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G protein-coupled receptor signaling: Role of PAR2 in breast carcinoma metastasis and regulation of agonist-promoted internalization of P2Y1 receptors.

机译：G蛋白偶联受体信号传导：PAR2在乳腺癌转移中的作用和激动剂促进的P2Y1受体内在化的调节。

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摘要

This dissertation is comprised of two projects investigating the function of GPCR signaling and regulation in cancer and/or cardiovascular physiology. The first study delineated the functional importance of PAR2 in breast cancer migration and invasion by using small interfering RNAs (siRNAs) to deplete highly invasive breast cancer cells of endogenous PAR2 protein. Our findings strongly suggest that PAR2 is critical for MDA-MB-231 and BT549 breast cancer cell migration and invasion towards NIH-3T3 fibroblast conditioned medium. We also examined the importance of PAR2 in mediating factors VIIa and Xa responses. We showed that MDA-MB-231 cells depleted of PAR2 exhibit a marked inhibition of VIIa and Xa signaling to phosphoinositide hydrolysis and ERK1/2 activation, whereas signaling by VIIa and Xa remained intact in PAR1 deficient cells. Factors VIIa and Xa-induced cellular migration was also impaired in MDA-MB-231 cells deficient in PAR2 but not in cells lacking PAR1. The results from these studies reveal the novel findings that PAR2 has a critical role in breast cancer cell migration and invasion and functions as the endogenous receptor for coagulant proteases VIIa and Xa in these cells.;For the second study, we investigated the regulation of agonist-promoted P2Y1 receptor internalization in Madin-Darby Canine Kidney cells. Our studies revealed that Ser336 within a highly conserved S336RAT 339 sequence regulates agonist-promoted P2Y1 internalization. We showed that mutation of Ser336 to Ala resulted in internalization rates faster than wild type receptors in response to agonist. Agonist-promoted [ 32P]phosphate incorporation studies indicated that increased phosphorylation was not the cause of enhanced internalization. Arrestin-GFP mobilization and internalization studies in arrestin2- and arrestin3-null mouse embryonic fibroblasts revealed that both wild type and mutant receptors required arrestins to undergo agonist-promoted internalization. We propose a model in which Ser336 regulates arrestin binding to an active conformation of the receptor. This model predicts that mutation of Ser336 to Ala increases the rate of internalization as a result of increased arrestin binding. These studies increase our understanding of the internalization and regulation of the P2Y1 receptor, a GPCR critical in regulation of various processes including ion and water transport across epithelia, smooth muscle relaxation, and platelet activation.
机译：本论文由两个研究GPCR信号传导和调控在癌症和/或心血管生理中的功能的项目组成。第一项研究描述了PAR2在乳腺癌迁移和侵袭中的功能重要性，方法是使用小干扰RNA（siRNA）消耗内源性PAR2蛋白的高侵袭性乳腺癌细胞。我们的发现强烈表明PAR2对于MDA-MB-231和BT549乳腺癌细胞向NIH-3T3成纤维细胞条件培养基的迁移和侵袭至关重要。我们还研究了PAR2在介导VIIa和Xa反应中的重要性。我们显示，耗尽PAR2的MDA-MB-231细胞对VIIa和Xa信号向磷酸肌醇水解和ERK1 / 2活化表现出明显的抑制作用，而VIIa和Xa的信号在PAR1缺陷细胞中保持完整。在缺乏PAR2的MDA-MB-231细胞中，因子VIIa和Xa诱导的细胞迁移也受到损害，而在缺乏PAR1的细胞中则没有。这些研究的结果揭示了新发现：PAR2在乳腺癌细胞迁移和侵袭中起关键作用，并在这些细胞中充当凝血蛋白酶VIIa和Xa的内源性受体。第二项研究是研究激动剂的调节作用。促进了Madin-Darby犬肾脏细胞中的P2Y1受体内在化。我们的研究表明，高度保守的S336RAT 339序列中的Ser336调节激动剂促进的P2Y1内部化。我们表明，Ser336突变为Ala导致内在化的速度快于野生型受体对激动剂的反应。激动剂促进的[32P]磷酸盐掺入研究表明，磷酸化的增加并不是内在化增强的原因。在restarin2和restingin3无效的小鼠胚胎成纤维细胞中进行arrestin-GFP动员和内在化研究，发现野生型和突变型受体均需要restarin进行激动剂促进的内在化。我们提出了一种模型，其中Ser336调节抑制蛋白与受体的活性构象结合。该模型预测，由于抑制素结合增加，Ser336突变为Ala会增加内在化的速率。这些研究增加了我们对P2Y1受体的内在化和调节的理解，P2Y1受体是调节各种过程（包括离子和水通过上皮运输，平滑肌松弛和血小板活化）的关键的GPCR。

著录项

  • 作者

    Glast, Dionne Renee.;

  • 作者单位

    The University of North Carolina at Chapel Hill.;

  • 授予单位 The University of North Carolina at Chapel Hill.;
  • 学科 Biology Molecular.;Biology Cell.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 172 p.
  • 总页数 172
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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