Deletion of the serotonin 5-HT2C receptor PDZ recognition motif prevents receptor phosphorylation and delays resensitization of receptor responses
Deletion of the serotonin 5-HT2C receptor PDZ recognition motif prevents receptor phosphorylation and delays resensitization of receptor responses
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DOI：
10.1074/jbc.m000922200
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发表时间：
2000-08-04
影响因子：
4.8
通讯作者：
Sanders-Bush, E
中科院分区：
文献类型：
--
作者：
Backstrom, JR;Price, RD;Sanders-Bush, E
产生磷酸化缺陷的5-羟色胺5-HT 2C受体以确定磷酸化是否促进受体应答的脱敏。缺乏羧基末端PDZ识别基序（Ser（458）-Ser-Val-COOH; Delta PDZ）的突变型5-HT 2C受体的磷酸化基于带移磷酸化测定和P-32的掺入是不可检测的。用5-羟色胺处理稳定表达Delta PDZ或野生型5-HT 2C受体的细胞产生相同的最大反应和诱导[H-3]-磷酸肌醇形成的EC 50值。在钙成像研究中，用100 nM 5-羟色胺处理表达Delta PDZ或野生型5-HT 2C受体的细胞引起了最初的最大反应和不可区分的衰减率。然而，在洗脱后2.5分钟第二次应用5-羟色胺引起的最大反应类似于相对于野生型5-HT 2C受体，Delta PDZ受体低5倍。10分钟后，Delta PDZ受体的反应恢复到野生型5-HT 2C受体水平。在Ser（458）（S458 A）或Ser（459）（S459 A）处具有单突变的受体将马槟榔宁介导的磷酸化降低至野生型受体水平的50%。此外，S459 A受体随后的钙反应相对于S458 A和野生型受体减少。这些结果表明，脱敏发生在5-HT 2C受体磷酸化的情况下，并表明Ser 459的受体磷酸化增强了5-HT 2C受体反应的再敏化。
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.