Deletion of the serotonin 5-HT2C receptor PDZ recognition motif prevents receptor phosphorylation and delays resensitization of receptor responses

Phosphorylation-deficient serotonin 5-HT2C receptors were generated to determine whether phosphorylation promotes desensitization of receptor responses. Phosphorylation of mutant 5-HT2C receptors that lack the carboxyl-terminal PDZ recognition motif (Ser(458)-Ser-Val-COOH; Delta PDZ) was not detectable based on a band-shift phosphorylation assay and incorporation of P-32. Treatment of cells stably expressing Delta PDZ or wild-type 5-HT2C receptors with serotonin produced identical maximal responses and EC50 values for eliciting [H-3]-inositol phosphate formation. In calcium imaging studies, treatment of cells expressing Delta PDZ or wild-type 5-HT2C receptors with 100 nM serotonin elicited initial maximal responses and decay rates that were indistinguishable. However, a second application of serotonin 2.5 min after washout caused maximal responses that were similar to 5-fold lower with Delta PDZ receptors relative to wild-type 5-HT2C receptors. After 10 min, responses of Delta PDZ receptors recovered to wild-type 5-HT2C receptor levels. Receptors with single mutations at Ser(458) (S458A) or Ser(459) (S459A) decreased serotonin-mediated phosphorylation to 50% of wild-type receptor levels. Furthermore, subsequent calcium responses of S459A receptors were diminished relative to S458A and wild-type receptors. These results establish that desensitization occurs in the absence of 5-HT2C receptor phosphorylation and suggest that receptor phosphorylation at Ser459 enhances resensitization of 5-HT2C receptor responses.