Date of Award

5-2006

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Cynthia B. Peterson

Committee Members

Ronald Wetzel, Mary Ann Handel, John W. Koontz, Elizabeth E. Howell

Abstract

Vitronectin is a human circulatory protein that is recruited to the extracellular matrix (ECM) during tissue remodeling pathways linked to injury, inflammation, and tumor metastasis. Whereas circulating vitronectin is monomeric, the tissue-associated form is multimeric and contains multivalent binding sites for both cell-surface receptors and components of the extracellular matrix. In addition, matrix-associated vitronectin directly regulates plasmin-regulated matrix proteolysis through the localization and stabilization of the serine protease inhibitor, plasminogen activator inhibitor type-1 (PAI-1). As a component of the ECM, vitronectin adopts a pro-adhesive role, providing a unique regulatory link between cell adhesion and pericellular proteolysis. The mechanism by which vitronectin interconverts between a monomeric, circulating protein and a multimeric ECM component remains to be established. Vitronectin shows a high degree of conformational flexibility, and in vitro studies demonstrate that a number of chaotropic agents and macromolecular ligands like PAI-1 are able to induce the multimerization of vitronectin into higher-order forms. The induced multimeric form of vitronectin shows similar ligand-binding properties to that of the matrix-associated form, with a potential for multivalent interactions with biological partners including cell- surface receptors and ECM components. The following dissertation serves to summarize a graduate research project designed to address the working hypothesis that PAI-1 represents a physiological cofactor for the conversion of vitronectin from a circulating, monomeric form to a matrix-associated, "activated" form. PAI-1-binding to vitronectin induces the formation of higher-order complexes that display altered adhesive properties distinct from the circulating (monomeric) form of vitronectin. These altered adhesive properties arise from the formation of multivalent binding sites for both cell surfaces and components of the ECM. Formation of vitronectin/PAI-1 complexes follows a step-wise assembly process that is dependent on protein concentration and time, ultimately leading to an increased accumulation of vitronectin at the cell-matrix interface. To address this hypothesis, biophysical studies were designed to evaluate the mechanism of assembly of PAI-1/vitronectin complexes and the pathway leading to multimerization of vitronectin. These studies were performed in parallel with cell- and matrix-binding experiments to establish a correlation between PAI-1-induced multimerization with enhanced adhesive properties and and conversion of vitronectin into a matrix-associated form.

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