Investigation of the mechanism of the dominant negative effect of mutations in the tyrosine kinase domain of the insulin receptor.

Mutations in the tyrosine kinase domain of the insulin receptor cause insulin resistance in a dominant fashion. It has been proposed that formation of hybrid dimers between normal and mutant receptors may explain the dominant negative effect of these mutations. To investigate this mechanism, we expressed two types of human insulin receptors in NIH-3T3 cells; wild type and the tyrosine kinase-deficient Ile1153 mutant. To distinguish the two types of receptors, 43 amino acids were deleted from the C-terminus of the wild type receptor (delta 43 truncation). If mutant and wild type receptors assemble in a random fashion, 50% of the receptors would be hybrid oligomers (alpha 2 beta beta mut). However, alpha 2 beta beta mut hybrids were undetectable. Nevertheless, insulin stimulated the kinase competent delta 43 receptors to transphosphorylate the kinase-deficient Ile1153 mutant receptor in co-transfected cells via an intermolecular mechanism. Furthermore, transphosphorylation of the Ile1153 mutant receptor is sufficient to trigger insulin-stimulated endocytosis. Despite the absence of alpha 2 beta beta mut hybrids, expression of the Ile1153 mutant receptor inhibited the ability of the delta 43 truncated receptor to mediate insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1). Evidence is presented to support the hypothesis that the Ile1153 mutant receptor retains the ability to bind IRS-1, and that sequestration of substrate may explain the dominant negative effect of the mutant receptor to inhibit phosphorylation of IRS-1.     

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.     

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.     

Assessment of the in situ tyrosine kinase activity of mutant insulin receptors lacking tyrosine autophosphorylation sites 1162 and 1163.

In the present studies mutant insulin receptors with regulatory tyrosine residues 1162 and 1163 changed to phenylalanines were tested for tyrosine kinase activity. In agreement with prior studies, this mutant receptor was found to exhibit almost no insulin-stimulated exogenous kinase activity when assayed in vitro. In contrast, this mutant receptor was found in situ to have a significant, albeit reduced, ability to mediate the tyrosine phosphorylation of various endogenous proteins, as assessed by Western blotting with antiphosphotyrosine antibodies. In addition, extracts of insulin-treated cells overexpressing this mutant receptor exhibited increased amounts of tyrosine phosphorylated phosphatidylinositol 3-kinase compared to control cells. Finally, this mutant receptor, like the wild-type receptor, was found to mediate an increase in the activity of a membrane-associated phosphatidylinositol 4,5-biphosphate kinase. These results indicate that 1) in vitro assessments of the tyrosine kinase activity of mutant insulin receptors may not accurately reflect their in vivo activities; and 2) the ability of the mutant receptor lacking tyrosine autophosphorylation sites 1162 and 1163 to mediate insulin-stimulated tyrosine phosphorylation of various endogenous substrates may account for the reported ability of this receptor to mediate various biological responses.     

The pathway mediating insulin's effects on pyruvate dehydrogenase bypasses the insulin receptor tyrosine kinase

The effect of insulin on pyruvate dehydrogenase activity was examined in two different cell types that over expressed either normal or defective human insulin receptors, RAT 1 embryonic fibroblasts and Chinese hamster ovary (CHO) cells. Insulin stimulated pyruvate dehydrogenase activity in cells that expressed normal insulin receptors (RAT 1 HIRc, and CHO-WT and CHO-T cells), or receptors in which lysine 1018 in the ATP-binding site of the tyrosine kinase domain was exchanged for alanine (RAT 1 A/K1018 and CHO-mut cells). For both rat and hamster cell lines, the insulin dose-response curves from cells that expressed the mutant receptors were identical to those from the appropriate controls that over expressed the normal insulin receptors. Insulin failed to stimulate pyruvate dehydrogenase activity in CHO-delta cells, which expressed a mutant human insulin receptor that was truncated by 112 amino acids at the carboxyl terminal of the beta chain. Control studies verified that all the cells used in this study exhibited the expected phenotypes with respect to the number of insulin receptors which they expressed, insulin-stimulated tyrosine kinase activity, and the biological consequences of inactivating the insulin receptor tyrosine kinase. These findings show that the insulin receptor tyrosine kinase does not play an obligatory role in the insulin signaling pathway that stimulates pyruvate dehydrogenase activity.     

