Serine-294 and threonine-295 in the exofacial loop domain between helices 7 and 8 of glucose transporters (GLUT) are involved in the conformational alterations during the transport process.
The role of a conserved polar motif (STS) in the exofacial loop between helices 7 and 8 of 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 photolabelling with the exofacial label 2-N4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1, 3-bis-(d-mannosyloxy)-2-propylamine (ATB-BMPA) were assayed in membranes from transfected cells and corrected for immunoreactivity of expressed transporters. Replacement of Ser-294 with Ala or Thr suppressed transport activity and cytochalasin B binding. ATB-BMPA photolabelling was normal in S294A mutants, and even increased in S294T mutants. Replacement of Thr-295 with Ala suppressed transport activity and cytochalasin B binding, whereas ATB-BMPA photolabelling was normal; substitution of Ser failed to alter the investigated parameters. Similarly, exchanging Ser-296 for Ala generated a normally functioning protein. The data suggest that Ser-294 and Thr-295 are involved in the conformational change in GLUT during the transport process, and that their substitution may arrest the transporter in an outward-facing conformation.
Doege
H
H
Institute of Pharmacology and Toxicology, Medical Faculty, Technical University of Aachen, Germany.
Schürmann
A
A
Ohnimus
H
H
Monser
V
V
Holman
G D
GD
Joost
H G
HG
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D051275:Glucose Transporter Type 4; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000235:genetics / Q000378:metabolism*
D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D013912:Threonine; D012694:Serine; D000409:Alanine; D005998:Glycine