Activation of insulin receptor signaling by a single amino acid substitution in the transmembrane domain.

The insulin receptor is a ligand-activated tyrosine kinase composed of two alpha and two beta subunits. A single transmembrane domain composed of 23 hydrophobic residues is contained in each beta subunit. We examined the role of the transmembrane domain in regulating insulin receptor signaling by inserting a negatively charged amino acid (Asp) for Val938 (V938D). Chinese hamster ovary (CHO) cells were stably transfected with a plasmid containing both the neomycin-resistance gene and either the wild-type or the mutant (V938D) insulin receptor cDNA. Insulin binding increased similarly in CHO cells stably transfected with the wild-type and the V938D-mutant insulin receptor cDNA. Insulin stimulated glucose transport and cell growth in cells expressing the normal insulin receptor. By contrast, in the absence of insulin, glucose transport and cell growth in CHO-V938D cells were as high as in insulin-stimulated control cells and no longer responsive to insulin stimulation. Phosphorylation of the beta subunit of the insulin receptor was also increased in CHO-V938D cells not exposed to insulin. These results support an essential role of the transmembrane domain of the insulin receptor in the transduction of insulin signaling.     

Evidence supporting a passive role for the insulin receptor transmembrane domain in insulin-dependent signal transduction

We previously have demonstrated that intramolecular interactions between alpha beta-alpha beta subunits are necessary for insulin-dependent activation of the protein kinase domain within a single alpha 2 beta 2 heterotetrameric insulin-receptor complex (Wilden, P. A., Morrison, B. D., and Pessin, J. E. (1989) Biochemistry 28, 785-792). To evaluate the role of the beta subunit transmembrane domain in the insulin-dependent signalling mechanism, mutant human insulin receptors containing a series of nested transmembrane domain deletions (amino acids 941-945) were generated and stable Chinese hamster ovary-transfected cell lines were obtained. In addition, a substitution of Val-938 for Glu (E/V938) similar to the oncogenic mutation found in the neu transmembrane domain was also introduced into the insulin receptor. Scatchard analysis of insulin binding to the stable Chinese hamster ovary cell lines expressing either wild type or mutant insulin receptors indicated equivalent receptor number (2-4 x 10(6)/cell) and similar high affinity binding constants (Kd 0.1-0.3 nM). 125I-Insulin affinity cross-linking demonstrated that all of the expressed insulin receptors were assembled and processed into alpha 2 beta 2 heterotetrameric complexes. Surprisingly, all the mutant insulin receptors retained insulin-stimulated autophosphorylation both in vivo and in vitro. Furthermore, endogenous substrate phosphorylation in vivo as well as insulin-stimulated thymidine incorporation into DNA were unaffected by the transmembrane domain mutations. These data demonstrate that marked structural alterations in the insulin receptor transmembrane domain do not interfere with insulin-dependent signal transduction.     

Insulin receptor kinase domain autophosphorylation regulates receptor enzymatic function.

We have studied a series of insulin receptor molecules in which the 3 tyrosine residues which undergo autophosphorylation in the kinase domain of the beta-subunit (Tyr1158, Tyr1162, and Tyr1163) were replaced individually, in pairs, or all together with phenylalanine or serine by in vitro mutagenesis. A single-Phe replacement at each of these three positions reduced insulin-stimulated autophosphorylation of solubilized receptor by 45-60% of that observed with wild-type receptor. The double-Phe replacements showed a 60-70% reduction, and substitution of all 3 tyrosine residues with Phe or Ser reduced insulin-stimulated tyrosine autophosphorylation by greater than 80%. Phosphopeptide mapping each mutant revealed that all remaining tyrosine autophosphorylation sites were phosphorylated normally following insulin stimulation, and no new sites appeared. The single-Phe mutants showed insulin-stimulated kinase activity toward a synthetic peptide substrate of 50-75% when compared with wild-type receptor kinase activity. Insulin-stimulated kinase activity was further reduced in the double-Phe mutants and barely detectable in the triple-Phe mutants. In contrast to the wild-type receptor, all of the mutant receptor kinases showed a significant reduction in activation following in vitro insulin-stimulated autophosphorylation. When studied in intact Chinese hamster ovary cells, insulin-stimulated receptor autophosphorylation and tyrosine phosphorylation of the cellular substrate pp185 in the single-Phe and double-Phe mutants was progressively lower with increased tyrosine replacement and did not exceed the basal levels in the triple-Phe mutants. However, all the mutant receptors, including the triple-Phe mutant, retained the ability to undergo insulin-stimulated Ser and Thr phosphorylation. Thus, full activation of the insulin receptor tyrosine kinase is dependent on insulin-stimulated Tris phosphorylation of the kinase domain, and the level of autophosphorylation in the kinase domain provides a mechanism for modulating insulin receptor kinase activity following insulin stimulation. By contrast, insulin stimulation of receptor phosphorylation on Ser and Thr residues by cellular serine/threonine kinases can occur despite markedly reduced tyrosine autophosphorylation.     

