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dc.contributor.authorXiong, Ying
dc.date.accessioned2018-07-12T17:35:05Z
dc.date.available2018-07-12T17:35:05Z
dc.date.issued2008
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 69-07, Section: B, page: 3997.;Advisors: E. Richard Stanley.
dc.identifier.urihttps://yulib002.mc.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:3323730
dc.identifier.urihttps://hdl.handle.net/20.500.12202/982
dc.description.abstractColony-stimulating factor-1 (CSF-1) is the primary regulator of the survival, proliferation and differentiation of mononuclear phagocytic cells. These effects are mediated via the CSF-1 receptor (CSF-1R), a class III receptor tyrosine kinase. In order to study CSF-1R-mediated signal transduction in the appropriate cellular context, a GM-CSF-dependent, CSF-1R-deficient bone marrow-derived macrophage line, MacCsf1r-/-, was developed.;CSF-1-induced CSF-1R tyrosine phosphorylation, ubiquitination and interchain disulfide bonding were studied in macrophages. Phosphorylation of CSF-1R Tyr559, required for the binding of Src family kinases (SFKs), was both necessary and sufficient for these responses and for c-Cbl tyrosine phosphorylation and all these responses were inhibited by SFK inhibitors. In c-Cbl-deficient macrophages, CSF-1R ubiquitination, interchain disulfide bonding and tyrosine phosphorylation were substantially inhibited. Reconstitution with wild-type c-Cbl, but not ubiquitin ligase-defective C381A c-Cbl rescued these responses, while expression of C381A c-Cbl in wild-type macrophages suppressed them. Cysteine mutagenesis indicated that the ligand-induced interchain disulfide linkages involve four cysteines in the major kinase domain. A CSF-1R bearing mutations in all four cysteines failed to form disulfide-bonded dimers, exhibited compromised receptor tyrosine phosphorylation, but retained wild-type receptor ubiquitination and degradation. These results indicate that a CSF-1-regulated Tyr559/SFK/Cbl pathway leads to CSF-1R interchain disulfide bonding that permits full receptor tyrosine phosphorylation in macrophages.;CSF-1R interchain disulfide bonding had been proposed to occur in the extracellular domain (ECD) of the receptor, but mutation of the only free cysteines in the ECD, Cys432 and Cys439, failed to affect it. Interestingly, macrophages expressing the C432,439S CSF-1R were able to proliferate in the absence of CSF-1 for two weeks and CSF-1-stimulated CSF-1R tyrosine phosphorylation, ubiquitination and internalization were compromised in these cells. Following removal of exogenous CSF-1, these macrophages were resistant to rounding-up and retained significantly more CSF-1 on the cell surface than cells expressing the wild-type CSF-1R. These results indicate that the C432,439S mutations increase the affinity of the CSF-1R for CSF-1. It is predicted that the soluble C432,439S CSF-1R ECD will be a therapeutically significant high-affinity inhibitor of CSF-1 binding to the CSF-1R.
dc.publisherProQuest Dissertations & Theses
dc.subjectMolecular biology.
dc.subjectCellular biology.
dc.titleStructure-function analysis of the colony -stimulating factor-1 receptor in macrophages
dc.typeDissertation


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