Mutagenesis of the Human type 1 glucose transporter exit site: A functional study

BIOL 64

Trista K Robichaud, Trista.Robichaud@umassmed.edu, Dept. of Biochemistry & Molecular Pharmacology, University of Mass. Medical School, Worcester, 364 Plantation St 970T LRB, Worcester, MA 01604 and Anthony Carruthers, anthony.carruthers@umassmed.edu, Department of Biochemsitry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St., LRB 926, Worcester, MA 01605.
Human erythrocyte glucose transport protein, GLUT1, isomerizes between two functional states - e1 (presenting a sugar export site) and e2 (import site). GLUT1-mediated sugar uptake is noncompetitively inhibited by mutually antagonistic ligands that trap GLUT1 in the e1 conformation by binding at or near the sugar export site. Low concentrations (<50nM) of some e1 ligands stimulate sugar uptake and binding of other inhibitors, suggesting that transport and binding is cooperative. This is consistent with the oligomeric state of GLUT1 (noncovalent tetramer). Clinical mutations of GLUT1 in GLUT1 deficiency syndrome and computer modeling suggest several amino acid residues (S80, R400 and C421) form crucial elements of the e1 export site. We initiated systematic analysis by targeted alanine mutagenesis expressed in HEK cells and subsequently analysed e1-ligand inhibition of GLUT1. S80A prevents surface expression. R400A and C421A mutant forms express at the cell surface and catalyze sugar transport, but have lost cooperative ligand binding.
 

Frontiers in Chemical Biology
5:00 PM-7:00 PM, Sunday, August 19, 2007 BCEC -- Exhibit Hall - B2, Poster

Division of Biological Chemistry

The 234th ACS National Meeting, Boston, MA, August 19-23, 2007