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FUNCTION OF SUBSTANCE P RECEPTOR

P物质受体的功能

基本信息

批准号：
2714546
负责人：
MADAN M KWATRA
金额：
$ 16.14万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-07-01 至 2001-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2714546
关键词：
G protein Sf9 cell line Xenopus oocyte guanosine triphosphate isozymes neuropeptide receptor phosphoproteins protein kinase C protein reconstitution protein structure function site directed mutagenesis substance P voltage /patch clamp

项目摘要

The long-term objective of this project is to understand the function of human substance P (and other tachykinin receptors. Within this context, the overall focus of this project is elucidation of mechanisms involved in regulation of the substance P receptor (SPR). SPRs mediate effects of the neuropeptide substance P (SP); SP (as well as glutamate, opiates, and alpha2-adrenergic agonists) are important modulators in pain pathways. SP also plays an important role in inflammation and neurogenic edema including the axon flare reaction, vasodilation, gut motility and secretion. Given the many important physiologic roles of SP, it is important to understand SPR function. Hence, the PI plans to test the hypothesis that SPR function is regulated by receptor phosphorylation, and that SPR phosphorylation is related to agonist exposure. To achieve this goal, the PI plans to evaluate SPR phosphorylation using in vitro reconstitution (aim 1), whole cell (aim 2), and Xenopus oocyte (aim 3) systems. The PI has recently demonstrated that phosphorylation of SPRs is catalyzed by G protein receptor kinase (GRK) isozymes 2 and 3, a novel finding for receptors coupled to phosphoinositide hydrolysis. Since other GRK isozymes and protein kinase C (PKC) may also be involved in SPR phosphorylation, the first specific aim is designed to characterize phosphorylation of SPRs by various kinases. The PI has established conditions to purify and reconstitute human SPRs recombinantly expressed in Sf9 cells. This in vitro reconstitution system will be used to analyze the effect of SPR phosphorylation on interactions with G proteins and arrestins, and elucidate sites of phosphorylation using mutagenesis approaches. In order to ascertain the physiologic relevance of SPR phosphorylation, specific aim 2 uses whole cell approaches and for these studies, the PI has engineered an epitope at the amino-terminus of the SPR, and receptor specific antibodies are currently being developed. These tools will be used to assess the role of SPR phosphorylation under desensitizing conditions and conditions known to activate PKC isozymes. In specific aim 3, the role of phosphorylation in SPR function will be further analyzed using a Xenopus oocyte system. In this model, mRNA of wild type and mutant SPRs (from specific aim 1) will be transcribed in vitro, injected into oocytes, and desensitization of SP-induced chloride currents measured using a two electrode voltage clamp technique recently established in the PI's laboratory. The effect of truncated (minus carboxyl terminus) and mutated (lacking PKC phosphorylation site[s]) SPRs, and PKC modulators, on SPR desensitization will be explored. Extensive experience in receptor phosphorylation and protein biochemistry, current availability of unique substrates such as purified reconstituted SPRs, epitope tagged SPRs, and various GRK and PKC isozymes, as well as ongoing development of SPR antibodies in the PI's laboratory, places the PI in a unique position to complete the proposed studies. Information derived from these studies should facilitate understanding mechanisms underlying many SP-mediated physiologic processes.

该项目的长期目标是了解人类 P 物质（和其他速激肽受体。在这种情况下，该项目的总体重点是阐明涉及的机制 P 物质受体 (SPR) 的调节。 SPR 介导的影响神经肽物质P(SP)； SP（以及谷氨酸盐、阿片类药物和 α2-肾上腺素能激动剂）是疼痛通路的重要调节剂。 SP 在炎症和神经源性水肿中也起着重要作用包括轴突耀斑反应、血管舒张、肠道蠕动和分泌。鉴于 SP 具有许多重要的生理作用，了解 SPR 功能很重要。因此，PI 计划测试假设 SPR 功能受受体磷酸化调节，并且 SPR 磷酸化与激动剂暴露有关。为了实现这一目标目标，PI 计划使用体外评估 SPR 磷酸化重建（目标 1）、全细胞（目标 2）和非洲爪蟾卵母细胞（目标 3）系统。 PI 最近证明 SPR 的磷酸化是由 G 蛋白受体激酶 (GRK) 同工酶 2 和 3 催化，一种新型寻找与磷酸肌醇水解偶联的受体。由于其他 GRK 同工酶和蛋白激酶 C (PKC) 也可能参与 SPR 磷酸化，第一个具体目标是设计表征 SPR 被各种激酶磷酸化。 PI 已建立纯化和重建重组表达的人类 SPR 的条件在Sf9细胞中。该体外重构系统将用于分析 SPR 磷酸化对与 G 蛋白相互作用的影响抑制蛋白，并通过诱变阐明磷酸化位点接近。为了确定 SPR 的生理相关性磷酸化，具体目标 2 使用全细胞方法，并且对于这些研究中，PI 在氨基末端设计了一个表位目前正在开发 SPR 和受体特异性抗体。这些工具将用于评估 SPR 磷酸化的作用脱敏条件和已知激活 PKC 同工酶的条件。在具体目标 3 中，磷酸化在 SPR 功能中的作用将是使用非洲爪蟾卵母细胞系统进一步分析。在该模型中，mRNA 野生型和突变型 SPR（来自特定目标 1）将被转录为体外，注射到卵母细胞中，以及 SP 诱导的氯化物脱敏最近使用两电极电压钳技术测量电流建立在PI的实验室内。截断的效果（减去羧基末端）和突变（缺乏 PKC 磷酸化位点）SPR，和 PKC 调制器，对 SPR 脱敏作用将进行探讨。广泛的在受体磷酸化和蛋白质生物化学方面的经验，目前独特底物的可用性，例如纯化的重组 SPR，表位标记的 SPR、各种 GRK 和 PKC 同工酶，以及正在进行的在 PI 实验室中开发 SPR 抗体，将 PI 置于完成拟议研究的独特地位。衍生信息这些研究应该有助于理解潜在的机制许多 SP 介导的生理过程。

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Human substance P receptor lacking the C-terminal domain remains competent to desensitize and internalize.

缺乏 C 末端结构域的人 P 物质受体仍然具有脱敏和内化的能力。

DOI：
10.1046/j.1471-4159.2003.01577.x
发表时间：
2003
期刊：
Journal of neurochemistry
影响因子：
4.7
作者：
Richardson,MarkD;Balius,AnastasiaM;Yamaguchi,Keisuke;Freilich,EmilyR;Barak,LarryS;Kwatra,MadanM
通讯作者：
Kwatra,MadanM

A constitutively active form of neurokinin 1 receptor and neurokinin 1 receptor-mediated apoptosis in glioblastomas.