Inhibition of the receptor-mediated endocytosis of diferric transferrin is associated with the covalent modification of the transferrin receptor with palmitic acid

The human transferrin receptor is post-translationally modified by the covalent attachment of palmitic acid to Cys62 and Cys67 via a thio-ester bond. To investigate the role of the acylation of the transferrin receptor, Cys62 and Cys67 were substituted with serine and alanine residues. The properties of the mutant receptors were compared with wild-type receptors after expression in Chinese hamster ovary cells that lack endogenous transferrin receptors. Rapid incorporation of [3H]palmitate into the wild-type transferrin receptor was observed, but the mutant receptors were found to be palmitoylation-defective. The kinetics of endocytosis and recycling of the wild-type and mutant receptors were compared. It was observed that the rate of endocytosis of the palmitoylation-defective transferrin receptors was significantly greater than the rate measured for the wild-type transferrin receptor. In contrast, the mutation of Cys62 and Cys67 was found to have no significant effect on the rate of transferrin receptor recycling. Consistent with these observations, it was found that cells expressing palmitoylation-defective transferrin receptors exhibited an increased rate of accumulation of [59Fe]diferric transferrin. Together, these data indicate that the palmitoylation of the transferrin receptor is associated with an inhibition of the rate of transferrin receptor endocytosis. Addition of insulin to cultured cells causes an increase in the palmitoylation of cell surface transferrin receptors and a decrease in the rate of transferrin receptor internalization. It was observed that the effect of insulin to inhibit the endocytosis of the acylation-defective [Ala62 Ala67]transferrin receptor was attenuated in comparison with the wild-type receptor. The decreased effectiveness of insulin to inhibit the internalization of the acylation-defective transferrin receptor is consistent with the hypothesis that palmitoylation represents a potential mechanism for the regulation of transferrin receptor endocytosis.     

The Drosophila insulin receptor activates multiple signaling pathways but requires insulin receptor substrate proteins for DNA synthesis.

The Drosophila insulin receptor (DIR) contains a 368-amino-acid COOH-terminal extension that contains several tyrosine phosphorylation sites in YXXM motifs. This extension is absent from the human insulin receptor but resembles a region in insulin receptor substrate (IRS) proteins which binds to the phosphatidylinositol (PI) 3-kinase and mediates mitogenesis. The function of a chimeric DIR containing the human insulin receptor binding domain (hDIR) was investigated in 32D cells, which contain few insulin receptors and no IRS proteins. Insulin stimulated tyrosine autophosphorylation of the human insulin receptor and hDIR, and both receptors mediated tyrosine phosphorylation of Shc and activated mitogen-activated protein kinase. IRS-1 was required by the human insulin receptor to activate PI 3-kinase and p70s6k, whereas hDIR associated with PI 3-kinase and activated p70s6k without IRS-1. However, both receptors required IRS-1 to mediate insulin-stimulated mitogenesis. These data demonstrate that the DIR possesses additional signaling capabilities compared with its mammalian counterpart but still requires IRS-1 for the complete insulin response in mammalian cells.     

Nonacylated human transferrin receptors are rapidly internalized and mediate iron uptake

The human transferrin receptor is post-translationally modified by the addition of a fatty acyl moiety. In earlier studies, transient expression in Cos cells of human transferrin receptors in which Cys62 or Cys67 was altered to serine provided evidence that Cys62 is the major acylation site of the receptor (Jing, S., and Trowbridge, I. S. (1987) EMBO J. 6, 327-331). To determine whether acylation of the receptor is required for high efficiency endocytosis and iron uptake, wild type and mutant human transferrin receptors have been stably expressed in chick embryo fibroblasts using a helper-independent retroviral vector. In marked contrast to Cos cells, both Cys62 and Cys67 of the wild type human transferrin receptor were acylated in chick embryo fibroblasts. Moreover, their modification to serine did not abolish palmitate labeling, implying that one or both of these serine residues could serve as alternative lipid attachment sites in these cells. The relative labeling of mutant receptors with palmitate and the susceptibility of their lipid moieties to cleavage by hydroxylamine were consistent with Ser67 but not Ser62 serving as a lipid attachment site. Consequently, to obtain human transferrin receptors lacking covalently bound lipid in the chick embryo fibroblasts, it was necessary to alter Cys62 and Cys67 to alanine. Functional studies indicated that these non-acylated mutant receptors were internalized efficiently and mediated iron uptake from human transferrin at a similar rate to that of wild type receptors. We conclude, therefore, that acylation of the human transferrin receptor is not essential for endocytosis and recycling.     

