The major objective of this project is to conduct in depth studies of the molecular mechanisms underlying cellular adhesion to the extracellular matrix. This will be accomplished by investigating the structure, function and biological properties of vitronectin receptor (VNR), a member of the integrin family of cell adhesion receptors. Integrins are expressed on virtually all cells examined from slime mold to man and mediate a divalent cation-dependent cellular adhesion to a wide variety of adhesive proteins present in the extracellular matrix. Structurally, integrins are heterodimes composed of one of three beta subunits with as many as 10 different alpha subunits. A number of integrins have ligand binding properties which depend on the recognition of the Arg-Gly-Asp (RGD) sequence in one or more adhesive proteins. In this regard, we have recently isolated an integrin termed VNR-1, from human endothelial and melanoma cells which binds vitronectin, fibrinogen and von Willebrand factor in an RGD-dependent manner. One of the goals of this proposal will be to define the minimal structural requirements for VNR's recognition of RGD-containing matrix components and to characterize the divalent cation regulation of this interaction. These studies will focus on receptor-ligand interactions involving VNR in solution, in liposomes and on the cell surface. We have recently isolated a novel member of the integrin family which shares some features with VNR-1. This receptor is expressed on cells of epithelial origin and is characterized by an antigenically similar alpha subunit to that present on VNR-1. Cells that express VNR-2 attach to vitronectin but fail to recognize fibrinogen or von Willebrand factor suggesting that this receptor is functionally distinct from vNR-1. Therefore, we will apply knowledge gained for VNR-1 to investigate the structure and function of VNR-2. This approach will attempt to explain the molecular differences between these integrins that accounts for their functional differences. This will be accomplished by initially comparing the primary sequences of VNR-1 and VNR-2, followed by a molecular approach using site-directed mutagenesis to identify functional sites and to exchange subunits between cells expressing either receptor by cDNA- mediated gene transfer. The approaches outlined in this project should provide basic molecular and cell biological information pertaining to the structure, function and regulation of integrin-dependent cell recognition of the extracellular matrix, thus furthering our understanding of the molecular mechanisms underlying cellular adhesion.
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