SUBSTITUTION OF CONSERVED TYROSINE RESIDUES IN HELIX 4(Y143) AND 7(Y293) AFFECTS THE ACTIVITY, BUT NOT IAPS-FORSKOLIN BINDING, OF THE GLUCOSE-TRANSPORTER GLUT4

Six tyrosine residues (Y28, Y143, Y292, Y293, Y308, Y432(1)) which are conserved in all mammalian glucose transporters were substituted for phenylalanine by site-directed mutagenesis, and mutant glucose transporters were transiently expressed in COS-7 cells. Glucose transport activity as assessed by reconstitution of the solubilized transporters into lecithin liposomes was reduced by 70% in the mutant Y143F and appeared to be abolished in Y293F, but was not affected by substitution of Y28, Y292, Y308 and Y432. In contrast, covalent binding of the photolabel (125)IAPS-forskolin was normal in all mutants. Stable expression of the mutants Y143F, Y293F, and Y292F in LTK cells yielded identical results. These data indicate that only two of the 6 conserved helical tyrosine residues, located in helices 4 and 7, are essential for full activity, but not for IAPS-forskolin binding of the GLUT4.