Mutation of the high cysteine region of the human insulin receptor alpha-subunit increases insulin receptor binding affinity and transmembrane signaling

Our previous studies indicated that amino acid residues 240-250 in the cysteine-rich region of the human insulin receptor alpha-subunit constitute a site in which insulin binds (Yip, C. C., Hsu, H., Patel, R. G., Hawley, D. M., Maddux, B. A., and Goldfine, I. D. (1988) Biochem. Biophys. Res. Commun. 157, 321-329). We have now constructed a human insulin receptor mutant in which 3 residues in this sequence were altered (Thr-Cys-Pro-Pro-Pro-Tyr-Tyr-His-Phe-Gln-Asp to Thr-Cys-Pro-Arg-Arg-Tyr-Tyr-Asp-Phe-Gln-Asp) and have expressed this mutant in rat hepatoma (HTC) cells. When compared with cells transfected with normal insulin receptors, cells transfected with mutant receptors had an increase in insulin-binding affinity and a decrease in the dissociation of bound 125I-insulin. Studies using solubilized receptors also demonstrated that mutant receptors had a higher binding affinity than normal receptors. In contrast, cells transfected with either mutant or normal receptors bound monoclonal antibodies against the receptor alpha-subunit with equal affinity. When receptor tyrosine kinase activity and alpha-aminoisobutyric acid uptake were measured, cells transfected with mutant insulin receptors were more sensitive to insulin than cells transfected with normal receptors. These findings lend further support therefore to the hypothesis that amino acid sequence 240-250 of the human insulin receptor alpha-subunit constitutes one site that interacts with insulin, and they indicate that mutations in this site can influence insulin receptor binding and transmembrane signaling.     

A bioluminescence resonance energy transfer 2 (BRET2) assay for monitoring seven transmembrane receptor and insulin receptor crosstalk

The angiotensin AT₁ receptor is a seven transmembrane (7TM) receptor, which mediates the regulation of blood pressure. Activation of angiotensin AT₁ receptor may lead to impaired insulin signaling indicating crosstalk between angiotensin AT₁ receptor and insulin receptor signaling pathways. To elucidate the molecular mechanisms behind this crosstalk, we applied the BRET² technique to monitor the effect of angiotensin II on the interaction between Rluc⁸ tagged insulin receptor and GFP² tagged insulin receptor substrates 1, 4, 5 (IRS1, IRS4, IRS5) and Src homology 2 domain-containing protein (Shc). We demonstrate that angiotensin II reduces the interaction between insulin receptor and IRS1 and IRS4, respectively, while the interaction with Shc is unaffected, and this effect is dependent on Gαq activation. Activation of other Gαq-coupled 7TM receptors led to a similar reduction in insulin receptor and IRS4 interactions whereas Gαs- and Gαi-coupled 7TM receptors had no effect. Furthermore, we used a panel of kinase inhibitors to show that angiotensin II engages different pathways when regulating insulin receptor interactions with IRS1 and IRS4. Angiotensin II inhibited the interaction between insulin receptor and IRS1 through activation of ERK1/2, while the interaction between insulin receptor and IRS4 was partially inhibited through protein kinase C dependent mechanisms. We conclude that the crosstalk between angiotensin AT₁ receptor and insulin receptor signaling shows a high degree of specificity, and involves Gαq protein, and activation of distinct kinases. Thus, the BRET² technique can be used as a platform for studying molecular mechanisms of crosstalk between insulin receptor and 7TM receptors.     

