Transmembrane signalling by insulin via an insulin receptor mutated at tyrosines 1158, 1162, and 1163

In order to study the role of tyrosine autophosphorylation in insulin receptor signalling, we investigated a mutant human insulin receptor whereby the three major tyrosine autophosphorylation sites at positions 1158, 1162, and 1163 in the receptor beta-subunit were mutated to phenylalanines. When these mutant receptors were expressed in HTC rat hepatoma cells, there was no enhanced beta-subunit autophosphorylation and tyrosine kinase activity. In these cells there was enhanced insulin stimulation of [3H]AIB uptake and [3H]thymidine incorporation when compared to wild type HTC cells. The present study suggests therefore that the presence of the major insulin autophosphorylation sites is not a requirement for insulin stimulation of amino acid transport and mitogenesis.     

Mutation of tyrosine residues 1162 and 1163 of the insulin receptor affects hormone and receptor internalization

Insulin internalization and degradation, insulin receptor internalization and recycling, as well as long term receptor down-regulation were comparatively studied in Chinese hamster ovary (CHO) cell lines, either parental or expressing the wild-type human insulin receptor (CHO.R) or a mutated receptor in which the tyrosine residues in positions 1162 and 1163 were replaced by phenylalanines (CHO.Y2). The two transfected cell lines presented very similar binding characteristics, and their pulse labeling with [35S]methionine revealed that the receptors were processed normally. As expected, the mutation of these twin tyrosines resulted in a defective insulin stimulation of both receptor kinase activity and glycogen synthesis. We now present evidence that compared to CHO.R cells, which efficiently internalized and degraded insulin, CHO.Y2 cells exhibited a marked defect in hormone internalization, leading to impaired insulin degradation. Moreover, the mutated receptors were found to be less effective than the wild-type receptors in transducing the hormone signal for receptor internalization, whereas the process of receptor recycling after internalization seemed not to be altered. In parental CHO cells, insulin induced long term receptor down-regulation, but was totally ineffective in both transfected cell lines. These results reveal that the tyrosines 1162 and 1163 in the kinase regulatory domain of the receptor beta-subunit play a pivotal role in insulin and receptor internalization.     

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.     

Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose

Insulin stimulates the autophosphorylation of tyrosine residues of the beta subunit of the insulin receptor (IR); this modified insulin-independent kinase has increased activity toward exogenous substrates in vitro. We show here that replacement of one or both of the twin tyrosines (residues 1162 and 1163) with phenylalanine results in a dramatic reduction in or loss of insulin-activated autophosphorylation and kinase activity in vitro. In vivo, these mutations not only result in a substantial decrease in insulin-stimulated IR autophosphorylation but also in a parallel decrease in the insulin-activated uptake of 2-deoxyglucose. Furthermore, a truncated IR protein (lacking the last 112 amino acids) has an unstable beta subunit; this mutant has no kinase activity in vitro or in vivo and does not mediate insulin-stimulated uptake of 2-deoxyglucose. IR autophosphorylation is thus implicated in the regulation of IR activities, with tyrosines 1162 and 1163 as major sites of this regulation.     

Insulin receptor tyrosine residues 1162 and 1163 control insulin stimulation of myristoyl-diacylglycerol generation and subsequent activation of glucose transport

Chinese hamster ovary (CHO) transfectants expressing human insulin receptors that were mutated at tyrosines 1162 and 1163 (CHO-Y2 cells) exhibit decreased insulin stimulation of both receptor tyrosine kinase and 2-deoxyglucose uptake compared with transfectants expressing wild-type human insulin receptors (CHO-R cells). We now provide evidence that insulin stimulation of myristoyl-diacylglycerol (DAG) production is also markedly impaired in CHO-Y2 cells; this is manifested as a decreased responsiveness and sensitivity to insulin as compared with CHO-R and parental CHO cells. Further, we report that (i) the concentration-response curves of insulin-stimulated myristoyl-DAG production and 2-deoxyglucose uptake were superimposable within each of the three cell lines. (ii) The insulin-induced increase in myristoyl-DAG production preceded that in 2-deoxyglucose uptake, and the time course was altered for both responses in CHO-Y2 cells. (iii) Insulin also increased the phosphorylation of a 40-kDa protein known to be a substrate for protein kinase C, but to a much lesser extent in CHO-Y2 cells than in CHO-R cells. (iv) Exogenously added 1,2-dimyristoyl-glycerol and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) again stimulated both the phosphorylation of the 40-kDa protein and 2-deoxyglucose uptake, but in contrast to insulin, they elicited the same level of response in both CHO-R and CHO-Y2 cells. (v) Finally, in protein kinase C-depleted CHO-R cells, insulin and PMA stimulation of 40-kDa protein phosphorylation as well as PMA stimulation of 2-deoxyglucose uptake were completely abolished whereas insulin-stimulated 2-deoxyglucose uptake was only partially decreased. Taken together, these results suggest that insulin stimulation of 2-deoxyglucose uptake involves myristoyl-DAG production and, at least in part, protein kinase C activation, all three of these processes being controlled by receptor tyrosines 1162 and 1163.     

Phosphorylation of tyrosines 1158, 1162 and 1163 on a synthetic dodecapeptide by the insulin receptor protein-tyrosine kinase.

To investigate the mechanism of tyrosine phosphorylation by the insulin receptor protein-tyrosine kinase, we utilized a synthetic dodecapeptide substrate (RRDIYETDYYRK; amino acids 1155-1165) containing the three major insulin receptor autophosphorylation sites. (1) We show that all three tyrosines on this peptide are rapidly phosphorylated and that phosphorylation is probably initiated at tyrosine 9. This peptide thus serves as a useful tool to study the mechanism of transphosphorylation by the insulin receptor. (2) A proteolytic activity was detected in purified receptor preparations that removed basic residues from the peptide and prevented it binding to phosphocellulose paper. Such activity could pose a serious problem when using peptide substrates to assay for protein kinases in other acellular systems.     

