We have expressed the human isoforms of the liver-type (GLUT2) and brain-type (GLUT3) facilitative glucose transporters in oocytes from Xenopus laevis via injection of in vitro transcribed mRNA. As reported previously [Gould, Thomas, Jess and Bell (1991) Biochemistry 30, 5139-5145], GLUT2 mediates the transport of fructose and galactose, and GLUT3 mediates the transport of galactose. We have examined the effects of D-glucose, D-fructose and maltose on deoxyglucose transport in oocytes expressing GLUT2, and D-glucose, D-galactose and maltose on deoxyglucose transport in oocytes expressing GLUT3, and show that each sugar is a competitive inhibitor of transport. Moreover, D-glucose and maltose competitively inhibit fructose transport by GLUT2 and galactose transport by GLUT3, indicating that the transport of the alternative substrates for these transporters is likely to be mediated by the same outward-facing sugar-binding site used by glucose. Cytochalasin B is a non-competitive inhibitor of glucose transport by the well-characterized GLUT1 isoform. We show here that cytochalasin B is also a non-competitive inhibitor of the transport of deoxyglucose and alternative substrates by GLUT2 and GLUT3 expressed in oocytes. Km and Ki values for each substrate and inhibitor are presented for each isoform, together with further analysis of the binding sites for alternative substrates for these transporter isoforms.

In this study we examined the regulation of the stressactivated protein (SAP) kinases and inhibitory B kinases (IKKs) through stimulation of the novel G-proteincoupled receptor proteinase-activated receptor-2 in the human keratinocyte cell line NCTC2544. Trypsin and the peptide SLIGKV stimulated a time-dependent increase in both c-Jun N-terminal kinase and p38 mitogenactivated protein kinase activity. Trypsin also stimulated NFB-DNA binding and the activation of the upstream kinases IKK␣ and -␤. Phorbol 12-myristate 13-acetate also strongly activated both SAP kinases and IKK isoforms, suggesting the potential for a protein kinase C-mediated regulatory mechanism underlying the effects of trypsin. Pre-incubation with selective protein kinase C (PKC) inhibitors GF109203X and Gö 6983, or transfection of dominant negative (DN)-PKC␣, abolished phorbol 12-myristate 13-acetate-mediated c-Jun N-terminal kinase activity, although it only partially inhibited the response to trypsin. In contrast, Gö 6983 reduced trypsin-stimulated p38 mitogen-activated protein kinase activity to a greater extent than GF109203X, although DN-PKC␣ or PKC had no substantial effect. Additionally, inhibitors of PKC partially reduced trypsin-stimulated IKK␣ activity but abolished that of IKK␤, whereas DN-PKC␣ but not DN-PKC substantially reduced trypsin-stimulated Flag-IKK␤ activity. This study shows for the first time proteinase-activated receptor-2-mediated stimulation of both SAP kinase and IKK signaling and differing roles for PKC isoforms in the regulation of each pathway.

The liver-type (GLUT2) and brain-type (GLUT3) human facilitative glucose transporters exhibit distinct kinetics (K(m) values for deoxyglucose transport of 11.2 +/- 1.1 and 1.4 +/- 0.06 mM, respectively) and patterns 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 chimeric glucose transporters composed of regions of GLUT2 and GLUT3 with a view to identifying the regions of the transporter which are involved in substrate recognition and binding. The functional characteristics of these chimeras were determined by expression in Xenopus oocytes after 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 GLUT2 resulted in a species which was unable to transport fructose and whose K(m) for 2-deoxyglucose was indistinguishable from wild-type GLUT3. We have determined the affinity for 2-deoxyglucose, D-fructose, and D-galactose of these and other chimeras. In addition, the Ki for maltose, a competitive inhibitor of 2-deoxyglucose transport, which binds to the exofacial sugar binding site was determined for these chimeras. The results obtained support a model in which the seventh putative transmembrane-spanning helix is intimately involved in the selection of transported 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-terminal half of the protein may also participate in substrate recognition and transport catalysis.

No abstract

We have expressed the liver (GLUT 2), brain (GLUT 3) and insulin-responsive (GLUT 4) glucose transporters in oocytes from Xenopus laevis by microinjection of in vitro-transcribed mRNA. Using a range of halogeno- and deoxy-glucose analogues, and other hexoses, we have studied the structural basis of sugar binding to these different isoforms. We show that a hydrogen bond to the C-3 position is involved in sugar binding for all three isoforms, but that the direction of this hydrogen bond is different in GLUT 2 from either GLUT 1, 3 or 4. Hydrogen-bonding at the C-4 position is also involved in sugar recognition by all three isoforms, but we propose that in GLUT 3 this hydrogen bond plays a less significant role than in GLUT 2 and 4. In all transporters we propose that the C-4 position is directed out of the sugar-binding pocket. The role of the C-6 position is also discussed. In addition, we have analysed the ability of fructopyranose and fructofuranose analogues to inhibit the transport mediated by GLUT2. We show that fructofuranose analogues, but not fructopyranose analogues, are efficient inhibitors of transport mediated by GLUT 2, and therefore suggest that GLUT 2 accommodates D-glucose as a pyranose ring, but D-fructose as a furanose ring. Models for the binding sites of GLUT 2, 3 and 4 are presented.

No abstract

The brain-type glucose transporter (GLUT3) is a high-affinity transporter for D-glucose and D-galactose and is a member of a family of mammalian sugar transporters, each of which are proposed to adopt a secondary structure containing 12 transmembrane helices. In an effort to understand structure-function relationships within such transporters, we have employed alanine-scanning mutagenesis to examine the functional importance of each residue within putative transmembrane helix VIII of the human GLUT3 isoform. Each residue in this helix was replaced individually with alanine, and the functional properties of the mutants were examined by microinjection of in vitro transcribed mRNA into Xenopus oocytes. We show that substitution of residues 305, 306, 308-314, and 316-325 with alanine had minimal effect on the functional activity of the transporter, as determined by measurement of the Km for deoxyglucose transport and the Ki for maltose. In contrast, Asn-315 > Ala-315 exhibited a significant increase in the Km for deoxyglucose independently of any effect on the Ki for maltose. This data suggests that, despite the strong sequence conservation in this helix among the GLUT family, no individual residue is absolutely required for transport catalysis by this isoform. We have also examined the role of proline residues in transport catalysis mediated by GLUT3. Substitution of Pro-203 (helix VI), Pro-206, Pro-209 (cytoplasmic loop between helices VI and VII), Pro-381, Pro-383 and Pro-385 (helix X), Pro-399 (intracellular loop between helices X and XI), or Pro-451 (in the carboxy terminus, close to the end of helix XII) with alanine did not change the Km for deoxyglucose transport for any mutant. However, both Pro-381 and Pro-385 when mutated to alanine exhibited a reduction in the Ki for cytochalasin B. In addition, the Ki for maltose inhibition of deoxyglucose transport was increased for mutants Pro206Ala, Pro381Ala, Pro383Ala, and Pro451Ala. These results will be discussed in terms of proposed structural models for the transporters.

No abstract