The type I and type II bovine scavenger receptors expressed in Chinese hamster ovary cells are trimeric proteins with collagenous triple helical domains comprising noncovalently associated monomers and Cys83-disulfide-linked dimers.

Scavenger receptors have been implicated in the development of atherosclerosis and other macrophage-associated functions. The structures and processing of type I and type II bovine macrophage scavenger receptors were examined using polyclonal anti-receptor antibodies. Pulse/chase metabolic labeling experiments showed that both types of scavenger receptors expressed in Chinese hamster ovary (CHO) cells behaved as typical cell surface membrane glycoproteins. They were synthesized as endoglycosidase H-sensitive precursors which were converted to endoglycosidase H-resistant mature forms expressed on the cell surface. The reduced precursor and mature forms were doublets on sodium dodecyl sulfate-gel electrophoresis, primarily because of heterogeneous N-glycosylation. The approximate molecular sizes were: type I precursor, 65/63 kDa; type I mature, 82/76 kDa; type II precursor, 57/53 kDa; and type II mature, 72/65 kDa. During post-translational processing, the cysteine-rich C terminus (SRCR domain) of some of the type I receptors was proteolytically removed to form a relatively stable, approximately 69-kDa degradation product. Type II receptors differ from type I receptors in that they do not have SRCR domains and an analogous proteolytic cleavage was not observed. Several experiments provided strong evidence that the Gly-X-Y-repeat domains in the scavenger receptors oligomerize into collagenous triple helices. For example, alpha,alpha'-dipyridyl, an inhibitor of the collagen-modifying enzymes prolyl and lysyl hydroxylases, interfered with both the kinetics and nature of post-translational receptor processing, and both precursor and mature forms of the receptors in intact cells could be cross-linked with difluorodinitrobenzene into reduction-resistant trimers. In intact cells, precursor receptor trimers (type I, 198 kDa; type II, 176 kDa) were assembled in the endoplasmic reticulum by the noncovalent association of monomers and Cys83-disulfide-linked dimers (type I, 129 kDa; type II, 119 kDa). When cells were lysed in the absence of the sulfhydryl trapping agent iodoacetamide, oxidation of the side chain of Cys17 in the cytoplasmic domain leads to the artifactual formation of reduction-sensitive covalently linked trimers. The approximate masses of the mature dimer and trimer forms were 162 and 237 kDa for type I receptors and 147 and 219 kDa for type II receptors. Cys83-disulfide-linked dimer formation was not required for function because mutant receptors (Cys83----Gly83) assembled into trimers of noncovalently associated monomers and exhibited normal receptor activity. Treatment of cells with difluorodinitrobenzene cross-linked some of the receptors into complexes larger than trimers, raising the possibility that the trimers may assemble into higher order oligomers.     

Structural and functional characterization of insulin receptor substrate proteins and the molecular mechanisms of their interaction with insulin superfamily tyrosine kinase receptors and effector proteins

The literature data on the role of IRS1/IRS2 proteins, endogenous substrates for insulin receptor tyrosine kinase, in transduction of signals generated by insulin superfamily peptides (insulin, insulin-like growth factor) were analyzed. The molecular mechanisms of the functional coupling of IRS proteins with peptide receptors possessing a tyrosine kinase activity and SH2 domain-containing proteins (phosphatidylinositol 3-kinase, Grb2 adaptor protein, protein phosphotyrosine phosphatase) were discussed. The structural and functional properties of IRS proteins (distribution of functional domains and sites for tyrosine phosphorylation; conservatism of amino acid sequences) were characterized. The data on the alternative pathways of transduction of signals which are generated by insulin and related peptides and do not involve IRS proteins were analyzed. These pathways are realized through Shc proteins or via direct interaction between receptors and SH2 proteins. Amino acid sequences of IRS proteins and insulin superfamily tyrosine kinase receptors were compared. The homologous regions in IRS proteins and receptors, which can be responsible for their coupling with phosphatidylinositol 3-kinase and protein phosphotyrosine phosphatases, were identified.     