The role of insulin receptor kinase domain autophosphorylation in receptor-mediated activities. Analysis with insulin and anti-receptor antibodies.

The role of specific tyrosine autophosphorylation sites in the human insulin receptor kinase domain (Tyr1158, Tyr1162, and Tyr1163) was analyzed using in vitro mutagenesis to replace tyrosine residues individually or in combination. Each of the three single-Phe, the three possible double-Phe a triple-Phe and a triple-Ser mutant receptors, stably expressed in Chinese hamster ovary cells, were compared with the wild-type receptor in their ability to mediate stimulation of receptor kinase activity, glycogen synthesis, and DNA synthesis by insulin or the human-specific anti-receptor monoclonal antibody 83-14. At a concentration of 0.1 nM insulin which produced approximately half-maximal responses with wild-type receptor, DNA synthesis and glycogen synthesis mediated by the three single-Phe mutants ranged from 52 to 88% and from 32 to 79% of the wild-type receptor, respectively. The corresponding figures for the double-Phe mutants averaged 15 and 6%, whereas the triple-mutants were unresponsive in both assays. The level of biological function approximately paralleled the insulin-stimulated tyrosine kinase activity in the intact cell as estimated by tyrosine phosphorylation of the insulin receptor and its endogenous substrate pp 185/IRS-1. Interestingly, all mutants showed a marked decrease in insulin-stimulated receptor internalization. Anti-receptor antibody stimulated receptor kinase activity and mimicked insulin action in these cells. In general, the impairment of the metabolic response was greater and impairment of the growth response was less when antibody was the stimulus. These experiments show that the level and specific sites of autophosphorylation are critical determinants of receptor function. The data are consistent with a requirement for the receptor tyrosine kinase either as an obligatory step or a modulator, in both metabolic and growth responses, and demonstrate the important role of the level of insulin receptor kinase domain autophosphorylation in regulating insulin sensitivity.     

The insulin receptor functions normally in Chinese hamster ovary cells after truncation of the C terminus.

We studied the structure and function of the human insulin receptor (IR) and a mutant which lacked the last 43 amino acids of the beta-subunit (IR delta ct). This deletion removed tyrosine (Tyr1322, Tyr1316) and threonine (Thr1336) phosphorylation sites. In Chinese hamster ovary (CHO) cells, insulin binding to the mutant receptor was normal, and [35S]methionine labeling indicated that both the IR and IR delta ct were processed normally; however, the beta-subunit of IR delta ct was 5 kDa smaller than that of the IR. The time course of insulin-stimulated autophosphorylation of the partially purified IR delta ct was normal, but the maximum autophosphorylation was reduced 20-30%. Tryptic phosphopeptide mapping confirmed the absence of the C-terminal phosphorylation sites and indicated that phosphorylation of the regulatory region (Tyr1146, Tyr1150, Tyr1151) occurred normally; kinase activity of the IR and IR delta ct was activated normally by insulin-stimulated autophosphorylation. In the intact CHO cells, insulin-stimulated serine and threonine phosphorylation of the IR delta ct was reduced 20%, suggesting that most Ser/Thr phosphorylation sites are located outside of the C terminus. During insulin stimulation, the wild-type and mutant insulin receptor activated the phosphatidylinositol 3-kinase. Moreover, insulin itself or human-specific anti-insulin receptor antibodies stimulated glycogen and DNA synthesis equally in both CHO/IR and CHO/IR delta ct cells. These data suggest that the C terminus plays a minimal role in IR function and signal transmission in CHO cells.     

