Role of Conserved Arginine and Glutamate Residues on the Cytosolic Surface of Glucose Transporters for Transporter Function

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Abstract

The role of conserved arginine and glutamic acid residues at the cytoplasmic surface of the GLUT4 for transporter function was investigated by site-directed mutagenesis and expression of the constructs in COS-7 cells. Reconstituted glucose transport activity, cytochalasin B binding, and photolabeling with the exofacial label 2-N4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1, 3-bis(d-mannosyloxy)-2-propylamine (ATB-BMPA) was assayed in membranes from transfected cells and corrected for immunoreactivity of expressed transporters. Exchange of Arg 92 (R92L amino acid residues are numbered according to the corresponding residues in the GLUT1) or Arg 333/334 (RR333/4LA) reduced or suppressed transport activity with no or very little effect on photolabeling with ATB-BMPA and cytochalasin B binding. It is suggested that the lack of these residues selectively disturbes the substrate-induced conformational change of the carrier during transport. Exchange of Glu 146 (E146D) or Arg 153 (R153L) markedly reduced transport activity, ATB-BMPA photolabeling, and cytochalasin B binding. Transport activity and ATB-BMPA labeling were abolished in the mutants E329Q, E393D, and R400L, whereas binding of cytochalasin B was normal. Thus, exchange of Glu 329, Glu 393, and Arg 400 appears to arrest the transporter in an inward facing conformation. It is concluded that the conserved arginine and glutamate residues at the cytoplasmic surface of the glucose transporter GLUT4 are essential for its appropriate conformation, and that it is the interaction of charged residues which mediates the oscillation between outward and inward facing states.

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