Construction and characterization of a monomeric insulin receptor

A mutant insulin receptor was constructed by replacing cysteine residues Cys(524), Cys(682), Cys(683), and Cys(685) with serine. The mutant was expressed in COS7 and Chinese hamster ovary cells, did not form covalently linked dimers, and was present at the cell surface. There was half as much insulin binding activity at the cell surface in cells expressing the mutant compared with that in cells expressing the wild type receptor. The intracellular processing of the mutant receptor was affected, since its beta-subunit migrated more slowly than that of the wild type receptor on SDS-PAGE. The mutant was capable of insulin-dependent autophosphorylation and phosphorylation of insulin receptor substrate-1 in vivo and could be cross-linked into receptor dimers when membrane-bound. The amount of insulin-dependent autophosphorylation of the mutant receptor was half that of the wild type receptor. However, after solubilization the monomeric insulin receptor had minimal autophosphorylation activity, and, unlike the naturally occurring monomeric receptor tyrosine kinases, the solubilized monomeric insulin receptor did not dimerize in response to insulin binding as determined by sucrose density gradient centrifugation.     

Insulin receptor substrate 4 supports insulin- and interleukin 4-stimulated proliferation of hematopoietic cells.

Signaling from the activated insulin receptor is initiated by its tyrosine phosphorylation of the insulin receptor substrates (IRSs). The IRSs then act as docking/effector proteins for various signaling proteins containing src homology 2 domains. Four members of the IRS family, designated IRS-1 through IRS-4, have been identified. Although these IRSs show considerable structural homology, the extent to which they overlap in functions has not been explored in detail. The 32D hematopoietic cell line, which contains no detectable amounts of any IRS, provides a system in which to determine whether an IRS supports cell proliferation. Previous studies have shown that introduction of IRS-1 or -2 into 32D cells overexpressing the insulin and IL-4 receptors (32D-R cells) enables the cells to undergo mitogenesis in response to insulin and IL-4. In the present study, we have examined IRS-4, a member of the IRS family that we recently discovered, in this system. Expression of IRS-4 in 32D-R cells permitted the cells to undergo mitogenesis and continuous proliferation in response to insulin and IL-4. Immunoblotting of phosphotyrosine proteins showed that insulin and IL-4 elicited the tyrosine phosphorylation of IRS-4 in these cells. Thus, IRS-4, like IRS-1 and -2, can function in the signal transduction pathways linking insulin and IL-4 receptors to cell proliferation.     

Insulin receptor substrates 1 and 2 but not Shc can activate the insulin receptor independent of insulin and induce proliferation in CHO-IR cells

Ligand-activated insulin receptor (IR) attracts and phosphorylates various substrates such as insulin receptor substrates 1-4 (IRS) and Shc. To investigate how binding affinity for substrate affects signalling we generated chimeric receptors with the beta-chain of the insulin receptor containing NPXY motives with different affinities for receptor substrates. We found that the extent of receptor tyrosine phosphorylation positively correlates with binding affinity towards IRS1/2 but not towards Shc. Moreover, overexpression of IRS1 or IRS2 but not of Shc increased IR tyrosine phosphorylation in a dose-dependent manner, also independent of insulin. Molecular truncations of IRS1 revealed that neither the isolated PH and PTB domains nor the C-terminus with the tyrosine phosphorylation sites alone are sufficient for substrate-dependent receptor activation. Overexpression of IRS1 and IRS2 impaired insulin-induced internalization of the IR in a dose-dependent manner suggesting that IRS proteins prevent endosome-associated receptor dephosphorylation/inactivation. IRS1 and IRS2 could therefore target the activated IR to different cellular compartments. Overexpression of IRS1 and IRS2 inhibited insulin-stimulated activation of the MAP kinases Erk1/2 while it increased/induced activation of Akt/PKB. Finally, overexpression of IRS1 and IRS2 but not of Shc induced DNA synthesis in starved CHO-IR cells independent of exogenous growth factors. Our results demonstrate that variations in cellular IRS1 and IRS2 concentration affect insulin signalling both upstream and downstream and that IRS proteins could play instructive rather than just permissive roles in signal transmission.     

