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 125IAPS-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.
Institut für Pharmakologie und Toxikologie, RWTH Aachen, Germany.
S
S
Wandel
A
A
Schürmann
W
W
Becker
S A
SA
Summers
M F
MF
Shanahan
H G
HG
Joost
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism*; D002460:Cell Line; D005576:Colforsin / Q000031:analogs & derivatives* / Q000378:metabolism; D017124:Conserved Sequence; D004224:Diterpenes; D051275:Glucose Transporter Type 4; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D009124:Muscle Proteins; D014443:Tyrosine / Q000737:chemistry*
D000345:Affinity Labels; D001386:Azides; D004224:Diterpenes; D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C054433:3-iodo-4-azidophenethylamido-7-O-succinyldeacetylforskolin; D005576:Colforsin; D014443:Tyrosine