Insulin stimulates phosphorylation of the beta 2-adrenergic receptor by the insulin receptor, creating a potent feedback inhibitor of its tyrosine kinase.

Insulin counterregulates catecholamine action at several levels, primarily in liver, fat, and adipose tissue. Herein we observe that expression of increased levels of beta(2)-adrenergic receptor increasingly inhibits insulin-stimulated phosphorylation of its primary downstream substrates (IRS-1,2). In Chinese hamster ovary cells, the insulin receptor phosphorylates dominantly Tyr(364) in the C-terminal cytoplasmic domain of the beta-receptor. A Y364A mutant form of the beta(2)-adrenergic, in contrast, loses it ability to inhibit insulin-stimulated phosphorylation of IRS-1,2. Upon phosphorylation, the C-terminal cytoplasmic domain of the beta(2)-adrenergic receptor demonstrates a potent inhibitory feedback action that can block both insulin-stimulated autophosphorylation of the insulin receptor and phosphorylation of IRS-1,2 in NIH mouse 3T3-L1 adipocyte membranes. Studies in vitro with purified insulin receptor and the C-terminal cytoplasmic domain of the beta(2)-adrenergic receptor demonstrate that the tyrosine-phosphorylated beta-receptor domain is a potent counterregulatory inhibitor of the insulin receptor tyrosine kinase.     

A mutation in the extracellular domain of the insulin receptor impairs the ability of insulin to stimulate receptor autophosphorylation.

Mutations of the insulin receptor gene have been shown to cause insulin-resistant diabetes in patients with genetic forms of insulin resistance. We have previously reported that a mutation substituting valine for Phe382 in the alpha-subunit of the insulin receptor is associated with impaired transport of the mutant receptor to the plasma membrane (Accili, D., Frapier, C., Mosthaf, L., McKeon, C., Elbein, S. C., Permutt, M. A., Ramos, E., Lander, E. S., Ullrich, A., and Taylor, S. I. (1989) EMBO J. 8, 2509-2517). In this study, we demonstrate that the Val382 mutation impairs the ability of insulin to activate receptor autophosphorylation. Furthermore, the Val382 receptor has reduced activity to phosphorylate other peptide substrates in the presence of insulin. Nevertheless, when the Val382 mutant and wild-type receptors are mixed together, the wild-type human insulin receptor is able to phosphorylate the Val382 mutant receptor, thereby activating the tyrosine kinase activity of the mutant receptor. Thus, the conformational change caused by the Val382 mutation compromises the ability of the receptor to transmit a signal across the plasma membrane. Furthermore, our observations suggest that receptor phosphorylation by an intermolecular mechanism (i.e. transphosphorylation) may play a role in mediating the action of insulin upon the target cell.     

A mutant insulin receptor with defective tyrosine kinase displays no biologic activity and does not undergo endocytosis

The cDNAs encoding the normal human insulin receptor (HIRc) and a receptor that had lysine residue 1018 replaced by alanine (A/K1018) were used to transfect Rat 1 fibroblasts. Lysine 1018 is a critical residue in the ATP binding site of the tyrosine kinase domain in the receptor beta-subunit. Untransfected Rat 1 cells express 1700 endogenous insulin receptors. Expressed HIRc receptors had levels of insulin-stimulable autophosphorylation in vitro comparable to normal receptors, whereas A/K1018 receptors had less than 1% of that activity. Stimulation by insulin of HIRc receptors in situ in intact cells led to phosphorylation of beta-subunit tyrosine residues and activation of tyrosine kinase activity that could be preserved and assayed in vitro after receptor purification. In contrast, A/K1018 receptors showed no such activation, either of autophosphorylation or of kinase activity toward histone. Cells expressing HIRc receptors display enhanced sensitivity to insulin of 2-deoxyglucose transport and glycogen synthase activity. This increased sensitivity was proportional to insulin receptor number at low but not at high levels of receptor expression. A/K1018 receptors were unable to mediate these biologic effects and actually inhibited insulin's ability to stimulate glucose transport and glycogen synthase through the endogenous Rat 1 receptors. Expressed HIRc receptors mediated insulin internalization and degradation, whereas A/K1018 receptors mediated little, if any. Endocytotic uptake of the expressed A/K1018 insulin receptors was also markedly depressed compared to normal receptors. Unlike HIRc receptors, A/K1018 receptors also fail to undergo down-regulation after long (24 h) exposures to high (170 nM) concentrations of insulin. We conclude the following. 1) Normal human insulin receptors expressed in Rat 1 fibroblasts display active tyrosine-specific kinase, normal intracellular itinerary after endocytosis, and normal coupling to insulin's biologic effects. 2) A receptor mutated to alter the ATP binding site in the tyrosine kinase domain had little if any tyrosine kinase activity. 3) This loss of kinase activity was accompanied by a nearly complete lack of both endocytosis and biologic activity.     