Insulin receptor substrate 2 and FoxO3a signalling are involved in E-cadherin expression and transforming growth factor-β1-induced repression in kidney epithelial cells

Insulin receptor substrate (IRS) proteins comprise a family of adaptor molecules that integrate extracellular signals from insulin and other ligands to intracellular effectors such as phosphoinositide 3-kinase and mitogen-activated protein kinase. The predominant forms of IRS protein in humans, IRS1 and IRS2, are widely expressed. Despite structural similarities, IRS1 and IRS2 display distinct signalling modalities, and mice lacking these proteins present with distinct phenotypes. Transforming growth factor (TGF)-β1 is the primary cytokine shown to induce epithelial-mesenchymal transition. Recent data have demonstrated a role for IRS1 in TGF-β1-induced epithelial-mesenchymal transition in lung epithelial cells. In the present study, we report data showing that TGF-β1 signals via IRS2 in kidney epithelial cells. Small interfering RNA (siRNA)-mediated targeting of IRS2 increased E-cadherin expression, although it did not alter TGF-β1-mediated E-cadherin repression. Phosphorylation of the downstream target of IRS2/Akt signalling, FoxO3a, was induced on Ser253 and, to a lesser extent, on Thr32. Transfection of FoxO3aThr32Ala mutant for 24?h greatly reduced FoxO3a phosphorylation on Ser253 but over-expression of FoxO3a Ser253Ala did not effect Thr32 phosphorylation, suggesting that a distinct order of phosphorylation of FoxO3a is required for physiological function in cells. Transfection of FoxO3a Ser253Ala mutant partially inhibited TGF-β1-mediated E-cadherin repression at 24?h. Taken together, these data highlight novel roles for IRS2 and FoxO3a in the regulation of kidney epithelial cells by E-cadherin.     

Biologic activities of naturally occurring human insulin receptor mutations. Evidence that metabolic effects of insulin can be mediated by a kinase-deficient insulin receptor mutant.

We have studied insulin receptor-mediated signaling in Chinese hamster ovary (CHO) cell transfectants that expressed either of two naturally occurring mutant human insulin receptors: Trp1200----Ser1200 and Ala1134----Thr1134. Compared with overexpressed normal human insulin receptors, both mutant receptors displayed normal processing and normal binding affinity; however, neither was capable of detectable insulin-stimulated autophosphorylation or tyrosine kinase activity toward endogenous (pp185) or exogenous substrates. Several biologic actions of insulin were evaluated in transfected cells. Compared with neomycin-only transfected CHO cells (CHO-NEO), cells expressing normal receptors demonstrated increased insulin sensitivity for 2-deoxyglucose uptake, [14C]glucose incorporation into glycogen, [3H]thymidine incorporation into DNA, and specific gene expression (accumulation of glucose transporter GLUT-1 mRNA). Cells expressing either Ser1200 or Thr1134 receptors showed no increase in insulin-stimulated thymidine incorporation or GLUT-1 mRNA accumulation compared with CHO-NEO. Surprisingly, cells expressing Ser1200 receptors showed increased insulin stimulation of 2-deoxyglucose uptake and glucose incorporation into glycogen compared with CHO-NEO, whereas Thr1134 receptors failed to signal these metabolic responses. We conclude that 1) transfected kinase-deficient insulin receptor mutants derived from insulin-resistant patients have distinct defects in the ability to mediate insulin action in vitro; 2) divergence of insulin signaling pathways may occur at the level of the receptor; and 3) normal activation of the receptor tyrosine kinase by insulin is not necessarily required for signaling of certain important biologic actions.     

Use of a disulfide cross-linking strategy to study muscarinic receptor structure and mechanisms of activation.

