Regulation of P2Y2 purinergic receptors

P2Y2 嘌呤能受体的调节

• 批准号: 6576227
• 负责人: T KENDALL HARDEN
• 金额: $ 7.38万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6576227
• 关键词: G protein coupled receptor kinase; arrestins; biological signal transduction; chimeric proteins; clathrin; confocal scanning microscopy; guanine nucleotide binding protein; human tissue; immunocytochemistry; ion transport; liposomes; phosphorylation; protein purification; protein structure function; purinergic receptor; pyrimidine nucleotides; receptor binding; receptor expression; receptor sensitivity; respiratory epithelium; tissue /cell culture

G protein-coupled P2Y receptors (P2Y-R) for extracellular nucleotides subserve important roles in lung epithelial cell biology and pathophysiology. The long-term goal of our research is to delineate the molecular basis for nucleotide action at airway P2Y-R. The human P2Y2- R will be purified to homogeneity and reconstituted in proteoliposomes with G1 and other G proteins to: (i) unequivocally define agonist binding efficacies and potencies for the P2Y2-R, (ii) develop insight into the nature of constitutive activity of the P2Y2-R, (iii) determine the specificities of interaction of the P2Y2-R with G proteins, and (iv) establish whether agonist and G protein selectivities of the P2Y2-R are regulated by protein kinase-promoted phosphorylation or RGS proteins. Functional chimeric proteins will be constructed fusing the P2Y2-R with Galphaq, Galphai1, or Galpha12. Our preliminary results indicate that the human P2Y2-R, P2Y4-R, and P2Y6-R exhibit very different phenotypes of agonist-induced desensitization, internalization, and down-regulation. These differences in occurrence and characteristics of desensitization of P2Y-R may provide highly relevant to drug therapy of obstructive lung disease and other pathophysiologies. As such the molecular mechanisms underlying these differences will be elucidated. Our studies will include biochemical analyses that take advantage of expression of wild-type and dominant negative constructs of various regulatory proteins, e.g. G protein-coupled receptor kinases, beta-arrestins, and proteins involved in clathrin-related internalization, thought to be involved in agonist- dependent regulation of receptors. These studies will be complemented by studies that apply confocal microscopy to assess agonist-promoted translocation of P2Y-R and regulatory proteins in living cells. Key domains and residues involved in agonist-dependent regulation of the P2Y-R will be revealed by mutational analysis, and potential sites of phosphorylation will be confirmed by in vivo phosphorylation studies. The kinetics and regulation of agonist-dependent phosphorylation of P2Y-R by G protein receptor kinase 2 will be studied in proteoliposomes reconstituted with P2Y-R and Gq. This in vitro system will be utilized to directly define the mechanism(s) whereby P2Y-R phosphorylation regulates P2Y-R system will be utilized to directly define the mechanism(s) whereby P2Y-R phosphorylation regulates P2Y-R G protein coupling. Taken together these studies will provide novel molecular insight into P2Y-R biology and will generate heuristics for therapeutic strategies targeting P2Y-R in airway epithelial cells and other tissues.

G蛋白偶联的P2 Y受体（P2 Y-R）在肺上皮细胞生物学和病理生理学中发挥重要作用。我们研究的长期目标是描绘核苷酸作用于气道P2 Y-R的分子基础。将人P2 Y2- R纯化至同质，并在蛋白脂质体中与G1和其他G蛋白复溶，以：（i）明确定义P2 Y2-R的激动剂结合效力和效力，（ii）深入了解P2 Y2-R的组成型活性的性质，（iii）确定P2 Y2-R与G蛋白相互作用的特异性，和（iv）确定P2 Y2-R的激动剂和G蛋白选择性是否由蛋白激酶促进的磷酸化或RGS蛋白调节。将构建融合P2 Y2-R与Galphaq、Galpha 11或Galpha 12的功能性嵌合蛋白。我们的初步结果表明，人类P2 Y2-R，P2 Y 4-R，和P2 Y 6-R表现出非常不同的表型激动剂诱导的脱敏，内化，和下调。P2 Y-R脱敏的发生和特征的这些差异可能为阻塞性肺疾病和其他病理生理学的药物治疗提供高度相关性。因此，这些差异的分子机制将得到阐明。我们的研究将包括利用各种调节蛋白的野生型和显性负性构建体的表达的生化分析，所述调节蛋白例如G蛋白偶联受体激酶、β-抑制蛋白和参与网格蛋白相关内化的蛋白，其被认为参与受体的激动剂依赖性调节。这些研究将通过应用共聚焦显微镜来评估激动剂促进的活细胞中P2 Y-R和调节蛋白的易位的研究来补充。参与激动剂依赖性调节P2 Y-R的关键结构域和残基将通过突变分析揭示，并且磷酸化的潜在位点将通过体内磷酸化研究确认。将在用P2 Y-R和Gq重构的蛋白脂质体中研究G蛋白受体激酶2对P2 Y-R的激动剂依赖性磷酸化的动力学和调节。该体外系统将用于直接限定P2 Y-R磷酸化调节P2 Y-R系统的机制，该体外系统将用于直接限定P2 Y-R磷酸化调节P2 Y-R G蛋白偶联的机制。总之，这些研究将为P2 Y-R生物学提供新的分子见解，并将为靶向气道上皮细胞和其他组织中的P2 Y-R的治疗策略产生药理学。