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 dete rmine whether phosphorylation promotes desensitization of receptor response s. Phosphorylation of mutant 5-HT2C receptors that lack the carboxyl-termin al PDZ recognition motif (Ser(458)-Ser-Val-COOH; Delta PDZ) was not detecta ble based on a band-shift phosphorylation assay and incorporation of P-32. Treatment of cells stably expressing Delta PDZ or wild-type 5-HT2C receptor s with serotonin produced identical maximal responses and EC50 values for e liciting [H-3]-inositol phosphate formation. In calcium imaging studies, tr eatment of cells expressing Delta PDZ or wild-type 5-HT2C receptors with 10 0 nM serotonin elicited initial maximal responses and decay rates that were indistinguishable. However, a second application of serotonin 2.5 min afte r washout caused maximal responses that were similar to 5-fold lower with D elta PDZ receptors relative to wild-type 5-HT2C receptors. After 10 min, re sponses of Delta PDZ receptors recovered to wild-type 5-HT2C receptor level s. Receptors with single mutations at Ser(458) (S458A) or Ser(459) (S459A) decreased serotonin-mediated phosphorylation to 50% of wild-type receptor l evels. Furthermore, subsequent calcium responses of S459A receptors were di minished relative to S458A and wild-type receptors. These results establish that desensitization occurs in the absence of 5-HT2C receptor phosphorylat ion and suggest that receptor phosphorylation at Ser459 enhances resensitiz ation of 5-HT2C receptor responses.