To gain insight into the molecular architecture of the cytoplasmic surface of G protein-coupled receptors, we have developed a disulfide cross-linking strategy using the m3 muscarinic receptor as a model system. To facilitate the interpretation of disulfide cross-linking data, we initially generated a mutant m3 muscarinic receptor (referred to as m3'(3C)-Xa) in which most native Cys residues had been deleted or substituted with Ala or Ser (remaining Cys residues Cys-140, Cys-220, and Cys-532) and in which the central portion of the third intracellular loop had been replaced with a factor Xa cleavage site. Radioligand binding and second messenger assays showed that the m3'(3C)-Xa mutant receptor was fully functional. In the next step, pairs of Cys residues were reintroduced into the m3'(3C)-Xa construct, thus generating 10 double Cys mutant receptors. All 10 mutant receptors contained a Cys residue at position 169 at the beginning of the second intracellular loop and a second Cys within the C-terminal portion of the third intracellular loop, at positions 484-493. Radioligand binding studies and phosphatidylinositol assays indicated that all double Cys mutant receptors were properly folded. Membrane lysates prepared from COS-7 cells transfected with the different mutant receptor constructs were incubated with factor Xa protease and the oxidizing agent Cu(II)-(1,10-phenanthroline)3, and the formation of intramolecular disulfide bonds between juxtaposed Cys residues was monitored by using a combined immunoprecipitation/immunoblotting strategy. To our surprise, efficient disulfide cross-linking was observed with 8 of the 10 double Cys mutant receptors studied (Cys-169/Cys-484 to Cys-491), suggesting that the intracellular m3 receptor surface is characterized by pronounced backbone fluctuations. Moreover, [35S]guanosine 5'-3-O-(thio)triphosphate binding assays indicated that the formation of intramolecular disulfide cross-links prevented or strongly inhibited receptor-mediated G protein activation, suggesting that the highly dynamic character of the cytoplasmic receptor surface is a prerequisite for efficient receptor-G protein interactions. This is the first study using a disulfide mapping strategy to examine the three-dimensional structure of a hormone-activated G protein-coupled receptor.     

Substitution of isoleucine for methionine at position 1153 in the beta-subunit of the human insulin receptor. A mutation that impairs receptor tyrosine kinase activity, receptor endocytosis, and insulin action.

The intracellular domain of the insulin receptor possesses activity as a tyrosine-specific protein kinase. The receptor tyrosine kinase is stimulated by insulin binding to the extracellular domain of the receptor. Previously, we have identified a patient with a genetic form of insulin resistance who is heterozygous for a mutation substituting Ile for Met1153 in the tyrosine kinase domain of the receptor near the cluster of the three major autophosphorylation sites (Tyr1158, Tyr1162, and Tyr1163). In this investigation, the Ile1153 mutant receptor was expressed by transfection of mutant cDNA into NIH-3T3 cells. The mutation impairs receptor tyrosine kinase activity and also inhibits the ability of insulin to stimulate 2-deoxyglucose uptake and thymidine incorporation. These data support the hypothesis that the receptor tyrosine activity plays a necessary role in the ability of the receptor to mediate insulin action in vivo. Furthermore, expression of the Ile1153 mutant receptor exerted a dominant negative effect to inhibit the ability of endogenous murine receptors for insulin and insulin-like growth factor I to mediate their actions upon the cell. This observation is consistent with previous suggestions that mutant receptors dimerize with wild type receptors, thereby creating hybrid molecules which lack biological activity. The dominant negative effect of the mutant receptor may explain the dominant mode of inheritance of insulin resistance caused by the Ile1153 mutation. Finally, the mutation inhibits the ability of insulin to stimulate receptor endocytosis. This may explain the normal number of insulin receptors on the surface of the patient's cells in vivo. Despite the presence of markedly elevated levels of insulin in the patient's plasma, the receptors were resistant to down-regulation.     

Postbinding characterization of five naturally occurring mutations in the human insulin receptor gene: impaired insulin-stimulated c-jun expression and thymidine incorporation despite normal receptor autophosphorylation.

