| Facilitated glucose transporters (GLUT's) mediate transport of sugars across cell membranes using the chemical gradient of sugars as the driving force. Improved cloning techniques and database analyses have expanded this family of proteins to a total of 14 putative members. In this I work present the characterization of a novel hexose transporter isoform, GLUT7, which has been cloned from a human intestinal cDNA library using a PCR-based strategy. The encoded protein is comprised of 524 amino acid residues and shares 68% similarity and 53% identity with GLUT5, its most closely related isoform. When GLUT7 was expressed in Xenopus oocytes it showed high affinity transport for glucose and fructose. Northern blotting indicated that the mRNA for GLUT7 is present in the human jejunum and ileum, but absent from stomach, duodenum, colon and lung. Western blotting and immunohistochemistry of rat tissues using an antibody raised against the predicted C-terminal sequence confirmed this distribution and indicated that the transporter is predominantly expressed in the brush-border membrane of enterocytes. The unusual substrate specificity and high level of sequence identity with GLUT5 suggest that GLUT7 represents an intermediate between class II GLUT's and the class I member GLUT2.; Sequence alignments indicated that GLUTs 2, 5, 7 & 9 all had an isoleucine residue at position '314' (GLUT7) at the exofacial border of the helix 7, whereas the non-fructose transporting isoforms, GLUT's 1, 3, & 4, had a valine at the equivalent position. Mutation of I314 to a valine in GLUT7 resulted in a loss of fructose transport, while glucose transport remained completely unaffected. Similar results were obtained with GLUT's 2, 5, 9 and 11. Energy minimization modeling of GLUT7 indicates that I314 projects from TM7 into the lumen of the aqueous pore where it could form a hydrophobic interaction with Tryptophan 89 from TM2. A valine residue at '314' appears to produce a narrowing of the vestibule compared to isoleucine. It is proposed that this hydrophobic interaction across the pore forms a selectivity filter restricting the access of some hexoses to the substrate binding site/s within the aqueous channel. |
