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dc.contributor.authorChen, Pei-Wen
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 68-12, Section: B, page: 7752.;Advisors: Glenn S. Kroog.
dc.description.abstractBombesin (BN)-like peptides stimulate growth and motility in fibroblasts and human tumors by activating a family of G protein-coupled receptors including gastrin-releasing peptide receptor (GRPr). We sought to study the structural requirement for GRPr-mediated growth and to identify novel GRPr signaling partners.;We found that BN induced two waves of ERK activation and one wave of ERK inhibition. An early wave of activation was GRPr carboxyl-terminal domain (CTD)-independent and occurred in cells with low GRPr expression and/or treated with low concentration of BN ([BN]). A later wave of activation required an intact GRPr CTD and occurred after addition of high [BN] in cells with high GRPr expression. The early wave of ERK activation was more sensitive than the later one to a protein kinase C inhibitor or hypertonic sucrose. A third pathway activated protein tyrosine phosphatase(s) to inhibit ERK phosphorylation after addition of high [BN] in cells with high GRPr expression. Therefore, we demonstrated that GRPr regulates ERK through multiple pathways in a single cell type depending upon receptor expression and agonist concentration.;We also studied leupaxin (LPXN), a protein structurally related to the focal adhesion (FA) adapter protein paxillin. GRPr activation by BN stimulated LPXN translocation from cytoplasm to FAs and LPXN tyrosine phosphorylation. Using mutagenesis, we identified LIM3 as the primary FA targeting domain for LPXN and showed BN-induced LPXN tyrosine phosphorylation on residues 22, 62 and 72. A LIM3 point mutant of LPXN failed to target to FAs and had no BN-stimulated tyrosine phosphorylation. Conversely, a non-phosphorylatable mutant (Y22/62/72F) still targets to FAs. Therefore, LPXN tyrosine phosphorylation requires translocation to FAs. LPXN and paxillin had opposite roles in adhesion to collagen I (CNI) in MDA-MB-231 breast cancer cells. LPXN knockdown stimulated whereas paxillin knockdown inhibited cell adhesion. Knockdown of both LPXN and paxillin behaved similarly to paxillin knockdown alone, suggesting LPXN's function in adhesion might depend on paxillin. Additionally, LPXN regulated spreading on CNI but not on fibronectin (FN) whereas paxillin knockdown suppressed spreading on both substrates. These results demonstrated that LPXN FA targeting and tyrosine phosphorylation are similar to paxillin, but it has distinct functions.
dc.publisherProQuest Dissertations & Theses
dc.subjectCellular biology.
dc.subjectMolecular biology.
dc.titleCharacterization of leupaxin, a novel gastrin-releasing peptide receptor regulated focal adhesion protein

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