Effect of phosphotyrosyl-IRS-1 level and insulin receptor tyrosine kinase activity on insulin-stimulated phosphatidylinositol 3, MAP,and S6 kinase activities

The role of tyrosine phosphorylation of the insulin receptor substrate 1 (IRS-1) was studied utilizing parental CHO cells or CHO cells that overexpress IRS-1, the insulin receptor, or both IRS-1 and the insulin receptor. Insulin stimulation of these four cell lines led to progressive levels of IRS-1 tyrosine phosphorylation of one, two, four, and tenfold. Maximal insulin-stimulated IRS-1 associated Ptdlns 3′-kinase activit in these cells was 1-, 1.5-, 3-, and 3-fold, while insulin sensitivity, as determined by ED₅₀, was 1-, 2.5-, 10-, and 10-fold. Both sensitivity and maximal response paralleled the increased level of phosphotyrosyl-IRS-1; however, the increased level of phosphotyrosyl-IRS-1 seen in CHO/IR/IRS-1 cells did not further increase these responses. Likewise, maximal insulin-stimulated MAP kinase activity in these cell lines increased in parallel with IRS-1 tyrosine phosphorylation except in the CHO/IR/IRS-1 cell lines with activity levels of one-, five-, nine-, and ninefold. However, insulin sensitivity of the MAP and S6 kinases and maximal insulin-stimulated S6 kinase activity was not changed by a twofold increase in phosphotyrosyl-IRS-1, but an increase was observed with insulin-stimulated receptor autophosphorylation and kinase activity in CHO/IR cells which led to a tenfold increase in insulin receptor autophosphorylation and a fourfold increase in IRS-1 tyrosine phosphorylation. Thus, these three kinase activities may be differentially coupled to the activation of the insulin receptor kinase activity via IRS-1 and other possible cellular substrates. © 1995 Wiley-Liss, Inc.     

Substitution of the insulin receptor transmembrane domain with that of glycophorin A inhibits insulin action.

To study the role of transmembrane (TM) domains interactions in the activation of the insulin receptor, we have replaced the insulin receptor TM domain with that of glycophorin A (GpA), an erythrocyte protein that spontaneously forms detergent-resistant dimers through TM-TM interactions. Insulin receptor cDNA sequences with the TM domain replaced by that of GpA were constructed and stably transfected in CHO cells. Insulin binding to cells and solubilized receptors was not modified. Electrophoresis after partial reduction of disulfide bonds revealed an altered structure for the soluble chimeric receptors, seen as an altered mobility apparently due to increased interactions between the beta subunits of the receptor. Insulin signaling was markedly decreased for cells transfected with chimeric receptors compared with cells transfected with normal receptors. A decrease in insulin-induced receptor kinase activity was observed for solubilized chimeric receptors. In conclusion, substitution by the native GpA TM domain of the insulin receptor results in structurally modified chimeric receptors that are unable to transmit the insulin signal properly. It is hypothesized that this substitution may impose structural constraints that prevent the proper changes in conformation necessary for activation of the receptor kinase. Other mutants modifying the structure or the membrane orientation of the glycophorin A TM domain are required to better understand these constraints.     

The tyrosine kinase activity of the chicken insulin receptor is similar to that of the human insulin receptor

The tyrosine kinase activity of a chimeric insulin receptor composed of the extracellular domain of the human insulin receptor (IR) and the intracellular domain of the chicken IR was compared with wild-type human IR. The degrees of autophosphorylation, phosphorylation of IRS-1, and in vitro phosphorylation of an exogenous substrate after stimulation by human insulin were similar to that seen with the human IR. We conclude that the insulin resistance of chickens is not attributable to a lower level of intrinsic tyrosine kinase activity of IR.     

Genistein differentially inhibits postreceptor effects of insulin in rat adipocytes without inhibiting the insulin receptor kinase.