Differential modulation of the tyrosine phosphorylation state of the insulin receptor by IRS (insulin receptor subunit) proteins.

In response to insulin, tyrosine kinase activity of the insulin receptor is stimulated, leading to autophosphorylation and tyrosine phosphorylation of proteins including insulin receptor subunit (IRS)-1, IRS-2, and Shc. Phosphorylation of these proteins leads to activation of downstream events that mediate insulin action. Insulin receptor kinase activity is requisite for the biological effects of insulin, and understanding regulation of insulin receptor phosphorylation and kinase activity is essential to understanding insulin action. Receptor tyrosine kinase activity may be altered by direct changes in tyrosine kinase activity, itself, or by dephosphorylation of the insulin receptor by protein-tyrosine phosphatases. After 1 min of insulin stimulation, the insulin receptor was tyrosine phosphorylated 8-fold more and Shc was phosphorylated 50% less in 32D cells containing both IRS-1 and insulin receptors (32D/IR+IRS-1) than in 32D cells containing only insulin receptors (32D/IR), insulin receptors and IRS-2 (32D/IR+IRS-2), or insulin receptors and a form of IRS-1 that cannot be phosphorylated on tyrosine residues (32D/IR+IRS-1F18). Therefore, IRS-1 and IRS-2 appeared to have different effects on insulin receptor phosphorylation and downstream signaling. Preincubation of cells with pervanadate greatly decreased protein-tyrosine phosphatase activity in all four cell lines. After pervanadate treatment, tyrosine phosphorylation of insulin receptors in insulin-treated 32D/IR, 32D/ IR+IRS-2, and 32D/IR+IRS-1F18 cells was markedly increased, but pervanadate had no effect on insulin receptor phosphorylation in 32D/IR+IRS-1 cells. The presence of tyrosine-phosphorylated IRS-1 appears to increase insulin receptor tyrosine phosphorylation and potentially tyrosine kinase activity via inhibition of protein-tyrosine phosphatase(s). This effect of IRS-1 on insulin receptor phosphorylation is unique to IRS-1, as IRS-2 had no effect on insulin receptor tyrosine phosphorylation. Therefore, IRS-1 and IRS-2 appear to function differently in their effects on signaling downstream of the insulin receptor. IRS-1 may play a major role in regulating insulin receptor phosphorylation and enhancing downstream signaling after insulin stimulation.     

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.     

Various proteins modulate the kinase activity of the insulin receptor

Previous studies of the substrate specificity of the purified insulin receptor tyrosine kinase using synthetic random polymers have demonstrated that the receptor kinase phosphorylates poly (Glu, Tyr) 4:1 but not poly (Glu, Tyr) 1:1. In the present study, insulin treatment of Chinese hamster ovary cells overexpressing the human insulin receptor was found to stimulate the ability of their membrane extracts to phosphorylate poly (Glu, Tyr) 1:1. It was concluded that this activity was due to the receptor itself because: 1) it was precipitated with a monoclonal antibody to the receptor; 2) the addition of various membrane extracts to purified insulin receptor preparations stimulated the ability of these preparations to phosphorylate poly (Glu, Tyr) 1:1; and 3) certain purified proteins, including bovine serum albumin and casein, were also capable of stimulating the purified receptor to phosphorylate poly (Glu, Tyr) 1:1. The effect of albumin was dose-dependent; 0.5 and 10 mg/ml bovine serum albumin stimulated the phosphorylation of poly (Glu, Tyr) 1:1 by 2- and 230-fold, respectively. In contrast, albumin had no effect on the phosphorylation of poly (Glu, Tyr) 4:1. These results indicate that the activity of the insulin receptor kinase on certain substrates can be modulated by the presence of other proteins.     

