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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033795-03
Application #
3283838
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1986-07-01
Project End
1991-06-30
Budget Start
1988-07-01
Budget End
1989-06-30
Support Year
3
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Miami School of Medicine
Department
Type
Schools of Medicine
DUNS #
City
Miami
State
FL
Country
United States
Zip Code
33101
Carraway, C A; Carvajal, M E; Carraway, K L (1999) Association of the Ras to mitogen-activated protein kinase signal transduction pathway with microfilaments. Evidence for a p185(neu)-containing cell surface signal transduction particle linking the mitogenic pathway to a membrane-microfilament association J Biol Chem 274:25659-67
Li, Y; Hua, F; Carraway, K L et al. (1999) The p185(neu)-containing glycoprotein complex of a microfilament-associated signal transduction particle. Purification, reconstitution, and molecular associations with p58(gag) and actin. J Biol Chem 274:25651-8
Juang, S H; Carvajal, M E; Whitney, M et al. (1996) Tyrosine phosphorylation at the membrane-microfilament interface: a p185neu-associated signal transduction particle containing Src, Abl and phosphorylated p58, a membrane- and microfilament-associated retroviral gag-like protein. Oncogene 12:1033-42
Carraway, K L; Carraway, C A (1995) Signaling, mitogenesis and the cytoskeleton: where the action is. Bioessays 17:171-5
Juang, S H; Huang, J; Li, Y et al. (1994) Molecular cloning and sequencing of a 58-kDa membrane- and microfilament-associated protein from ascites tumor cell microvilli with sequence similarities to retroviral Gag proteins. J Biol Chem 269:15067-75
Carraway, C A; Carvajal, M E; Li, Y et al. (1993) Association of p185neu with microfilaments via a large glycoprotein complex in mammary carcinoma microvilli. Evidence for a microfilament-associated signal transduction particle. J Biol Chem 268:5582-7
Carothers Carraway, C A; Fang, H; Ye, X H et al. (1991) Membrane-microfilament interactions in ascites tumor cell microvilli. Identification and isolation of a large microfilament-associated membrane glycoprotein complex. J Biol Chem 266:16238-46
Ye, X H; Metcalf 3rd, T N; Andrews, D M et al. (1989) Strong association of bovine IgM with microvilli and their microfilament core from 13762 ascites tumor cells. Exp Cell Res 182:160-72
Sheng, Z; Vanderpuye, O A; Hull, S R et al. (1989) Topography and microfilament core association of a cell surface glycoprotein of ascites tumor cell microvilli. J Cell Biochem 40:453-66
Carraway, K L; Carraway, C A (1989) Membrane-cytoskeleton interactions in animal cells. Biochim Biophys Acta 988:147-71

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