Genistein, an isoflavone putative tyrosine kinase inhibitor, was used to investigate the coupling of insulin receptor tyrosine kinase activation to four metabolic effects of insulin in the isolated rat adipocyte. Genistein inhibited insulin-stimulated glucose oxidation in a concentration-dependent manner with an ID50 of 25 micrograms/ml and complete inhibition at 100 micrograms/ml. Genistein also prevented insulin's (10(-9) M) inhibition of isoproterenol-stimulated lipolysis with an ID50 of 15 micrograms/ml and a complete effect at 50 micrograms/ml. The effect of genistein (25 micrograms/ml) was not reversed by supraphysiological (10(-7) M) insulin levels. In contrast, genistein up to 100 micrograms/ml had no effect on insulin's (10(-9) M) stimulation of either pyruvate dehydrogenase or glycogen synthase activity. We determined whether genistein influenced insulin receptor beta-subunit autophosphorylation or tyrosine kinase substrate phosphorylation either in vivo or in vitro by anti-phosphotyrosine immunoblotting. Genistein at 100 micrograms/ml did not inhibit insulin's (10(-7) M) stimulation of insulin receptor tyrosine autophosphorylation or tyrosine phosphorylation of the cellular substrates pp185 and pp60. Also, genistein did not prevent insulin-stimulated autophosphorylation of partially purified human insulin receptors from NIH 3T3/HIR 3.5 cells or the phosphorylation of histones by the activated receptor tyrosine kinase. In control experiments using either NIH 3T3 fibroblasts or partially purified membranes from these cells, genistein did inhibit platelet-derived growth factor's stimulation of its receptor autophosphorylation. These findings indicate the following: (a) Genistein can inhibit certain responses to insulin without blocking insulin's stimulation of its receptor tyrosine autophosphorylation or of the receptor kinase substrate tyrosine phosphorylation. (b) In adipocytes genistein must block the stimulation of glucose oxidation and the antilipolytic effects of insulin at site(s) downstream from the insulin receptor tyrosine kinase. (c) The inhibitory effects of genistein on hormonal signal transduction cannot necessarily be attributed to inhibition of tyrosine kinase activity, unless specifically demonstrated.     

In vitro phosphorylation of the adipocyte lipid-binding protein (p15) by the insulin receptor. Effects of fatty acid on receptor kinase and substrate phosphorylation.

Phosphorylation of the adipocyte lipid-binding protein (ALBP) isolated from 3T3-L1 cells has been studied in vitro utilizing the wheat germ agglutinin-purified 3T3-L1 adipocyte insulin receptor and the soluble kinase domain of the human insulin receptor. Following insulin-stimulated, ATP-dependent autophosphorylation of the wheat germ agglutinin-purified receptor beta-subunit, ALBP was phosphorylated exclusively on tyrosine 19 in the sequence Glu-Asn-Phe-Asp-Asp-Tyr19, analogous to the substrate phosphorylation consensus sequence observed for several tyrosyl kinases. The concentration of insulin necessary for half-maximal receptor autophosphorylation (KIR0.5) was identical to that necessary for half-maximal ALBP phosphorylation (KALBP0.5), 10 nM. Kinetic analysis indicated that stimulation of ALBP phosphorylation by insulin was attributable to a 5-fold increase in the Vmax (to 0.33 fmol/min/fmol insulin-binding sites) while the Km for ALBP was largely unaffected. By utilizing the soluble kinase domain of the human receptor beta-subunit, the presence of oleate bound to ALBP increased the kcat/Km greater than 3-fold. Oleate dramatically inhibited autophosphorylation of the 38-kDa fragment of the soluble receptor kinase in a concentration dependent fashion (I0.5 approximately 4 microM). The 48-kDa kinase exhibited much less sensitivity to the effects of oleate (I0.5 approximately 190 microM). The inhibition of autophosphorylation of the 48-kDa soluble kinase by oleate was reversed by adding saturating levels of ALBP. These results demonstrate that in vitro the murine adipocyte lipid-binding protein is phosphorylated on tyrosine 19 in an insulin-stimulated fashion by the insulin receptor and that the presence of a bound fatty acid on ALBP increases the affinity of insulin receptor for ALBP. Inhibition of insulin receptor kinase activity by unbound fatty acids suggests that the end products of the lipogenic pathway may feedback inhibit the tyrosyl kinase and that fatty acid-binding proteins have the potential to modulate such interaction.     