Some patients with extreme insulin resistance have mutations in their insulin receptor gene. We previously identified five such mutations located in the extracellular domain of the insulin receptor (Asn-->Lys15, His-->Arg209, Phe-->Val382, Lys-->Glu460, and Asn-->Ser462) and studied the effects of these mutations upon posttranslational processing, insulin binding, and tyrosine autophosphorylation. We now characterize the ability of these mutant receptors to mediate biological actions of insulin in transfected NIH-3T3 fibroblasts. All cell lines expressing mutant receptors showed marked impairment in insulin-stimulated c-jun expression and thymidine incorporation when compared with cells expressing wild-type human insulin receptors. The most severe impairment was seen in cells expressing the Val382 mutant (a mutation which causes an intrinsic defect in receptor autophosphorylation). These cells had insulin responses similar to the untransfected cells (used as a negative control). In contrast, cells expressing the Lys15 mutant have the ability to achieve a normal level of maximal autophosphorylation but require an abnormally high concentration of insulin to do so (as the result of decreased insulin binding affinity). These cells show a higher basal rate and much lower insulin stimulation of both c-jun expression and thymidine incorporation when compared with the cells expressing the wild-type human insulin receptors. This pattern is also seen in the cells expressing the other mutants with normal autophosphorylation (Arg209, Glu460, and Ser462). Although the most severe defects in insulin action are seen with the mutation which has an intrinsic defect in receptor autophosphorylation, the ability to undergo normal autophosphorylation does not seem to preclude mutations from impairing the ability of receptors to mediate some of the actions of insulin.     

Disruption of the SH2-B gene causes age-dependent insulin resistance and glucose intolerance

Insulin regulates glucose homeostasis by binding and activating the insulin receptor, and defects in insulin responses (insulin resistance) induce type 2 diabetes. SH2-B, an Src homology 2 (SH2) and pleckstrin homology domain-containing adaptor protein, binds via its SH2 domain to insulin receptor in response to insulin; however, its physiological role remains unclear. Here we show that SH2-B was expressed in the liver, skeletal muscle, and fat. Systemic deletion of SH2-B impaired insulin receptor activation and signaling in the liver, skeletal muscle, and fat, including tyrosine phosphorylation of insulin receptor substrate 1 (IRS1) and IRS2 and activation of the phosphatidylinositol 3-kinase/Akt and the Erk1/2 pathways. Consequently, SH2-B-/- knockout mice developed age-dependent hyperinsulinemia, hyperglycemia, and glucose intolerance. Moreover, SH2-B directly enhanced autophosphorylation of insulin receptor and tyrosine phosphorylation of IRS1 and IRS2 in an SH2 domain-dependent manner in cultured cells. Our data suggest that SH2-B is a physiological enhancer of insulin receptor activation and is required for maintaining normal insulin sensitivity and glucose homeostasis during aging.     

Cysteine 981 of the human insulin receptor is required for covalent cross-linking between beta-subunit and a thiol-reactive membrane-associated protein

The cytoplasmic domain of the insulin receptor (IR) beta-subunit contains cysteine (Cys) residues whose reactivity and function remain uncertain. In this study, we examined the ability of the bifunctional cross-linking reagent 1,6-bismaleimidohexane (BMH) to covalently link IR with interacting proteins that possess reactive thiols. Transfected Chinese hamster ovary cells expressing either the wild-type human IR, C-terminally truncated receptors, or mutant receptors with Cys --> Ala substitutions and mouse 3T3-L1 adipocytes were used to compare the BMH effect. The results showed the formation of a large complex between the wild-type human receptor beta-subunit and molecule X, a thiol-reactive membrane-associated protein, in both intact and semipermeabilized cells in response to BMH. Prior cell stimulation with insulin had only a modest effect in this process. Western blot analysis revealed that the receptor alpha-subunit was not present in the beta-X complex. The BMH cross-linking did not inhibit in vitro tyrosine phosphorylation of the receptor complexed with molecule X. Both the human IR Cys981Ala mutant and murine IR, that lacks the equivalent of human Cys(981), failed to react with BMH. Finally, no covalent association between IR beta-subunit and IRS-1, the protein tyrosine phosphatase LAR or SHP-2 was observed in BMH-treated cells expressing the wild-type human IR. These results demonstrate a striking difference in reactivity among the cytoplasmic IR beta-subunit thiols and clearly show that Cys(981) of human IR beta-subunit is in close proximity to a thiol-reactive membrane-associated protein under basal and insulin-stimulated conditions.     

