STRUCTURE-FUNCTION ANALYSIS OF LIVER-TYPE (GLUT2) AND BRAIN-TYPE (GLUT3) GLUCOSE TRANSPORTERS - EXPRESSION OF CHIMERIC TRANSPORTERS IN XENOPUS OOCYTES SUGGESTS AN IMPORTANT ROLE FOR PUTATIVE TRANSMEMBRANE HELIX-7 IN DETERMINING SUBSTRATE SELECTIVITY

The liver-type (GLUT2) and brain-type (GLUT3) human facilitative gluco se transporters exhibit distinct kinetics (K-m values for deoxyglucose transport of 11.2 +/- 1.1 and 1.4 +/- 0.06 mM, respectively) and patt erns of substrate transport (GLUT2 is capable of D-fructose transport, GLUT3 is not) [Gould, G. W., Thomas, H. M., Jess, T. J., & Bell, G. I . (1991) Biochemistry 30, 5139-5145]. We have generated a range of chi meric glucose transporters composed of regions of GLUT2 and GLUT3 with a view to identifying the regions of the transporter which are involv ed in substrate recognition and binding. The functional characteristic s of these chimeras were determined by expression in Xenopus oocytes a fter microinjection of cRNA. Replacement of the region from the start of putative transmembrane helix 7 to the C-terminus of GLUT3 with the corresponding region from GLUT2 results in a chimera with the ability to transport fructose and exhibits a K-m for 2-deoxyglucose transport of close to that observed for wild-type GLUT2 (8.3 +/- 0.3 mM compared to 11.2 +/- 1.1 mM). Replacement of the region in GLUT3 from the end of helix 7 to the C-terminus with the corresponding region from GLUT:! resulted in a species which was unable to transport fructose and whos e K-m for 2-deoxyglucose was indistinguishable from wild-type GLUT3. W e have determined the affinity for 2-deoxyglucose, D-fructose, and D-g alactose of these and other chimeras. In addition, the K-i for maltose , a competitive inhibitor of 2-deoxyglucose transport, which binds to the exofacial sugar binding site was determined for these chimeras. Th e results obtained support a model in which the seventh putative trans membrane-spanning helix is intimately involved in the selection of tra nsported substrate and in which this region plays an important role in determining the K-m for 2-deoxyglucose. Additional data is presented which suggests that a region between the end of putative transmembrane helix 7 and the end of helix 10, together with sequences in the N-ter minal half of the protein may also participate in substrate recognitio n and transport catalysis.