Pertussis toxin inhibits autophosphorylation and activation of the insulin receptor kinase.

Pertussis toxin is an ADP-ribosyltransferase which alters the function of some of the GTP-binding proteins and inhibits some actions of insulin. In vivo, pertussis toxin (2 micrograms/ml/2h) inhibited insulin-stimulated tyrosyl autophosphorylation of the insulin receptor by 50% in FaO cells, and nearly completely inhibited phosphorylation of the cellular insulin receptor substrate pp185. Similarly, insulin-stimulated autophosphorylation and kinase activity of the insulin receptor purified on wheat germ agglutinin-agarose from pertussis toxin-treated FaO cells was diminished 50%; however, treatment of cells with the catalytically inactive B-oligomer of the toxin had no effect on receptor tyrosine kinase activity in vitro. Pertussis toxin did not alter insulin binding or the cellular levels of ATP, cAMP, and cGMP. Furthermore, immunoprecipitation of the insulin receptor from intact cells with anti-insulin receptor antibodies showed that pertussis toxin did not increase the phosphorylation of serine or threonine residues in the insulin receptor. These results suggest that pertussis toxin can modulate signal transduction of insulin at the level of the insulin receptor kinase.     

Regulation of insulin receptor function by a small molecule insulin receptor activator

In type 2 diabetes mellitus, impaired insulin signaling leads to hyperglycemia and other metabolic abnormalities. TLK19780, a non-peptide small molecule, is a new member of a novel class of anti-diabetic agents that function as activators of the insulin receptor (IR) beta-subunit tyrosine kinase. In HTC-IR cells, 20 microm TLK19780 enhanced maximal insulin-stimulated IR autophosphorylation 2-fold and increased insulin sensitivity 2-3-fold. In contrast, TLK19780 did not potentiate the action of insulin-like growth factor-1, indicating the selectivity of TLK19780 toward the IR. The predominant effect of TLK19780 was to increase the number of IR that underwent autophosphorylation. Kinetic studies indicated that TLK19780 acted very rapidly, with a maximal effect observed 2 min after addition to insulin-stimulated cells. In 3T3-L1 adipocytes, 5 microm TLK19780 enhanced insulin-stimulated glucose transport, increasing both the sensitivity and maximal responsiveness to insulin. These studies indicate that at low micromolar levels small IR activator molecules can enhance insulin action in various cultured cells and suggest that this effect is mediated by increasing the number of IR that are tyrosine-phosphorylated in response to insulin. These studies suggest that these types of molecules could be developed to treat type 2 diabetes and other clinical conditions associated with insulin resistance.     

An extracellular domain of the insulin receptor beta-subunit with regulatory function on protein-tyrosine kinase

Anti-insulin receptor monoclonal antibody MA-10 inhibits insulin receptor autophosphorylation of purified rat liver insulin receptors without affecting insulin binding (Cordera, R., Andraghetti, G., Gherzi, R., Adezati, L., Montemurro, A., Lauro, R., Goldfine, I. D., and De Pirro, R. (1987) Endocrinology 121, 2007-2010). The effect of MA-10 on insulin receptor autophosphorylation and on two insulin actions (thymidine incorporation into DNA and receptor down-regulation) was investigated in rat hepatoma Fao cells. MA-10 inhibits insulin-stimulated receptor autophosphorylation, thymidine incorporation into DNA, and insulin-induced receptor down-regulation without affecting insulin receptor binding. We show that MA-10 binds to a site of rat insulin receptors different from the insulin binding site in intact Fao cells. Insulin does not inhibit MA-10 binding, and MA-10 does not inhibit insulin binding to rat Fao cells. Moreover, MA-10 binding to down-regulated cells is reduced to the same extent as insulin binding. In rat insulin receptors the MA-10 binding site has been tentatively localized in the extracellular part of the insulin receptor beta-subunit based on the following evidence: (i) MA-10 binds to insulin receptor in intact rat cells; (ii) MA-10 immunoprecipitates isolated insulin receptor beta-subunits labeled with both [35S]methionine and 32P; (iii) MA-10 reacts with rat insulin receptor beta-subunits by the method of immunoblotting, similar to an antipeptide antibody directed against the carboxyl terminus of the insulin receptor beta-subunit. Moreover, MA-10 inhibits autophosphorylation and protein-tyrosine kinase activity of reduced and purified insulin receptor beta-subunits. The finding that MA-10 inhibits insulin-stimulated receptor autophosphorylation and reduces insulin-stimulated thymidine incorporation into DNA and receptor down-regulation suggests that the extracellular part of the insulin receptor beta-subunit plays a role in the regulation of insulin receptor protein-tyrosine kinase activity.     

