Regulation of P2Y2 purinergic receptors

胞外核苷酸G蛋白偶联P2Y受体（P2Y- r）在肺上皮细胞生物学和病理生理中起着重要作用。我们研究的长期目标是描述核苷酸作用于气道P2Y-R的分子基础。人P2Y2- R将被纯化至均匀性，并与G1和其他G蛋白在蛋白脂质体中重组，以：(i)明确定义激动剂与P2Y2-R结合的有效性和效力，（ii）深入了解P2Y2-R组成活性的性质，（iii）确定P2Y2-R与G蛋白相互作用的特异性，以及（iv）确定P2Y2-R的激动剂和G蛋白选择性是否受蛋白激酶促进的磷酸化或RGS蛋白的调节。将P2Y2-R与Galphaq、galpha1或Galpha12融合，构建功能性嵌合蛋白。我们的初步结果表明，人类P2Y2-R、P2Y4-R和P2Y6-R在激动剂诱导的脱敏、内化和下调方面表现出非常不同的表型。这些P2Y-R脱敏的发生和特征的差异可能与阻塞性肺疾病的药物治疗和其他病理生理具有高度相关性。因此，这些差异背后的分子机制将被阐明。我们的研究将包括生化分析，利用各种调节蛋白的野生型和显性阴性结构的表达，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质，这些蛋白质被认为参与受体的激动剂依赖调节。这些研究将通过使用共聚焦显微镜来评估激动剂促进的P2Y-R易位和活细胞中调节蛋白的研究来补充。P2Y-R激动剂依赖性调控的关键结构域和残基将通过突变分析揭示，潜在的磷酸化位点将通过体内磷酸化研究确认。在用P2Y-R和Gq重组的蛋白脂质体中，G蛋白受体激酶2对激动剂依赖性P2Y-R磷酸化的动力学和调控将被研究。该体外系统将用于直接定义P2Y-R磷酸化调控P2Y-R的机制，该系统将用于直接定义P2Y-R磷酸化调控P2Y-R G蛋白偶联的机制。综上所述，这些研究将为P2Y-R生物学提供新的分子视角，并将为针对气道上皮细胞和其他组织中的P2Y-R的治疗策略提供启发。

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.