The overall goal is to understand the control of the organization of cell surface molecules. Regulation of this organization has been implicated in the escape of tumors from immune defense mechanisms and is believed to involve interactions of cell surface glycoproteins with submembrane microfilaments. As a model system for studying these interactions, we use two ascites sublines of the 13762 rat mammary adenocarcinoma. MAT-C1 subline has immobile cell surface receptors and branched microvilli and is xenotransplantable. MAT-B1 has mobile receptors and unbranched microvilli and is not xenotransplantable. Microvilli and microvillar membranes have been isolated from both sublines for biochemical and ultrastructural analyses. From MAT-C1 membranes we have isolated a transmembrane complex containing a cell surface glycoprotein (CAG), actin and a 58,000 MW polypeptide. The complex isolated from MAT-B1 membranes has only CAG and actin. On the basis of biochemical analyses we have proposed that this complex is a microvillar membrane-microfilament interaction site. We propose to use three approaches to study the molecular interactions and functions of this complex. 1) CAG and 58K will be isolated for biochemical and ultrastructural studies of their structures, analyses of their actin binding and nucleation of actin polymerization and reconstitution of the membrane-microfilament interaction site. 2) Polyclonal antibodies against CAG and 58K will be prepared for analysis of their possible functions in controlling redistribution of other cell surface components and in the formation of microvilli, localization of these components in the tumor cells, identification of these components in other cell types and studies of the biosynthesis of the transmembrane complex. 3) Chemical crosslinking studies will be performed to analyze nearest neighbors in the isolated transmembrane complexes or complexes in microvilli. These studies should provide the first molecular description of a membrane-cytoskeleton interaction site in a cell other than the erythrocyte and the first molecular description of a direct microfilament-membrane interaction. They will also provide a molecular understanding of one mechanism for controlling the organization and mobility of cell surface components of tumor cells.
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