Proteinase-activated receptor-2-mediated activation of stress-activated protein kinases and inhibitory kappa B kinases in NCTC 2544 keratinocytes.

In this study we examined the regulation of the stress-activated protein (SAP) kinases and inhibitory kappa B kinases (IKKs) through stimulation of the novel G-protein-coupled 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 mitogen-activated protein kinase activity. Trypsin also stimulated NF kappa B-DNA binding and the activation of the upstream kinases IKK alpha and -beta. 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 Gö6983, or transfection of dominant negative (DN)-PKC alpha, abolished phorbol 12-myristate 13-acetate-mediated c-Jun N-terminal kinase activity, although it only partially inhibited the response to trypsin. In contrast, Gö6983 reduced trypsin-stimulated p38 mitogen-activated protein kinase activity to a greater extent than GF109203X, although DN-PKC alpha or PKC zeta had no substantial effect. Additionally, inhibitors of PKC partially reduced trypsin-stimulated IKK alpha activity but abolished that of IKK beta, whereas DN-PKC alpha but not DN-PKC zeta substantially reduced trypsin-stimulated Flag-IKK beta 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.

Proteinase-activated receptors.

Proteinase-activated receptors are a recently described, novel family of seven-transmembrane G-protein-coupled receptors. Rather then being stimulated through ligand receptor occupancy, activation is initiated by cleavage of the N terminus of the receptor by a serine protease resulting in the generation of a new tethered ligand that interacts with the receptor within extracellular loop-2. To date, four proteinase-activated receptors (PARs) have been identified, with distinct N-terminal cleavage sites and tethered ligand pharmacology. In addition to the progress in the generation of PAR-1 antagonists, we describe the role of thrombin in such processes as wound healing and the evidence implicating PAR-1 in vascular disorders and cancer. We also identify advances in the understanding of PAR-1-mediated intracellular signaling and receptor desensitization. The cellular functions, signaling events, and desensitization processes involved in PAR-2 activation are also assessed. However, other major aspects of PAR-2 are highlighted, in particular the ability of several serine protease enzymes, in addition to trypsin, to function as activators of PAR-2. The likely physiological and pathophysiological roles for PAR-2 in skin, intestine, blood vessels, and the peripheral nervous system are considered in the context of PAR-2 activation by multiple serine proteases. The recent discovery of PAR-3 and PAR-4 as additional thrombin-sensitive PARs further highlights the complexity in assessing the effects of thrombin in several different systems, an issue that remains to be fully addressed. These discoveries have also highlighted possible PAR-PAR interactions at both functional and molecular levels. The future identification of other PARs and their modes of activation are an important future direction for this expanding field of study.

QLS motif in transmembrane helix VII of the glucose transporter family interacts with the C-1 position of D-glucose and is involved in substrate selection at the exofacial binding site.

The liver-type (GLUT2) and brain-type (GLUT3) human facilitative glucose transporters exhibit distinct kinetics (Km values for deoxyglucose transport of approximately 11 mM and approximately 1.5 mM, respectively) and patterns of substrate transport (GLUT2 is capable of D-fructose transport, while GLUT3 is not). Using a range of chimeric glucose transporters comprised of regions of GLUT2 and GLUT3 studied by expression in Xenopus oocytes after microinjection of cRNA, we have proposed that the seventh putative transmembrane helix is intimately involved in the selection of transported substrate and that this region plays an important role in determining the Km for 2-deoxyglucose [Arbuckle, M. I., Kane, S., Porter, L. M., Seatter, M. J., and Gould, G. W. (1996) Biochemistry 35, 16519-16527]. Inspection of the predicted amino acid sequence of this region reveals that GLUTs 1, 3, and 4 (high-affinity glucose transporters) contain a conserved QLS motif in this helix (residues 277-279 in human GLUT3). In the glucose/fructose transporter (GLUT2) this motif is replaced by HVA. To study the role of the QLS motif in substrate selection, we have engineered substitutions in this region between GLUT2 and GLUT3. GLUT3 (QLS > HVA) exhibits a Km for deoxyglucose transport identical to that of native GLUT3 but increased sensitivity for inhibition of deoxyglucose transport by D-fructose. However, unlike native GLUT3, this species is capable of transporting D-fructose. Compared to wild-type GLUT2, GLUT2 (HVA > QLS) exhibits a lower Km for deoxyglucose transport (approximately 3 mM vs approximately 11 mM), the ability to transport D-fructose is reduced, and D-fructose is a less efficient inhibitor of deoxyglucose transport. Analysis of the ability of a range of glucose epimers and analogues to inhibit transport by these species suggests that the QLS motif interacts with the incoming D-glucose at the C-1 position; this may be a key interaction in the high-affinity recognition of the transported substrate. We further argue that this interaction acts as a molecular filter that is involved in the selection of the transported substrate.

Functional studies of human GLUT5: effect of pH on substrate selection and an analysis of substrate interactions.

The adsorption of D-fructose within the lumen of the human small intestine is thought to be mediated by the GLUT5 isoform of the human facilitative sugar transporter family. This isoform has been expressed in oocytes and shown to be capable of D-fructose transport. Some debate remains regarding the absolute substrate specificity of this isoform. To that end, we have undertaken an analysis of the functional properties of this protein when expressed in Xenopus oocytes. We have examined the pH dependence of transport activity, the ability to transport D-fructose versus deoxyglucose, and employed a range of sugar analogues to probe the nature of the exofacial substrate binding site. Our data show that the human GLUT5 isoform functions exclusively as a D-fructose transporter between pH 4.5 and 8. The Km for D-fructose was found to be 15 +/- 4 mM at pH 7. 5, and was relatively unaltered even at pH 4.5. Analysis of the effects of a range of compounds on GLUT5 function suggests that this isoform transports D-fructose preferentially in the furanose ring form.

Structure-function studies of the brain-type glucose transporter, GLUT3: alanine-scanning mutagenesis of putative transmembrane helix VIII and an investigation of the role of proline residues in transport catalysis.

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.

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 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.

Analysis of the structural requirements of sugar binding to the liver, brain and insulin-responsive glucose transporters expressed in oocytes.

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.

Kinetic analysis of the liver-type (GLUT2) and brain-type (GLUT3) glucose transporters in Xenopus oocytes: substrate specificities and effects of transport inhibitors.

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.

Pancreatic beta-cells express a low affinity glucose transporter: functional consequences in normal and diabetic states.

The application of molecular biology to the study of membrane transport proteins has led to a rapid advance in our understanding of the mechanisms behind the regulation of blood glucose levels. Moreover the demonstration of lesions in the expression of GLUT2 in the islets from diabetic models has provided a focus for research efforts aimed at addressing the defects responsible for the development and onset of both type I and perhaps type II diabetes. The recent demonstration that an 'artificial beta-cell' can be engineered from anterior pituitary-derived cell lines by transfection with both the insulin cDNA and the cDNA encoding GLUT2 represents a significant advance in the development of potential therapies for type I diabetes [24].