Glucose transport activity and photolabelling with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl (IAPS)-forskolin of two mutants at tryptophan-388 and -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 GLUT1 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-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl (125IAPS)-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.
Schürmann
A
A
Institut für Pharmakologie und Toxikologie, Medizinische Fakultät der RWTH Aachen.
Keller
K
K
Monden
I
I
Brown
F M
FM
Wandel
S
S
Shanahan
M F
MF
Joost
H G
HG
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D001665:Binding Sites; D001692:Biological Transport; D002478:Cells, Cultured; D005576:Colforsin / Q000031:analogs & derivatives / Q000378:metabolism*; D004247:DNA; D004224:Diterpenes; D005947:Glucose / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D004224:Diterpenes; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; C054433:3-iodo-4-azidophenethylamido-7-O-succinyldeacetylforskolin; D005576:Colforsin; D014364:Tryptophan; D004247:DNA; D005947:Glucose