Identification of an autoinhibitory domain in the insulin receptor tyrosine kinase

We have tested the hypothesis that activation of the insulin receptor tyrosine kinase is due to autophosphorylation of tyrosines 1146, 1150 and 1151 within a putative autoinhibitory domain. A synthetic peptide corresponding to residues 1134–1162, with tyrosines substituted by alanine or phenylalanine, of the insulin receptor subunit was tested for its inhibitory potency and specificity towards the tyrosine kinase activity. This synthetic peptide gave inhibition of the insulin receptor tyrosine kinase autophosphorylation and phosphorylation of the exogenous substrate poly(Glu, Tyr) with an approximate IC₅₀ of 100 M. Inhibition appeared to be independent of the concentrations of insulin or the substrate poly(Glu, Tyr) but was decreased by increasing concentrations of ATP. This same peptide also inhibited the EGF receptor tyrosine kinase but not a serine/threonine protein kinase. These results are consistent with the hypothesis that this autophosphorylation domain contains an autoinhibitory sequence. (Mol Cell Biochem120: 103–110, 1993)Abbreviations IR Insulin Receptor - SDS/PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis - CaM Calmodulin - HEPES 4-(2-Hydroxyethyl)-Piperazineethane-Sulfonic Acid - DMEM Dulbecco's Modified Eagle' Medium - PMSF Phenylmethyl-Sulfonyl Fluoride - HPLC High Performance Liquid Chromatography - PKC Protein Kinase C - PKI Inhibitory Peptide for cAMP-Kinase - CaMK II Ca²⁺/Calmodulin-Dependent Protein Kinase II - CaN A A Subunit of Calcineurin     

Internalization and activation of the rat liver insulin receptor kinase in vivo

The preparation of clearly delineated plasmalemma (PM) and endosomal subcellular fractions from rat liver has allowed us to compare insulin receptor (IR) kinase activity at the cell surface and in hepatic endosomes (ENs) as a function of dose and time after injected insulin. Tyrosine kinase activity in PM and ENs was measured, after solubilization and partial purification by wheat germ agglutinin chromatography (lectin-purified), using poly(Glu:Tyr) as substrate. Following the injection of a subsaturating dose of insulin (1.5 micrograms/100 g body weight), lectin-purified receptor showed peak activation at 30 s in PM and at 2 min in ENs. As observed previously (Khan, M. N., Savoie, S., Bergeron, J. J. M., and Posner, B. I. (1986) J. Biol. Chem. 261, 8462-8472) autophosphorylation activity was also augmented following insulin injection. In a pattern virtually identical to that of exogenous kinase activity, autophosphorylation attained peak activity at 30 s in PM and at 2 min in ENs. The time course of IR autophosphorylation in intact membranes was very similar to that observed for lectin purified receptors and was seen with an injected insulin dose as low as 150 ng/100 g body weight. Phosphatase treatment of the solubilized endosomal receptor abolished its enhanced activity. Hence, insulin treatment led to in vivo receptor phosphorylation which was reflected in the enhancement of both tyrosine kinase and autophosphorylation activities. Significant differences in the phosphorylation activities of PM and ENs were observed. Phosphoamino acid analyses revealed that the activated IR of intact PM was autophosphorylated in vitro, at both serine (55%) and tyrosine (45%) residues; whereas the activated IR of intact ENs was phosphorylated in vitro exclusively on tyrosine autophosphorylation specific activity for the activated IR of ENs was 3- to 4-fold that of the IR of PM. This was observed for the lectin purified IRs as well as for IRs of intact cell fractions. The reduced level of IR autophosphorylation in PM was not due to occlusion of tyrosine acceptor sites by prior in vivo phosphorylation. The rapidity with which activated IR accumulates in ENs as well as the sensitivity of endosomal IR kinase to activation by injected insulin are consistent with the endosomal apparatus serving a physiologically significant site for the regulation of transmembrane signaling.     

