FUNCTION OF SUBSTANCE P RECEPTOR

本项目的长期目标是了解 人P物质(和其他速激肽受体)。在此背景下， 本项目的总体重点是阐明 P物质受体(SPR)的调节。SPRs对大鼠心脏功能的调节作用 神经肽P物质(SP)；SP(以及谷氨酸、阿片类和 α2-肾上腺素能激动剂)是痛觉通路的重要调节剂。服务提供商 也在炎症和神经源性水肿中起着重要作用 包括轴突闪光反应、血管扩张、肠道运动和 分泌物。鉴于SP的许多重要生理作用，它是 对理解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，以及 SPR的磷酸化与激动剂的暴露有关。要做到这一点 Goal，PI计划在体外评估SPR的磷酸化 重组(目标1)、全细胞(目标2)和非洲爪哇卵母细胞(目标3) 系统。PI最近证明了SPRs的磷酸化是 由G蛋白受体激酶(GRK)同工酶2和3催化的一种新的 寻找偶联到肌醇磷脂水解物的受体。因为其他人 GRK同工酶和蛋白激酶C(PKC)也可能参与SPR 磷酸化，第一个特定的目标是为了表征 不同类型的激酶对SPRs的磷酸化作用。公安部已经建立了 重组人SPRs纯化和重组条件的研究 在Sf9细胞中。这个体外重建系统将被用来分析 SPR磷酸化对G蛋白和G蛋白相互作用的影响 阻滞素，并利用突变来阐明磷酸化的位置 接近了。为了确定SPR的生理相关性 磷酸化，特殊目标2使用全细胞方法，对于这些 研究表明，PI在氨基酸末端设计了一个表位。 SPR和受体特异性抗体目前正在开发中。 这些工具将用于评估SPR磷酸化在以下条件下的作用 脱敏条件和已知的激活PKC同工酶的条件。 在特定目标3中，磷酸化在SPR功能中的作用将是 使用非洲爪哇卵母细胞系统进行了进一步的分析。在这个模型中，mRNA的表达 野生型和突变型SPR(来自特定目标1)将在 体外注射入卵母细胞及SP诱导的氯离子脱敏 最近用双电极电压钳技术测量的电流 在PI的实验室里建立起来的。截断(减号)的效果 羧基末端)和突变(缺少PKC磷酸化位点[S])SPRs， 并对PKC调节剂对SPR脱敏作用进行了探讨。广泛性 受体磷酸化和蛋白质生物化学方面的经验 独特底物的可用性，例如纯化的重组SPR， 表位标记的SPR，以及各种GRK和PKC同工酶以及正在进行的 在PI的实验室中开发出SPR抗体，将PI置于 完成拟议研究的独特地位。派生的信息 从这些研究中应该有助于理解 许多由SP介导的生理过程。

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.