Identification of 5-HT1A Receptor Phosphorylation Sites

5-HT1A 受体磷酸化位点的鉴定

• 批准号: 6630119
• 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 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6630119
• 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.

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.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（5-羟色胺，5-HT）上的磷酸化位点是一种生物胺神经素养剂，是一种在1948年的强有力的血管生物学因素。 （5-HTN，其中n = 1-7），至少包含14个亚型。克隆的第一个5-HT受体，到目前为止，特征最佳的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很重要，尤其是涉及记忆，抑郁，侵略和焦虑的生理过程。 5-HT受体是G蛋白偶联受体（GPCR）。 GPCR的特征在于激活后通过G蛋白（鸟嘌呤核苷酸结合蛋白）信号传导。长期暴露于刺激（例如激素或神经递质）破坏G蛋白信号途径的刺激后，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的磷酸化。 这项研究的总体目标是通过使用肽质谱法确定5-羟色胺受体1a（5- HT1A）上的一些磷酸化位点。最初的研究将集中于代表受体的第三个假定细胞内环的纯化肽的磷酸化，并将涉及开发质谱技术，以明确鉴定磷酸化位点。 该提案的具体目的是：（1）识别，表达和分离一个模仿5-HTIA受体的第三个细胞内环（5-HTLA-I3）的肽，这是大约135个氨基酸区域，从R216到M350作为PKC的靶标。 （2）为了开发特定条件，从刺激的细胞裂解物中纯化的PKC或激活的PKC可以磷酸化5-HT1A-I3回路，并且（3）通过MALDI和HPLC-ESI质谱法鉴定5-HT1A-I3上的特定磷酸化位点。