Identification of 5-HT1A Receptor Phosphorylation Sites

5-HT1A 受体磷酸化位点的鉴定

• 批准号: 6723758
• 负责人: ANDREW K GELASCO
• 金额: $ 6.3万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6723758
• 关键词: G protein coupled receptor kinase; SDS polyacrylamide gel electrophoresis; affinity chromatography; high performance liquid chromatography; mass spectrometry; molecular site; peptides; phosphorylation; polymerase chain reaction; protein kinase C; serotonin; serotonin receptor; western blottings

DESCRIPTION (provided by applicant): This small grant application is submitted to support preliminary studies on the mechanism of phosphorylation of the serotonin 5-HT1A receptor by protein kinase C. The principal investigator is a new investigator in the field of receptor biochemistry and will incorporate peptide mass spectrometric methods to identify the putative protein kinase C phosphorylation sites on the i3 loop of the receptor. The long-term goal of this grant is to establish methods that will allow us to develop projects to investigate the phosphorylation sites on the intact membrane bound receptor 5-Hydroxytryptamine (serotonin, 5-HT) is a biogenic amine neurotransmitter first discovered as a potent vasotonic factor in 1948. The cellular and physiological effects of serotonin are mediated by seven major families of receptors (5-HTn, where n= 1-7), containing at least 14 subtypes. The first 5-HT receptor cloned and by far the best-characterized 5-HT receptor is the 5-HT1A receptor. The 5-HT1A receptor has been shown to be important in a number of neurochemically related physiological processes especially those involving memory, depression, aggression, and anxiety. The 5-HT receptors are G-protein coupled receptors (GPCRs). GPCRs are characterized by their signaling via G proteins (guanine nucleotide binding proteins) upon activation. Desensitization of a receptor can occur after prolonged exposure to a stimulus such as a hormone or neurotransmitter disrupting the G protein signal pathway. One of the cellular events implicated in receptor desensitization is the kinase-directed phosphorylation of the associated receptor. The overall goal of this study is to identify some of the phosphorylation sites on the serotonin receptor 1A (5- HT1A) through the use of peptide mass spectrometry. Initial studies will focus on phosphorylation of purified peptides representing the third putative intracellular loop of the receptor and will involve development of mass spectrometric techniques for unambiguous identification of the sites of phosphorylation. The specific aims of this proposal are: (1) to identify, express, and isolate a peptide that mimics the third intracellular loop of the 5-HTIA receptor (5-HTla-i3), an approximately 135 amino acid region from R216 to M350 as a target for PKC; (2) to develop specific conditions under which either purified PKC or activated PKC from stimulated cell lysates can phosphorylate the 5-HT1A-i3 loop, and (3) to identify the specific phosphorylation sites on 5-HT1A-i3 by MALDI and HPLC-ESI mass spectrometry.

描述（由申请人提供）：提交此小额资助申请是为了支持蛋白激酶 C 磷酸化血清素 5-HT1A 受体机制的初步研究。主要研究人员是受体生物化学领域的新研究人员，将纳入肽质谱法鉴定受体 i3 环上推定的蛋白激酶 C 磷酸化位点。这笔赠款的长期目标是建立方法，使我们能够开发项目来研究完整膜结合受体 5-羟色胺（血清素，5-HT）上的磷酸化位点，它是一种生物胺神经递质，最初被发现是一种有效的神经递质。 1948 年血管紧张因子。血清素的细胞和生理效应由七个主要受体家族（5-HTn，其中 n= 1-7）介导，包含至少 14 个亚型。第一个克隆的 5-HT 受体，也是迄今为止特征最清楚的 5-HT 受体，是 5-HT1A 受体。 5-HT1A 受体已被证明在许多神经化学相关的生理过程中非常重要，尤其是涉及记忆、抑郁、攻击性和焦虑的生理过程。 5-HT 受体是 G 蛋白偶联受体 (GPCR)。 GPCR 的特点是激活后通过 G 蛋白（鸟嘌呤核苷酸结合蛋白）发出信号。长时间暴露于刺激物（例如激素或神经递质）破坏 G 蛋白信号通路后，受体可能会发生脱敏。与受体脱敏有关的细胞事件之一是相关受体的激酶定向磷酸化。 本研究的总体目标是通过使用肽质谱法来鉴定血清素受体 1A (5-HT1A) 上的一些磷酸化位点。初步研究将集中于代表第三个假定的受体细胞内环的纯化肽的磷酸化，并将涉及质谱技术的开发，以明确识别磷酸化位点。 该提案的具体目标是：(1) 鉴定、表达和分离模拟 5-HTIA 受体 (5-HTla-i3) 的第三个细胞内环的肽，该肽是从 R216 到 M350 的大约 135 个氨基酸区域作为 PKC 的目标； (2) 开发特定条件，使纯化的 PKC 或来自受刺激细胞裂解物的活化 PKC 能够磷酸化 5-HT1A-i3 环，以及 (3) 通过 MALDI 和 HPLC 鉴定 5-HT1A-i3 上的特定磷酸化位点ESI 质谱。