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.     

Relationship of site-specific beta subunit tyrosine autophosphorylation to insulin activation of the insulin receptor (tyrosine) protein kinase activity

The ability of insulin to activate the insulin receptor protein kinase is shown to be completely dependent on prior beta subunit tyrosine autophosphorylation. Autophosphorylation in the presence of insulin is a highly concerted reaction; tryptic digestion of insulin receptor beta subunits derived from preparations whose kinase activation ranges from under 5% to 100% of maximal yields the same array of [32P]Tyr(P)-containing peptides over the entire range. Of special note is the significant contribution of multiply phosphorylated forms of tryptic peptides corresponding to proreceptor residues 1144-1152 (from the "tyrosine kinase" domain) and 1314-1329 (near the carboxyl terminus) to overall beta subunit phosphorylation at kinase activations of 5% and under. Thus, partially activated/autophosphorylated receptor preparations consist of mixtures of unactivated unphosphorylated receptors and activated fully (or nearly fully) phosphorylated receptors. The latter can be selectively removed by adsorption to antiphosphotyrosine antibodies. This abrupt multiple phosphorylation of individual receptor molecules explains why, in the presence of insulin, overall beta subunit tyrosine phosphorylation tracks closely with kinase, up to approximately 90% activation. Insulin stimulates phosphorylation into all domains (involving at least 6 of the 13 tyrosines on the intracellular portion of the beta subunit) but does not cause the appearance of "new" 32P-labeled species. Rather, insulin directs 32P incorporation preferentially into those domains most productive of kinase activation. Phosphorylation of the tyrosine residues at 1146, 1150, and 1151 correlates most closely with kinase activation. These residues show the largest 32P incorporation during rapid kinase activation; moreover, in comparisons of receptors with similar overall autophosphorylation but very different activations (or similar activations but different extents of autophosphorylation), achieved by omitting insulin or varying [ATP], the phosphorylation of peptide 1144-1152 tracks closely with kinase activation, and phosphorylation of sites and Mr 4000-5000 tryptic peptide (presumably Tyr 953 and/or 960) tract nearly as well. By contrast the extent of phosphorylation of the carboxy-terminal peptide is frequently dissociated from the extent of kinase activation. Phosphorylation of this latter domain probably underlies a beta subunit function other than tyrosine kinase activity.     

Substitution of the insulin receptor transmembrane domain with the c-neu/erbB2 transmembrane domain constitutively activates the insulin receptor kinase in vitro.

To examine the role of the transmembrane domain (TM) of the insulin receptor in insulin-induced receptor kinase activation, we prepared four mutated insulin receptors: 1) a Val938----Asp substitution (IR/TMv----D), 2) insertion of a 3-amino acid repeat (Val938-Phe939-Leu940) (IR/TM+3), or the entire TM was replaced by the corresponding domain of either the 3) platelet-derived growth factor (PDGF) receptor (IR/TMPDGFR) or 4) c-neu/erbB2 proto-oncogene product (IR/TMc-neu). Each mutant receptor was stably expressed in Chinese hamster ovary cells, assessed by fluorescence-activated cell sorting, insulin binding, and biosynthetic labeling. All mutant receptors exhibited normal affinity for insulin. Pulse-chase experiments showed that each proreceptor was processed into alpha- and beta-subunits, although the rate of IR/TMV----D conversion was reduced approximately 3-fold. With IR/TMPDGFR, IR/TMV----D, and IR/TM+3 basal and insulin-stimulated levels of autophosphorylation and tyrosine kinase activation were normal, both in wheat germ agglutinin (WGA)-purified receptor preparations and intact cells. By contrast, following WGA purification or isolation of crude membranes, IR/TMc-neu was a constitutively active autokinase and substrate kinase in vitro. However, in intact cells insulin-stimulated autophosphorylation and kinase activity appeared normal. We conclude that although there is considerable latitude in acceptable structure, residues within the insulin receptor transmembrane domain can play a functional role in regulation of insulin receptor tyrosine kinase activity.     

