| Using the preparation of purified glucose transporter from human erythrocytes as antigen, six monoclonal antibodies were prepared and characterized. Three of these antibodies have been shown to be to the glucose transporter by several criteria: they immunoprecipitate the transport activity, the cytochalasin B binding activity, and 75% of the protein from the solubilized purified preparation. The remaining three antibodies were shown to recognize the same polypeptide by a Western blot procedure. All of the antibodies reacted with the deglycosylated transporter and are thus against peptide determinants; most bound to the cytoplasmic domain of the transporter. Western blot analysis of erythrocyte membranes prepared in the presence of protease inhibitors showed that all six antibodies bound to a polypeptide of average M(,r) 55,000. Moreover, by immunological assay this polypeptide accounted for 5.3% of the membrane protein, a value similar to that given by cytochalasin B binding. These results show that the proposal in the literature that the native glucose transporter is a polypeptide of M(,r) 100,000 is incorrect.;The monoclonal antibodies have been used to examine the relationship between increased hexose uptake and the phosphorylation state of the glucose transporter in human fibroblasts treated with phorbol myristate acetate (PMA), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or insulin. Exposure to each agonist stimulated transport by 30% to 80%, as measured by the uptake of 2-deoxyglucose and 3-O-methylglucose. Maximal increases in 2-deoxyglucose uptake were stimulated within 30 min in all cases, and the concentration dependencies of the increased uptake into fibroblasts treated with each agonist have been determined. Phosphorylation of the glucose transporter was measured in fibroblasts cultured under identical conditions as those used for the assay of transport. Fibroblasts were incubated with a maximal concentration of PMA, PDGF, EGF, or insulin for 30 min, and the glucose transporter was immunoprecipitated from cell lysates using a monoclonal antibody. Under these conditions, no basal phosphorylation of the transporter was detected, and only phorbol ester stimulated significant incorporation of phosphate into the transport protein. Preliminary experiments to quantitate the stoichiometry of transporter phosphorylation suggested that about 11% of the glucose transporters are phosphorylated in fibroblasts treated with phorbol ester as compared with an average of 1.5% or less in the basal state or after treatment with PDGF, EGF, or insulin. These results show that while the transporter is a substrate for protein kinase C in vivo, phosphorylation of the transporter is probably not required for increased transport in response to growth factors and insulin. |
