Cloning and functional characterization of Xenopus laevis facilitated hexose transporters GLUT2 and GLUT4 (XL‐GLUT2, XL‐GLUT4).

RT‐PCR was used to clone members of the facilitated hexose transporter gene family (SLC2A) from Xenopus oocyte total RNA. Gene specific primers were designed using the NIH Xenopus Gene Collection. We have identified two Xenopus GLUT sequences, XL‐GLUT2 and XL‐GLUT4 (gene accession numbers EF102088 & EF102089 ) which have 62% & 69% identity and 78% & 83% similarity with their human orthologues, respectively. Functional characterization of XL‐GLUT2 shows that this protein transports 2‐deoxyglucose > galactose > glucose > 3‐O‐methyl glucose >> fructose. In contrast, hGLUT2 has an equal affinity for both glucose and fructose (Km > 20mM) while XL‐GLUT2 has a much higher affinity, for glucose alone with a Km of 2.5 ± 0.5 mM. Sequence alignment and comparison of the Xenopus and human transporters in the region of their predicted 7th transmembrane helix shows that several motifs are highly conserved, in particular the QLS and the NXV/I sequences. This latter hydrophobic motif has previously been found to influence substrate specificity in human GLUTs. hGLUT2 has the motif NGI which when mutated to NGV results in an almost complete loss of fructose transport, however, the Xenopus WT protein has very low fructose transport capacity, yet still has the NGI motif. This suggests that this hydrophobic region found near the exofacial opening of the transport pore of GLUT proteins is not the sole determinant of substrate specificity.