Monoclonal antibodies recognizing a mouse cell surface glycoprotein Of Mr 90,000 were found to coprecipitate the Mr 70,000 and 72,000 heat shock-induced proteins of NIH/3T3 cells. These two smaller proteins were among the most abundant components of heat-treated NIH/3T3 cells. The Mr 70,000 component was not detected in normal cells whereas there was a low rate of incorporation of [3S]methionine into the Mr 72,000 polypeptide in the absence ofheat shock. Tryptic peptide mapping and two-dimensional gel electrophoresis indicated that the coprecipitated and heat shock-induced polypeptides were identical and that the Mr 70,000 and 72,000 components contained homologous peptides. Also, the heat shock proteins had extensive structural homology with a cytoskeleton-associated protein of HeLa cells. The results suggest that the Mr 90,000 cell surface glycoprotein and the Mr 70,000 and 72,000 heat shock-inducible proteins mediate an association between the plasma membrane and the cell cytoskeleton.The remarkable changes in the protein composition of cells induced by sudden increases in temperature include the increased synthesis of a limited set of gene products and a concomitant decrease in the rate of synthesis of most cellular proteins (1, 2). This phenomenon has been observed with cells from various species (3-10). A similar response was also observed with other experimental regimens that disturb normal cell metabolism (11)(12)(13). Recent studies of the chemical properties of avian and mammalian heat shock proteins (8,14) showed that the major heat-induced protein, Mr 68,000, was a methylated cell component identical to a highly conserved polypeptide previously shown to copurify with intermediate filaments (15) and microtubules (16,17). The association of this Mr 68,000 heat shock protein with myofibrils, microtubules, and intermediate filaments suggested a possible cytoarchitectural role for the polypeptide (14).We have found that six independently derived monoclonal antibodies recognizing a mouse cell surface glycoprotein of 90,000 daltons (gp90) coprecipitated the major heat shock-induced proteins OfMr 70,000 and 72,000 (HSP 70/72) from NIH/ 3T3 cells. The peptide map of HSP 70/72 closely resembled that of a reported 68,000-dalton cytoskeleton-associated protein of HeLa cells (16,17). The data provide evidence for an association between the 90,000-dalton major plasma membrane glycoprotein and the cell cytoskeleton.
METHODSAll cells were grown without antibiotics in Dulbecco's modified Eagle's medium with glucose (GIBCO) at 4.5 g/liter and 10% (vol/vol) heat-inactivated fetal calf serum (Sterile Systems, Logan, UT) in 5% C02/95% 02 at 37°C. The anti-mouse fibroblast (AMF) monoclonal antibodies (18) used for these studies were anti-p220 (AMF-1), anti-gpllO (AMF-9), anti-gp90 (AMF4, AMF-5, AMF-6, AMF-7, AMF-17, and AMF-18), anti-gp8C (AMF-12), and anti-p72 (AMF-13). RESULTS (Fig. 1). The control immunoprecipitate with preimmune rat serum contained a Mr 65,000 polypeptide, a nonspecific precipitate of [3S...