Isolation of actin-containing transmembrane complexes from ascites adenocarcinoma sublines having mobile and immobile receptors

Abstract

The molecular nature of the cell surface-cytoskeleton interaction in microvilli isolated from ascites 13762 rat mammary adenocarcinoma sublines with immobile (MAT-Cl) and mobile (MAT-Bi) receptors was investigated by extraction and fractionation studies on the microvillar membranes. Extraction of membranes from MAT-Cl cells with Triton X-l00-containing buffers gave insoluble residues showing three major components by NaDodSO4/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 [31H]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/buffertreated 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 NaDodSO4 electrophoresis and by two-dimensional isoelectric focusing/ NaDodSO4 electrophoresis. The glycoprotein-actin association could be disrupted only under strongly denaturing conditions. Complex prepared from MAT-BI 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-Cl microvilli, thereby stabilizing the microvilli and restricting cell surface receptor mobility.Although the organization of cell surface proteins is widely believed to be controlled by interactions between these surface proteins and a submembrane cytoskeleton (1), via a transmembrane complex (2), there is little information about the molecular nature of these interactions in cells other than erythrocytes. One problem has been the difficulty in obtaining defined cell surface fractions that compare to the erythrocyte membrane (3), the best available model system. Cell surface specializations, such as microvilli, offer obvious advantages in such studies, because they are identified readily by their morphology. Moreover, some cell surface components are concentrated on microvilli (4), suggesting stabilized membrane-cytoskeleton interactions. We have been investigating the nature of membrane-cytoskeleton interactions by using ascites sublines ofthe 13762 rat mammary adenocarcinoma. The MAT-Cl subline is strikingly different in morphology and cell surface behavior. (12) showed that the glycopro-tein was firmly associated with the microvillar cytoskeleton (10). In light of these results the glycoprotein was termed a cytoskeleton-associated glycoprotein ...

c-Src association with and phosphorylation of p58gag, a membrane- and microfilament-associated retroviral Gag-like protein in a xenotransplantable rat mammary tumor

“…(4) The p58 gag in the microvilli of MAT-C1 cells interacts with actin and glycoproteins in a microfilament-associated TMC (Carraway et al, 1983). (5) Puri®ed p58 gag binds actin, phospholipid (Liu et al, 1989) and the TMC-glycoprotein complex (11,30), consistent with the hypothesis that it plays a role in stabilizing membrane-micro®lament interactions in the MAT-C1 cells (Carraway et al, 1983).…”

“…The (proto)oncogene tyrosine kinase receptor p185 neu is a component of the TMC glycoprotein complex, suggesting that this docking site with micro®laments is a large signal transduction particle (Carraway et al, 1983). The presence of all of the components of the Ras to MAP kinase pathway (Carraway, et al, 1999, in press), as well as the intracellular kinases c-Src and cAbl (Juang et al, 1996), supports this hypothesis.…”

“…The p58 gag protein is present in microvilli of the MAT-C1 ascites subline but not in the closely related MAT-B1 subline (Carraway et al, , 1983, derived from the same solid tumor. Several salient observations have been made on the expression of p58 gag and the unusual properties of the MAT-C1 subline.…”

“…One model system which is particularly amenable to these studies is microvilli isolated from ascites sublines of the 13 762 rat mammary adenocarcinoma . The microvilli can be isolated using minimally perturbing conditions and puri®ed in quantities su cient for biochemical characterization (Carraway et al, , 1983. A 58 kDa protein which associated with both membranes and micro®laments Liu et al, 1989) was implicated in the stabilization of the microvilli and their receptors in the sublines which expressed it.…”

“…A 58 kDa protein which associated with both membranes and micro®laments Liu et al, 1989) was implicated in the stabilization of the microvilli and their receptors in the sublines which expressed it. The anchorage mechanism was proposed to be the association of p58 with a large, micro®lament-associated glycoprotein complex, the transmembrane complex (TMC) (Carraway et al, 1983(Carraway et al, , 1991. Biochemical and immunological studies showed that the p58-containing TMC comprises a large, stably associated cell surface signal transduction particle (Carraway et al 1999, in press) containing the receptor tyrosine kinase p185 neu /ErbB2 (Carraway et al, 1993) and the cytoplasmic tyrosine kinases c-Src and c-Abl (Juang et al, 1996).…”