scholarly journals Ligand-induced conformational changes modify proteolytic cleavage of the adipocyte insulin-sensitive glucose transporter

1993 ◽  
Vol 295 (1) ◽  
pp. 183-188 ◽  
Author(s):  
Y Yano ◽  
J M May
Keyword(s):  
Conformational Changes ◽  
Human Erythrocyte ◽  
Glucose Transporter ◽  
Proteolytic Cleavage ◽  
Specific Antiserum ◽  
Tryptic Cleavage ◽  
Glut1 Protein ◽  
Membrane Bound ◽  
Glucose Transporter Glut4 ◽  
Glucose Transporter Glut1

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.

scholarly journals Proteolytic dissection as a probe of conformational changes in the human erythrocyte glucose transport protein

1988 ◽  
Vol 256 (2) ◽  
pp. 421-427 ◽  
Author(s):  
A F Gibbs ◽  
D Chapman ◽  
S A Baldwin
Keyword(s):  
Conformational Change ◽  
Glucose Transport ◽  
Conformational Changes ◽  
Steric Hindrance ◽  
Human Erythrocyte ◽  
Glucose Transporter ◽  
Cytoplasmic Surface ◽  
Tryptic Cleavage ◽  
Hydrophilic Region ◽  
Glucose Transport Protein

Tryptic digestion has been used to investigate the conformational changes associated with substrate translocation by the human erythrocyte glucose transporter. The effects of substrates and inhibitors of transport on the rates of tryptic cleavage at the cytoplasmic surface of the membrane have confirmed previous observations that this protein can adopt at least two conformations. In the presence of phloretin or 4,6-O-ethylidene-D-glucose, the rate of cleavage is slowed. Because these inhibitors bind preferentially at the extracellular surface of the transporter, their effects must result from a conformational change rather than from steric hindrance. A conformational change must also be responsible for the effect of the physiological substrate D-glucose, which is to increase the rate of cleavage. The regions of the protein involved in the conformational changes include both of the large cytoplasmic regions that are cleaved by trypsin: these are the central hydrophilic region of the sequence (residues 213-269) and the hydrophilic C-terminal region (residues 457-492).

scholarly journals Proteolytic and chemical dissection of the human erythrocyte glucose transporter

1984 ◽  
Vol 221 (1) ◽  
pp. 179-188 ◽  
Author(s):  
M T Cairns ◽  
D A Elliot ◽  
P R Scudder ◽  
S A Baldwin
Keyword(s):  
Human Erythrocyte ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Diffuse Band ◽  
Cytoplasmic Surface ◽  
Cytoplasmic Face ◽  
Membrane Bound ◽  
Beta Galactosidase

Treatment of the purified, reconstituted, human erythrocyte glucose transporter with trypsin lowered its affinity for cytochalasin B more than 2-fold, and produced two large, membrane-bound fragments. The smaller fragment (apparent Mr 18000) ran as a sharp band on sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis. When the transporter was photoaffinity labelled with [4-3H]cytochalasin B before tryptic digestion, this fragment became radiolabelled and so probably comprises a part of the cytochalasin B binding site, which is known to lie on the cytoplasmic face of the erythrocyte membrane. In contrast, the larger fragment was not radiolabelled, and ran as a diffuse band on electrophoresis (apparent Mr 23000-42000). It could be converted to a sharper band (apparent Mr 23000) by treatment with endo-beta-galactosidase from Bacteroides fragilis and so probably contains one or more sites at which an oligosaccharide of the poly(N-acetyl-lactosamine) type is attached. Since the transporter bears oligosaccharides only on its extracellular domain, whereas trypsin is known to cleave the protein only at the cytoplasmic surface, this fragment must span the membrane. Cleavage of the intact, endo-beta-galactosidase-treated, photoaffinity-labelled protein at its cysteine residues with 2-nitro-5-thiocyanobenzoic acid yielded a prominent, unlabelled fragment of apparent Mr 38000 and several smaller fragments which stained less intensely on SDS/polyacrylamide gels. Radioactivity was found predominantly in a fragment of apparent Mr 15500. Therefore it appears that the site(s) labelled by [4-3H]cytochalasin B lies within the N-terminal or C-terminal third of the intact polypeptide chain.

scholarly journals Characterization of functional human erythrocyte-type glucose transporter (GLUT1) expressed in insect cells using a recombinant baculovirus

1992 ◽  
Vol 283 (3) ◽  
pp. 643-646 ◽  
Author(s):  
C K Yi ◽  
B M Charalambous ◽  
V C Emery ◽  
S A Baldwin
Keyword(s):  
Human Erythrocyte ◽  
Binding Sites ◽  
Glucose Transporter ◽  
Cytochalasin B ◽  
Insect Cells ◽  
Recombinant Baculovirus ◽  
Transport Inhibitor ◽  
Glucose Transporter Glut1 ◽  
Transporter Glut1

The human erythrocyte-type glucose transporter (GLUT1) has been abundantly expressed in insect cells by using a recombinant baculovirus. At 4 days after infection with the virus, the insect cell-surface and intracellular membranes were found to contain greater than 200 pmol of D-glucose-sensitive binding sites for the transport inhibitor cytochalasin B per mg of protein. The characteristics of binding were identical with those of the erythrocyte transporter, although the two proteins differed substantially in apparent Mr, probably as a result of glycosylation differences.

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