Inhibition of the insulin receptor tyrosine kinase by sphingosine.

Sphingosine inhibits autophosphorylation of the insulin receptor tyrosine kinase in vitro and in situ. This lysosphingolipid has been shown previously to inhibit the Ca2+/lipid-dependent protein kinase C. Here we show that insulin-dependent autophosphorylation of partially purified insulin receptor is half-maximally inhibited by 145 microM sphingosine (9 mol %) in Triton X-100 micelles. Half-maximal inhibition of protein kinase C autophosphorylation occurs with 60 microM sphingosine (3.4 mol %) in Triton X-100 mixed micelles containing phosphatidylserine and diacylglycerol. Sphingomyelin does not inhibit significantly the insulin receptor, suggesting that, as with protein kinase C, the free amino group may be essential for inhibition. Similar to the effects observed for protein kinase C, inhibition of the insulin receptor kinase by sphingosine is reduced in the presence of other lipids. However, the reduction displays a marked dependence on the lipid species: phosphatidylserine, but not a mixture of lipids compositionally similar to the cell membrane, markedly reduces the potency of sphingosine inhibition. The inhibition occurs at the level of the protein/membrane interaction: a soluble form of the insulin receptor comprising the cytoplasmic kinase domain is resistant to sphingosine inhibition. Lastly, sphingosine inhibits the insulin-stimulated rate of tyrosine phosphorylation of the insulin receptor in NIH 3T3 cells expressing the human insulin receptor. These results suggest that sphingosine alters membrane function independently of protein kinase C.     

Bidirectional regulation of insulin receptor autophosphorylation and kinase activity by peroxynitrite

Accumulating evidence suggests that enhanced peroxynitrite formation occurs during diabetes. This report describes the effect of peroxynitrite on insulin receptor (IR) function. Addition of peroxynitrite to purified IR resulted in concentration-dependent tyrosine nitration and thiol oxidation. Interestingly, the basal and insulin-stimulated IR autophosphorylation and tyrosine kinase activity were upregulated at low peroxynitrite concentrations, but downregulated at high peroxynitrite concentrations. Concomitantly, peroxynitrite dramatically reduced ¹²⁵I-insulin binding capacity and phosphotyrosine phosphatase activity of IR preparations. Moreover, SIN-1 administration decreased blood glucose levels in normal mice via upregulation of IR/IRS-1 tyrosine phosphorylation. In contrast, SIN-1 markedly increased blood glucose levels in diabetic mice concomitant with downregulation of IR/IRS-1 tyrosine phosphorylation. Taken together, these data provide new insights regarding how peroxynitrite influences IR function in vitro and in vivo, suggesting that peroxynitrite plays a dual role in regulation of IR autophosphorylation and tyrosine kinase activity, and SIN-1 has hyperglycemic effect in diabetic mice.     

Mechanism of transmembrane signaling: insulin binding and the insulin receptor

Transmembrane signaling via receptor tyrosine kinases generally requires oligomerization of receptor monomers, with the formation of ligand-induced dimers or higher multimers of the extracellular domains of the receptors. Such formations are expected to juxtapose the intracellular kinase domains at the correct distances and orientations for transphosphorylation. For receptors of the insulin receptor family that are constitutively dimeric, or those that form noncovalent dimers without ligands, the mechanism must be more complex. For these, the conformation must be changed by the ligand from one that prevents activation to one that is permissive for kinase phosphorylation. How the insulin ligand accomplishes this action has remained a puzzle since the discovery of the insulin receptor over 2 decades ago, primarily because membrane proteins in general have been refractory to structure determination by crystallography. However, high-resolution structural evidence on individual separate subdomains of the insulin receptor and of analogous proteins has been obtained. The recently solved quaternary structure of the complete dimeric insulin receptor in the presence of insulin has now served as the structural envelope into which such individual domains were fitted. The combined structure has provided answers on the details of insulin/receptor interactions in the binding site and on the mechanism of transmembrane signaling of this covalent dimer. The structure explains many observations on the behavior of the receptor, from greater or lesser binding of insulin and its variants, point and deletion mutants of the receptor, to antibody-binding patterns, and to the effects on basal and insulin-stimulated autophosphorylation under mild reducing conditions.     

