Specificity in ligand binding and intracellular signalling by insulin and insulin-like growth factor receptors.

The physiological roles of insulin and insulin-like growth factors (IGFs) are distinct, with insulin acting to regulate cellular uptake and metabolism of fuels, whereas IGFs promote cell growth, survival and differentiation. The only components of signalling pathways known to be unique to insulin and IGFs are their respective receptors, and even these display substantial structural and functional similarity. Specificity of action in vivo must in part reflect relative levels of receptor expression in different tissues. The extent to which the receptors differ in intrinsic signalling capacity remains unclear, but specificity might in principle arise from differences in ligand-binding mechanism or properties of intracellular domains. To identify ligand binding determinants we expressed receptor fragments as soluble proteins. Both N-terminal domains and a C-terminal peptide sequence from the alpha-subunit are essential for ligand binding with moderate affinity. However, binding of ligand with high affinity and specificity requires higher-order structure. To compare signalling capacities, we constructed chimaeras containing intracellular domains of insulin or IGF receptors fused to the extracellular portion of TrkC. Expression and activation of these chimaeras in cell lines reveals subtle differences in signalling and end-point responses, which may depend on cell background.

Identification of further important residues within the Glut4 carboxy-terminal tail which regulate subcellular trafficking.

The insulin-responsive glucose transporter, Glut4, exhibits a unique subcellular distribution such that in the absence of insulin >95% of the protein is stored within intracellular membranes. In response to insulin, Glut4 exhibits a large mobilisation to the plasma membrane. Studies of the amino acid motifs which regulate the unique trafficking of Glut4 have identified several key residues within the soluble cytoplasmic N- and C-terminal domains of Glut4. Of particular note is a Leu-498Leu-499 motif within the C-terminal domain that has been proposed to regulate both internalisation from the plasma membrane and sorting to an insulin-sensitive compartment. In this study, we have examined the role of the adjacent amino acids (Glu-491, Gln-492 and Glu-493) by their sequential replacement with Ala. Our results are consistent with the notion that Glu-491 and Glu-493 play an important role in the sub-endosomal trafficking of Glut4, as substitution of these residues with Ala results in increased levels of these proteins at the cell surface, reduced insulin-stimulated translocation and increased susceptibility to endosomal ablation. These residues, together with other identified sequences within the C-terminus of Glut4, are likely to be crucial targeting elements that regulate Glut4 subcellular distribution.

Differences in signaling properties of the cytoplasmic domains of the insulin receptor and insulin-like growth factor receptor in 3T3-L1 adipocytes.

Insulin and insulin-like growth factors (IGFs) elicit distinct but overlapping biological effects in vivo. To investigate whether differences in intrinsic signaling capacity of receptors contribute to biological specificity, we constructed chimeric receptors containing the extracellular portion of the neurotrophin receptor TrkC fused to the intracellular portion of the insulin or IGF-I receptors. Chimeras were stably expressed in 3T3-L1 adipocytes at levels comparable to endogenous insulin receptors and were efficiently activated by neurotrophin-3. The wild-type insulin receptor chimera mediated approximately 2-fold greater phosphorylation of insulin receptor substrate 1 (IRS-1), association of IRS-1 with phosphoinositide 3-kinase, stimulation of glucose uptake, and GLUT4 translocation, compared with the IGF-I receptor chimera. In contrast, the IGF-I receptor chimera mediated more effective Shc phosphorylation, association of Shc with Grb2, and activation of mitogen-activated protein kinase compared with the insulin receptor chimera. The two receptors elicited similar activation of protein kinase B, p70S6 kinase, and glycogen synthesis. We conclude that the insulin receptor mediates some aspects of metabolic signaling in adipocytes more effectively than the IGF-I receptor, as a consequence of more efficient phosphorylation of IRS-1 and greater recruitment/activation of phosphoinositide 3-kinase.

Familial distal renal tubular acidosis is associated with mutations in the red cell anion exchanger (Band 3, AE1) gene.

All affected patients in four families with autosomal dominant familial renal tubular acidosis (dRTA) were heterozygous for mutations in their red cell HCO3-/Cl- exchanger, band 3 (AE1, SLC4A1) genes, and these mutations were not found in any of the nine normal family members studied. The mutation Arg589--> His was present in two families, while Arg589--> Cys and Ser613--> Phe changes were found in the other families. Linkage studies confirmed the co-segregation of the disease with a genetic marker close to AE1. The affected individuals with the Arg589 mutations had reduced red cell sulfate transport and altered glycosylation of the red cell band 3 N-glycan chain. The red cells of individuals with the Ser613--> Phe mutation had markedly increased red cell sulfate transport but almost normal red cell iodide transport. The erythroid and kidney isoforms of the mutant band 3 proteins were expressed in Xenopus oocytes and all showed significant chloride transport activity. We conclude that dominantly inherited dRTA is associated with mutations in band 3; but both the disease and its autosomal dominant inheritance are not related simply to the anion transport activity of the mutant proteins.

Compartment-ablation studies of GLUT4 distribution in adipocytes: evidence for multiple intracellular pools.

The available data suggest that GLUT4 does populate the recycling endosomal system to some extent, but that a large proportion of the intracellular GLUT4 resides in a compartment that is devoid of transferrin receptors and may have properties more akin to specialized secretory vesicles. The study of the nature and biogenesis of this compartment will provide important insight into the mechanism by which insulin stimulates glucose transport. Further study of the role of the synaptobrevins in these distinct subcellular compartments will probably shed further light on the mechanism by which insulin stimulates GLUT4 translocation.

Domain assembly of the GLUT1 glucose transporter.

A full-length construct of the glucose transporter isoform GLUT1 has been expressed in Sf9 (Spodoptera frugiperida Clone 9) insect cells, and a photolabelling approach has been used to show that the expressed protein binds the bismannose compound 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2-propylamine (ATB-BMPA) and cytochalasin B at its exofacial and endofacial binding sites respectively. Constructs of GLUT1 which produce either the N-terminal (amino acids 1-272) or C-terminal (amino acids 254-492) halves are expressed at levels in the plasma membrane which are similar to that of the full-length GLUT1 (approximately 200 pmol/mg of membrane protein), but do not bind either ATB-BMPA or cytochalasin B. When Sf9 cells are doubly infected with virus constructs producing both the C- and N-terminal halves of GLUT1, then the ligand labelling is restored. Only the C-terminal half is labelled, and, therefore, the labelling of this domain is dependent on the presence of the N-terminal half of the protein. These results suggest that the two halves of GLUT1 can assemble to form a stable complex and support the concept of a bilobular structure for the intact glucose transporters in which separate C- and N-domain halves pack together to produce a ligand-binding conformation.