Characterization of Drosophila insulin receptor substrate

Insulin receptor substrate (IRS) proteins are phosphorylated by multiple tyrosine kinases, including the insulin receptor. Phosphorylated IRS proteins bind to SH2 domain-containing proteins, thereby triggering downstream signaling pathways. The Drosophila insulin receptor (dIR) C-terminal extension contains potential binding sites for signaling molecules, suggesting that dIR might not require an IRS protein to accomplish its signaling functions. However, we obtained a cDNA encoding Drosophila IRS (dIRS), and we demonstrated expression of dIRS in a Drosophila cell line. Like mammalian IRS proteins, the N-terminal portion of dIRS contains a pleckstrin homology domain and a phosphotyrosine binding domain that binds to phosphotyrosine residues in both human and Drosophila insulin receptors. When coexpressed with dIRS in COS-7 cells, a chimeric receptor (the extracellular domain of human IR fused to the cytoplasmic domain of dIR) mediated insulin-stimulated tyrosine phosphorylation of dIRS. Mutating the juxtamembrane NPXY motif markedly reduced the ability of the receptor to phosphorylate dIRS. In contrast, the NPXY motifs in the C-terminal extension of dIR were required for stable association with dIRS. Coimmunoprecipitation experiments demonstrated insulin-dependent binding of dIRS to phosphatidylinositol 3-kinase and SHP2. However, we did not detect interactions with Grb2, SHC, or phospholipase C-gamma. Taken together with published genetic studies, these biochemical data support the hypothesis that dIRS functions directly downstream from the insulin receptor in Drosophila.     

Binding of mutant insulins to a mutated insulin receptor.

We studied the binding of mutant insulins to both the normal human insulin receptor and an insulin receptor in which the sequence 240-250 of the receptor alpha subunit was mutated to provide an additional net positive charge. One mutant insulin (AspB10), which has an additional negative charge, bound to both types of receptors with a higher affinity than native insulin. Moreover, this mutant insulin was more effective in activating the tyrosine kinase activity of both types of receptors. This study suggests, therefore, that charge interactions between insulin and its receptor may play a role in insulin receptor binding and action.     

Functional properties of a naturally occurring Trp1200----Ser1200 mutation of the insulin receptor

Based on the sequence of cDNA encoding the intracellular domain of the insulin receptor beta-subunit, we recently defined a heterozygous point mutation causing a Ser for Trp substitution at position 1200 in the tyrosine kinase domain of a patient (BI-2) with the type A syndrome of insulin resistance. We have now sequenced the remainder of BI-2's insulin receptor cDNA-coding region and find no additional alterations in the encoded proreceptor protein. The nucleotide sequence of cDNA encoding the portion of the beta-subunit which includes Trp1200 was normal in BI-2's unaffected mother. Hybridization of a mutant allele-specific oligonucleotide to polymerase chain reaction-amplified cDNA confirmed the presence of the mutant allele in the proband and excluded it in her unaffected sister and mother, 18 normal control subjects, and six other subjects with insulin resistance. To determine whether this mutation had functional consequences for receptor signalling, we reconstructed it into a full-length insulin receptor cDNA expression vector. Chinese hamster ovary cells were transfected with mutant cDNA, and the expressed insulin receptors were compared to receptors expressed by cells transfected with wild-type receptor cDNA. Both mutant and wild-type receptors were properly processed into receptor alpha- and beta-subunits, were expressed on the cell surface, and displayed similar insulin-binding affinity. In contrast, insulin-stimulated autophosphorylation of the mutant receptors was severely impaired, whether assessed in intact cells or with a partially purified receptor preparation.(ABSTRACT TRUNCATED AT 250 WORDS)