title: Ligand-induced conformational changes modify proteolytic cleavage of the adipocyte insulin-sensitive glucose transporter.
abstract: The transport conformation of the human erythrocyte glucose transporter (GLUT1) modifies rates of proteolytic cleavage of this protein by a variety of enzymes. We investigated the effects of ligand-induced conformational change on the susceptibility to enzymic cleavage of the insulin-sensitive rat adipocyte glucose transporter (GLUT4). A GLUT4-enriched slow sedimenting microsomal fraction was prepared from basal adipocytes and subjected to PAGE and immunoblotting. The GLUT4 protein was detected in these immunoblots with a C-terminal-specific antiserum as an M(r)-46,000-50,000 doublet. GLUT1 protein was not detected by a GLUT1-specific antiserum in these membranes. Tryptic digestion caused loss of the GLUT4 signal in immunoblots in a time- and concentration-dependent fashion. Low-M(r) membrane-bound fragments were not observed in electrophoretic gels, whether detection was attempted by immunoblotting or by counting radioactivity in gel slices following photolabelling with [3H]cytochalasin B. Transport-specific ligands known to induce an outward-facing conformation in the human erythrocyte GLUT1 protein retarded cleavage of the GLUT4 protein by submaximal concentrations of trypsin, 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 known behaviour of GLUT1, in which D-glucose accelerates cleavage. Cleavage of GLUT4 by thermolysin was also retarded by the outward-binding analogue 4,6-O-ethylidene glucose. These results show that the conformational sensitivity to proteolysis of GLUT4 mirrors that of GLUT1, except that the glucose-loaded GLUT4 has a different steady-state configuration, which may reflect underlying kinetic differences between the two proteins.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-2230.
authors.forename: Y
authors.initials: Y
authors.lastname: Yano
authors.forename: J M
authors.initials: JM
authors.lastname: May
mesh_terms: D017667:Adipocytes / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D003571:Cytochalasin B / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D004822:Epididymis; D005947:Glucose / Q000031:analogs & derivatives / Q000494:pharmacology; D051275:Glucose Transporter Type 4; D007668:Kidney; D008024:Ligands; D008297:Male; D008566:Membranes / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013820:Thermolysin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D051275:Glucose Transporter Type 4; D008024:Ligands; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495694:Slc2a4 protein, rat; C013984:ethylidene glucose; D003571:Cytochalasin B; D010450:Endopeptidases; D014357:Trypsin; D013820:Thermolysin; D005947:Glucose