mediated responses has been observed in assays that examine phosphoinositide hydrolysis (7, 17), release of intracellular calcium (18,19), and Ca 2ϩ -activated currents (20), it is unknown whether phosphorylation of the 5-HT 2C receptor is involved in desensitization. To address this issue, we identified a 5-HT 2C receptor domain that is required for receptor phosphorylation and performed functional assays with phosphorylation-deficient receptor mutants. First, we determined that phosphorylation of the 5-HT 2C receptor requires the carboxyl-terminal PDZ (PSD-95 discs-large ZO-1) recognition motif, a domain present in nonedited and edited isoforms of the 5-HT 2C receptor. Next, we found that phosphorylation-deficient receptors display identical initial responses as the wild-type 5-HT 2C receptor in phosphoinositide hydrolysis and calcium release assays. However, phosphorylation-deficient receptors exhibit diminished secondary responses and a delayed recovery relative to wild-type 5-HT 2C receptors. Cells stably expressing 5-HT 2C receptors with a single serine-to-alanine mutation also display decreased receptor phosphorylation and diminished secondary calcium responses. We therefore propose that 5-HT 2C receptor phosphorylation promotes resensitization of 5-HT 2C re...
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