The protein-tyrosine kinase activity of the insulin receptor is necessary for insulin-mediated receptor down-regulation

We have previously demonstrated that the human insulin receptor, mutated in the ATP-binding domain of the beta-subunit, is kinase-defective and fails to mediate multiple post-receptor actions of insulin in stably transfected Chinese hamster ovary cells (Chou, C.-K., Dull, T. J., Russell, D. S., Gherzi, R., Lebwohl, D., Ullrich, A., and Rosen, O. M. (1987) J. Biol. Chem. 262, 1842-1847). This study addresses the role of protein-tyrosine kinase activity in insulin-mediated receptor down-regulation. Although the mutant insulin proreceptor was properly processed and able to bind insulin like the wild-type human receptor, it differed from the latter in the following respects: 1) it failed to mediate internalization of surface-bound radiolabeled ligand; 2) it did not undergo short- or long-term down-regulation in response to 1 microM insulin; 3) it did not exhibit ligand-promoted receptor turnover; and 4) it was not phosphorylated on either tyrosine or serine residues in response to insulin. Although the cells transfected with the mutant receptor failed to respond to insulin-mediated insulin receptor down-regulation, they were able to down-regulate their insulin-like growth factor I receptors in response to insulin-like growth factor I or high concentrations of insulin and were sensitive to monoclonal antibody-induced down-regulation of their insulin receptors. Antibody-mediated receptor internalization alone, however, was unable to mimic at least one action of insulin, thymidine incorporation into DNA, and did not lead to any phosphorylation of the receptor. It is concluded that either the protein-tyrosine kinase activity of the insulin receptor or its phosphorylation state is essential for ligand-mediated receptor down-regulation.     

Mutation of tyrosine 960 within the insulin receptor juxtamembrane domain impairs glucose transport but does not inhibit ligand-mediated phosphorylation of insulin receptor substrate-2 in 3T3-L1 adipocytes

CSF-1 is equipotent to insulin in its ability to stimulate 2-[3H]deoxyglucose uptake in 3T3-L1 adipocytes expressing the colony stimulating factor-1 receptor/insulin receptor chimera (CSF1R/IR). However, CSF-1-stimulated glucose uptake and glycogen synthesis is reduced by 50% in comparison to insulin in 3T3-L1 cells expressing a CSF1R/IR mutated at Tyr960 (CSF1R/IRA960). CSF-1-treated adipocytes expressing the CSF1R/IRA960 were impaired in their ability to phosphorylate insulin receptor substrate 1 (IRS-1) but not in their ability to phosphorylate IRS-2. Immunoprecipitation of IRS proteins followed by Western blotting revealed that the intact CSF1R/IR co-precipitates with IRS-2 from CSF-1-treated cells. In contrast, the CSF1R/IRA960 co-precipitates poorly with IRS-2. These observations suggest that Tyr960 is important for interaction of the insulin receptor cytoplasmic domain with IRS-2, but it is not essential to the ability of the insulin receptor tyrosine kinase to use IRS-2 as a substrate. These observations also suggest that in 3T3-L1 adipocytes, tyrosine phosphorylation of IRS-2 by the insulin receptor tyrosine kinase is not sufficient for maximal stimulation of receptor-regulated glucose transport or glycogen synthesis.     

Analysis of the order of autophosphorylation of human insulin receptor tyrosines 1158, 1162 and 1163.

Insulin receptor tyrosines 1158, 1162 and 1163 are the most rapidly autophosphorylated residues following insulin binding. Although progression of these tyrosines from a bis- to tris-phosphorylated state leads to activation of the receptor tyrosine kinase towards added substrates, rather paradoxically, a receptor with a Y1158F mutation has been reported to be capable of normal activation. In the present study we demonstrate that autophosphorylation of the insulin receptor probably initiates on either of tyrosines 1158 and 1162 while autophosphorylation of tyrosine 1163 occurs predominantly late in the autophosphorylation cascade. Our results are compatible with tyrosines 1162 and 1163 being the major determinants of kinase activity and explain why wild-type insulin receptors only become active after all three of tyrosines 1158, 1162 and 1163 have been phosphorylated.     