Mutations in the juxtamembrane region of the insulin receptor impair activation of phosphatidylinositol 3-kinase by insulin.

CHO/IRF960/T962 cells express a mutant human insulin receptor in which Tyr960 and Ser962 in the juxtamembrane region of the receptor's beta-subunit are replaced by Phe and Thr, respectively. The mutant insulin receptor undergoes autophosphorylation normally in response to insulin; however, insulin fails to stimulate thymidine incorporation into DNA, glycogen synthesis, and tyrosyl phosphorylation of an endogenous substrate pp185 in these cells. Another putative substrate of the insulin receptor tyrosine kinase is phosphatidylinositol 3-kinase (Ptdlns 3-kinase). We have previously shown that Ptdlns 3-kinase activity in Chinese hamster ovary cells expressing the wild-type human insulin receptor (CHO/IR) increases in both antiphosphotyrosine [anti-Tyr(P)] immunoprecipitates and intact cells in response to insulin. In the present study a new technique (detection of the 85-kDa subunit of Ptdlns 3-kinase using [32P]phosphorylated polyoma virus middle T-antigen as probe) is used to monitor the Ptdlns 3-kinase protein. The 85-kDa subunit of Ptdlns 3-kinase is precipitated by anti-Tyr(P) antibodies from insulin-stimulated CHO/IR cells, but markedly less protein is precipitated from CHO/IRF960/T962 cells. The amount of Ptdlns 3-kinase activity in the immunoprecipitates was also reduced in the CHO/IRF960/T962 cells compared to CHO/IR cells. In intact CHO/IRF960/T962 cells, insulin failed to stimulate phosphate incorporation into one of the products of activated Ptdlns 3-kinase, phosphatidylinositol-3,4-bisphosphate [Ptdlns(3,4)P2], whereas it caused a 12-fold increase in CHO/IR cells. In contrast, phosphate incorporation into another product, phosphatidylinositol trisphosphate [PtdlnsP3], was only partially depressed in the CHO/IRF960/T962 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autophosphorylation within insulin receptor beta-subunits can occur as an intramolecular process.

The insulin receptor is a complex membrane-spanning glycoprotein composed of two alpha-subunits and two beta-subunits connected to form an alpha 2 beta 2 holoreceptor. Insulin binding to the extracellular alpha-subunits activates intracellular beta-subunit autophosphorylation and substrate kinase activity. The current study was designed to differentiate mechanisms of transmembrane signaling by the insulin receptor, specifically whether individual beta-subunits undergo cis- or trans-phosphorylation. We compared relative kinase activities of trypsin-truncated receptors, alpha beta-half receptors, and alpha 2 beta 2 holoreceptors under conditions that allowed us to differentiate intermolecular and intramolecular events. Compared to the insulin-stimulated holoreceptors, the trypsin-truncated receptor undergoes autophosphorylation at similar tyrosine residues and catalyzes substrate phosphorylation in the absence of insulin at a comparable rate. The truncated receptor sediments on a sucrose gradient at a position consistent with a structure comprising a single beta-subunit attached to a fragment of the alpha-subunit and undergoes autophosphorylation in this form in the absence of insulin. Autophosphorylation of the truncated insulin receptor is independent of receptor concentration, and immobilization of the truncated receptor on a matrix composed of an anti-receptor antibody bound to protein A-Sepharose diminishes neither autophosphorylation nor receptor-catalyzed substrate phosphorylation. Therefore, true intramolecular (cis) phosphorylations, which occur within individual beta-subunits derived from trypsin-truncated receptors, lead to kinase activation. However, insulin-stimulated autophosphorylation of insulin receptor alpha beta heterodimers is concentration-dependent, and both autophosphorylation and kinase activity are markedly reduced following immobilization.(ABSTRACT TRUNCATED AT 250 WORDS)