Characterization of a mutant GLUT4 lacking the N-glycosylation site: studies in transfected rat adipose cells.
GLUT4, the insulin-responsive glucose transporter expressed primarily in muscle and adipose tissue, contains a single N-glycosylation site. We characterized a mutant GLUT4 lacking the N-glycosylation site (Asn57-->Gln) in primary cultures of rat adipose cells. We transiently transfected cells with expression vectors for epitope-tagged GLUT4 containing either wild-type (GLUT4-HA) or mutant (GLN57-HA) cDNA sequences. Expression of GLN57-HA in adipose cells was approximately 10-fold lower than for GLUT4-HA even though mRNA levels for both recombinant transporters were comparable. Biosynthetic labeling studies showed markedly decreased incorporation of [35S]-methionine/cysteine into GLN57-HA relative to GLUT4-HA consistent with either a decreased synthetic rate or accelerated degradation of GLN57-HA. Interestingly, transient transfection of GLUT4-HA and GLN57-HA in COS-7 cells (which do not express endogenous GLUT4) resulted in comparable levels of protein expression for both transporters. Thus, in the physiologically relevant adipose cell, glycosylation of GLUT4 appears to play an important functional role.
Ing
B L
BL
Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Chen
H
H
Robinson
K A
KA
Buse
M G
MG
Quon
M J
MJ
D000273:Adipose Tissue / Q000378:metabolism*; D000818:Animals; D001216:Asparagine; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D017931:DNA Primers; D018274:Electroporation; D004822:Epididymis; D000939:Epitopes; D015870:Gene Expression / Q000187:drug effects; D051275:Glucose Transporter Type 4; D005973:Glutamine; D006031:Glycosylation; D007328:Insulin / Q000494:pharmacology; D008297:Male; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D009124:Muscle Proteins; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014162:Transfection
D017931:DNA Primers; D000939:Epitopes; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C495694:Slc2a4 protein, rat; D005973:Glutamine; D001216:Asparagine