Targeting of GLUT1-GLUT5 chimeric proteins in the polarized cell line Caco-2.
Caco-2, a human differentiated intestinal epithelial cell line, is a promising model for investigating the mechanism of polarized targeting of apical and basolateral membrane proteins. We stably transfected rat GLUT5 cDNA and rabbit GLUT1 cDNA into Caco-2 cells with an expression vector. Immunohistochemical study revealed that the GLUT5 protein expressed was localized at apical membranes and that the GLUT1 expressed was present primarily in the basolateral membranes of cells grown on permeable support. Next, to investigate the domain responsible for determining apical vs. basolateral sorting in glucose transporters, we prepared several GLUT1-GLUT5 chimeric cDNAs and transfected them into Caco-2 cells. A GLUT1 [N terminus approximately sixth transmembrane domain (TM6)]-GLUT5 [intracellular loop (IL) approximately C terminus] chimera was observed exclusively at the apical membrane, while GLUT1 (N terminus approximately IL)-GLUT5 (TM7 approximately C terminus) and GLUT1 (N terminus approximately TM12)-GLUT5 (C-terminal domain) chimeras were observed mainly at the basolateral membrane, a localization similar to that of GLUT1. Moreover, using a recombinant adenovirus expression system, we expressed a GLUT5 (N terminus approximately TM6)-GLUT1(IL)-GLUT5(TM7 approximately C-terminus) chimera, which was observed at the basolateral membrane. Based on these results, the C-terminal domain does not determine isoform-specific targeting of GLUT1 and GLUT5. Rather, it is the intracellular loop in glucose transporters that appears to play a pivotal role in apical-basolateral sorting signals in Caco-2 cells.
Inukai
K
K
Institute for Adult Disease, Asahi Life Foundation, Shinjuku-ku, Tokyo, Japan.
Takata
K
K
Asano
T
T
Katagiri
H
H
Ishihara
H
H
Nakazaki
M
M
Fukushima
Y
Y
Yazaki
Y
Y
Kikuchi
M
M
Oka
Y
Y
D000818:Animals; D018938:Caco-2 Cells; D016764:Cell Polarity; D018076:DNA, Complementary / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D051272:Glucose Transporter Type 1; D051296:Glucose Transporter Type 5; D006801:Humans; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D018076:DNA, Complementary; D051272:Glucose Transporter Type 1; D051296:Glucose Transporter Type 5; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C495677:SLC2A1 protein, human; C495679:Slc2a1 protein, rat