LIGAND-INDUCED CONFORMATIONAL-CHANGES MODIFY PROTEOLYTIC CLEAVAGE OF THE ADIPOCYTE INSULIN-SENSITIVE GLUCOSE-TRANSPORTER

The transport conformation of the human erythrocyte glucose transporte r (GLUT1) modifies rates of proteolytic cleavage of this protein by a variety of enzymes. We investigated the effects of ligand-induced conf ormational change on the susceptibility to enzymic cleavage of the ins ulin-sensitive rat adipocyte glucose transporter (GLUT4). A GLUT4-enri ched slow sedimenting microsomal fraction was prepared from basal adip ocytes and subjected to PAGE and immunoblotting. The GLUT4 protein was detected in these immunoblots with a C-terminal-specific antiserum as an M(r)-46000-50000 doublet. GLUT1 protein was not detected by a GLUT 1-specific antiserum in these membranes. Tryptic digestion caused loss of the GLUT4 signal in immunoblots in a time- and concentration-depen dent fashion. Low-M(r) membrane-bound fragments were not observed in e lectrophoretic gels, whether detection was attempted by immunoblotting or by counting radioactivity in gel slices following photolabelling w ith [H-3]cytochalasin B. Transport-specific ligands known to induce an outward-facing conformation in the human erythrocyte GLUT1 protein re tarded cleavage of the GLUT4 protein by submaximal concentrations of t rypsin, whereas ligands known to induce an inward-facing conformation increased the extent of cleavage. The transported substrate D-glucose retarded tryptic cleavage of GLUT4. This result contrasts with the kno wn behaviour of GLUT1, in which D-glucose accelerates cleavage. Cleava ge of GLUT4 by thermolysin was also retarded by the outward-binding an alogue 4,6-0-ethylidene glucose. These results show that the conformat ional sensitivity to proteolysis of GLUT4 mirrors that of GLUT1, excep t that the glucose-loaded GLUT4 has a different steady-state configura tion, which may reflect underlying kinetic differences between the two proteins.