Deletion of the serotonin 5-HT(2c) receptor PDZ recognition motif prevents receptor phosphorylation and delays resensitization of receptor responses

Abstract

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 (Ser458-Ser-Val-COOH; ΔPDZ) was not detectable based on a band-shift phosphorylation assay and incorporation of 32p. Treatment of cells stably expressing ΔPDZ or wild-type 5-HT(2C) receptors with serotonin produced identical maximal responses and EC50 values f0r ellciting [3H-inositol phosphate formation. In calcium imaging studies, treatment of cells expressing ΔPDZ or wild-type 5-HT(2C) 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 ~5-fold lower with ΔPDZ receptors relative to wild-type 5-HT(2C) receptors. After 10 min, responses of ΔPDZ receptors recovered to wild-type 5-HT(2C) receptor levels. Receptors with single mutations at Ser458 (S458A) or Ser459 (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-HT(2C) receptor phosphorylation and suggest that receptor phosphorylation at Ser459 enhances resensitization of 5-HT(2C) receptor responses.