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。 在这方面， 该项目的总体重点是阐明 P物质受体（SPR）。 SPRs介导了 神经肽物质P（SP）; SP（以及谷氨酸，阿片类药物， α 2肾上腺素能激动剂）是疼痛通路中的重要调节剂。 SP 在炎症和神经源性水肿中也起重要作用 包括轴突闪光反应、血管舒张、肠蠕动和 分泌物 鉴于SP的许多重要生理作用， 了解SPR功能很重要。 因此，PI计划测试 SPR功能受受体磷酸化调节的假说， SPR磷酸化与激动剂暴露有关。 实现这一 目的，PI计划使用体外方法评估SPR磷酸化 重组（目的1）、全细胞（目的2）和非洲爪蟾卵母细胞（目的3） 系统. PI最近证明，SPRs的磷酸化是 由G蛋白受体激酶（GRK）同工酶2和3催化， 发现与磷酸肌醇水解偶联的受体。 因为其他 GRK同工酶和蛋白激酶C（PKC）也可能参与SPR的发生 磷酸化，第一个具体目标是设计表征 SPRs被各种激酶磷酸化。 PI已经建立了 纯化和重建重组表达的人SPR的条件 在Sf 9细胞中。 该体外复溶系统将用于分析 SPR磷酸化对与G蛋白相互作用的影响， 抑制蛋白，并使用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 在研究中，PI已经在抗体的氨基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。 这些工具将被用来评估SPR磷酸化的作用， 脱敏条件和已知激活PKC同工酶的条件。 在具体的目标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， 野生型和突变型SPR（来自特定目的1）将在 体外，注射到卵母细胞中，和SP诱导的氯脱敏 最近使用双电极电压钳技术测量的电流 在PI的实验室里。 截断（减去）的效果 羧基末端）和突变的（缺乏PKC磷酸化位点）SPR， 和PKC调节剂对SPR脱敏的影响。 广泛 受体磷酸化和蛋白质生物化学经验，目前 独特底物如纯化的重构SPR的可用性， 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实验室开发SPR抗体，将PI置于 完成拟议研究的独特位置。 导出的信息 这些研究有助于理解 许多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.