Regulation of insulin receptor kinase by multisite phosphorylation

The regulation of the insulin receptor kinase by phosphorylation and dephosphorylation has been examined. Under in vitro conditions, the tyrosine kinase activity of the insulin receptor toward histone is markedly activated when the receptor either undergoes autophosphorylation or is phosphorylated by a purified preparation of src tyrosine kinase on tyrosine residues of its beta subunit. The elevated kinase activity of the phosphorylated insulin receptor is readily reversed when the receptor is dephosphorylated with alkaline phosphatase. Analysis of tryptic digests of phosphorylated insulin receptor using reverse-phase high pressure liquid chromatography suggests that phosphorylation of a specific tyrosine site on the receptor beta subunit may be involved in the mechanism of the receptor kinase activation. Further studies indicate that tyrosine phosphorylation-mediated increase in insulin receptor activity also occurs in intact cells. Thus, when the histone kinase activities of insulin receptor from control and insulin-treated H-35 hepatoma cells are assayed in vitro following the purification of the receptors under conditions which preserve the phosphorylation state of the receptors, the insulin receptors extracted from insulin-treated cells exhibit histone kinase activities 100% higher than those from control cells. The elevated receptor kinase activity from insulin-treated cells appears to result from the increase in phosphotyrosine content of the receptor. Taken together, these results indicate that tyrosine phosphorylation of the insulin receptor beta subunit exerts a major stimulatory effect on the kinase activity of the receptor. Insulin receptor partially purified by specific immunoprecipitation from detergent extracts of control and isoproterenol-treated cells have similar basal but diminished insulin-stimulated beta subunit autophosphorylation activities when incubated with [gamma-32 P]ATP. Similarly, the ability of insulin to stimulate the receptor beta subunit phosphorylation in intact isoproterenol-treated adipocytes is greatly attenuated, whereas, the basal phosphorylation of the insulin receptor is slightly increased by the beta-catecholamine. These data indicate that in rat adipocytes, a cyclic AMP-mediated mechanism, possibly through serine and threonine phosphorylation of the receptor or its regulatory components, may uncouple the receptor tyrosine kinase activity from activation by insulin. Treatment of 32P-labeled H-35 hepatoma cells with phorbol myristate acetate (PMA) results in a marked increase in serine phosphorylation of the insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of biological functions by an insulin receptor monoclonal antibody in insulin receptor beta-subunit mutants.

We investigated the effects of MA-5, a human-specific monoclonal antibody to the insulin receptor alpha-subunit, on transmembrane signaling in cell lines transfected with and expressing both normal human insulin receptors and receptors mutated in their beta-subunit tyrosine kinase domains. In cell lines expressing normal human insulin receptors, MA-5 stimulated three biological functions: aminoisobutyric acid (AIB) uptake, thymidine incorporation, and S6 kinase activation. Under conditions where these biological functions were stimulated, there was no detectable stimulation of receptor tyrosine kinase. We then combined the use of this monoclonal antibody with cells expressing insulin receptors with mutations in the beta-subunit tyrosine kinase domain; two of ATP binding site mutants V1008 (Gly----Val) and M1030 (Lys----Met) and one triple-tyrosine autophosphorylation site mutant F3 (Tyr----Phe at 1158, 1162, and 1163). In cells expressing V1008 receptors, none of the three biological functions of insulin was stimulated. In cells expressing M1030 receptors, AIB uptake was stimulated to a small, but significant, extent whereas the other two functions were not. In cells expressing F3 receptors, AIB uptake and S6 kinase activation, but not thymidine incorporation, were fully stimulated. The data suggest, therefore, that (1) activation of insulin receptor tyrosine kinase may not be a prerequisite for signaling of all the actions of insulin and (2) there may be multiple signal transduction pathways to account for the biological actions of insulin.