GLUCOSE-TRANSPORT ACTIVITY AND PHOTOLABELING WITH 3-[I-125]IODO-4-AZIDOPHENETHYLAMIDO-7-O-SUCCINYLDEACETYL (IAPS)-FORSKOLIN OF 2 MUTANTS AT TRYPTOPHAN-388 AND TRYPTOPHAN-412 OF THE GLUCOSE TRANSPORTER GLUT1 - DISSOCIATION OF THE BINDING DOMAINS OF FORSKOLIN AND GLUCOSE

The tryptophan residues 388 and 412 in the glucose transporter GLUT] were altered to leucine (L) by site-directed mutagenesis and were transiently expressed in COS-7 cells. As assessed by immunoblotting, comparable numbers of glucose transporters were present in plasma membranes from cells transfected with wild-type GLUT1, GLUT1-L388 or GLUT1-L412. Transfection of the wild-type GLUT1 gave rise to a 3-fold increase in the reconstituted glucose transport activity recovered from plasma membranes. In contrast, transfection of GLUT1-L412 failed to increase the reconstituted transport activity, whereas transfection of GLUT1-L388 produced only a 70% increase. Photolabelling of GLUT1-L412 with 3-[I-125]iodo-4-azidophenethylamido-7-O-succinyldeacetyl (I-125APS)-forskolin was not different from that of the wild-type GLUT1, whereas the GLUT1-L388 incorporated 70% less photolabel than did the wild-type GLUT1. These data suggest a dissociation of the binding sites of forskolin and glucose in GLUT1. Whereas both tryptophan-388 and tryptophan-412 appear indispensable for the function of the transporter, only tryptophan-388 is involved in the binding of the inhibitory ligand forskolin.