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

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Last Modified
  • March 20, 2019
Creator
  • Glast, Dionne Renee
    • Affiliation: School of Medicine, Department of Pharmacology
Abstract
  • 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 S336RAT339 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.
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  • "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology."
Advisor
  • Nicholas, Robert
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  • Chapel Hill, NC
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  • Open access
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