Isolation of actin-containing transmembrane complexes from ascites adenocarcinoma sublines having mobile and immobile receptors.
The molecular nature of the cell surface-cytoskeleton interaction in microvilli isolated from ascites 13762 rat mammary adenocarcinoma sublines with immobile (MAT-C1) and mobile (MAT-B1) receptors was investigated by extraction and fractionation studies on the microvillar membranes. Extraction of membranes from MAT-C1 cells with Triton X-100-containing buffers gave insoluble residues showing three major components by NaDodSO(4)/polyacrylamide gel electrophoresis: actin, a 58,000-dalton polypeptide, and a cell surface glycoprotein of 75,000-80,000 daltons. The ratio of these components in Triton X-100-insoluble residues, as determined by scintillation counting of bands from gels of [(3)H]leucine-labeled microvillar membranes, approached equimolar, suggesting a specific complex of the components. The three components of the putative complex cosedimented on sucrose density gradients of Triton X-100/buffer-treated membranes. Gel filtration on Sepharose 2B gave a peak included in the column that contained only the glycoprotein, actin, and 58,000-dalton polypeptide by one-dimensional NaDodSO(4) electrophoresis and by two-dimensional isoelectric focusing/NaDodSO(4) electrophoresis. The glycoprotein-actin association could be disrupted only under strongly denaturing conditions. Complex prepared from MAT-B1 microvillar membranes by Sepharose 2B gel filtration in Triton X-100-containing buffers contained actin and the glycoprotein but no 58,000-dalton polypeptide. From these results we propose that the cell surface-cytoskeleton interactions in the 13762 tumor cell microvilli involve direct association of actin with the cell surface glycoprotein. We further suggest that the 58,000-dalton polypeptide stabilizes the association of this complex with the microfilaments in the MAT-C1 microvilli, thereby stabilizing the microvilli and restricting cell surface receptor mobility.
D000199:Actins / Q000302:isolation & purification / Q000378:metabolism*; D000230:Adenocarcinoma / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats
D000199:Actins; D006023:Glycoproteins