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.     

Differential role of insulin receptor autophosphorylation sites 1162 and 1163 in the long-term insulin stimulation of glucose transport, glycogenesis, and protein synthesis.

The long-term regulatory effect of insulin on glucose transport activity and glucose transporter expression was examined in Chinese hamster ovary (CHO) transfectants that overexpress either human insulin receptors of the wild type (CHO-R cells) or human insulin receptors mutated at two major autophosphorylation sites, Tyr1162 and Tyr1163 (CHO-Y2 cells). Previous studies showed that, when acutely stimulated by insulin, CHO-Y2 cells exhibit decreased receptor kinase activity along with decreased signaling of several pathways, including that for glucose transport, as compared with CHO-R cells. We now report the following. (i) When treated for 24 h with insulin (10(-10) to 10(-6) M), CHO-R and CHO-Y2 cells displayed closely similar concentration-dependent increases in 2-deoxyglucose uptake. In both transfectants, the maximal insulin-induced increase (approximately 3.5-fold) in uptake was cycloheximide-sensitive and was paralleled by equivalent increases in the levels of GLUT-1 immunoreactive protein and mRNA. (ii) By contrast, under similar conditions, CHO-Y2 cells exhibited a marked decrease in their response to insulin for [U-14C]glucose incorporation into glycogen (decreased sensitivity and maximal responsiveness) and for [U-14C]leucine incorporation into protein (decreased sensitivity) as compared with CHO-R cells. (iii) After a 24-h treatment with 10(-7) M insulin, CHO-R (but not CHO-Y2) cells showed a decreased ability to respond to a subsequent acute insulin stimulation of either receptor exogenous kinase activity or 2-deoxyglucose uptake as compared with respective untreated controls. These results indicate that (i) insulin receptors mutated at Tyr1162 and Tyr1163 retain normal signaling of the long-term stimulatory effect of insulin on glucose transport activity and GLUT-1 expression, but not on glycogenesis and overall protein synthesis; (ii) these three insulin signaling pathways may be triggered by distinct domains of the insulin receptor beta-subunit; and (iii) wild-type (but not twin-tyrosine mutant) receptors undergo negative regulation by chronic insulin treatment for subsequent signaling of acute biological actions of insulin.     

Excess tyrosine restores the morphology and maturation of melanosomes affected by the murine slaty mutation

The slaty (Dct(slt)) mutation is known to reduce the activity of dopachrome tautomerase in melanocytes and to reduce the melanin content in the skin, hairs, and eyes. The slaty gene is known to be important for maximizing melanin deposition in melanosomes. However, it was not known whether the slaty mutation affects the morphology of melanosomes. Moreover, it was unknown whether melanosome development is modulated by melanogenic factors. In this study, the characteristics of melanosomes of slaty melanocytes in serum-free primary culture were investigated in detail under the electron microscope. In slaty melanocytes, melanosome maturation was blocked at stage III, and numerous spherical melanosomes with globular depositions of pigment in addition to elliptical melanosomes were observed. L-tyrosine (Tyr), the starting material of melanin synthesis, is known to stimulate melanin synthesis. To clarify whether L-Tyr restores the reduced production of melanin, L-Tyr was added to the culture medium and tested for its melanogenic effect. L-Tyr greatly increased the number and percentage of mature stage IV melanosomes. Moreover, L-Tyr increased elliptical melanosomes, but decreased spherical melanosomes. These results suggest that the slaty mutation inhibits the development of elliptical stage IV melanosomes, and that L-Tyr restores the development of elliptical stage IV melanosomes. L-Tyr seems to restore both the morphology and maturation of melanosomes affected by the slaty mutation.     

Protein tyrosine phosphatase regulation in fibroblasts from patients with an insulin receptor gene mutation

Tyrosine phosphorylation of the insulin receptor is the initial event following receptor binding to insulin, and it induces further tyrosine phosphorylation of various intracellular molecules. This signaling is countered by protein tyrosine phosphatases (PTPases), which reportedly are associated with insulin resistance that can be reduced by regulation of PTPases. Protein tyrosine phosphatase 1B (PTP1B) and leukocyte antigen-related PTPase (LAR) are the PTPases implicated most frequently in insulin resistance and diabetes mellitus. Here, we show that PTP1B and LAR are expressed in human fibroblasts, and we examine the regulation of PTPase activity in fibroblasts from patients with an insulin receptor gene mutation as an in vitro model of insulin resistance. Total PTPase activity was significantly lower in the cytosolic and membrane fractions of fibroblasts with mutations compared with controls (p<0.05). Insulin stimulation of fibroblasts with mutations resulted in a significantly smaller increase in PTP1B activity compared with stimulation of wild-type fibroblasts (p<0.05). This indicates that insulin receptor gene mutations blunt increases in PTPase activity in response to insulin, possibly via a negative feedback mechanism. Our data suggest that the PTPase activity in patients with insulin receptor gene mutation and severe insulin resistance may differ from that in ordinary type 2 diabetes.     

Monoclonal antibodies to the insulin receptor stimulate the intrinsic tyrosine kinase activity by cross-linking receptor molecules.

The effect of monoclonal anti-insulin receptor antibodies on the intrinsic kinase activity of solubilized receptor was investigated. Antibodies for six distinct epitopes stimulated receptor autophosphorylation and kinase activity towards exogenous substrates. This effect of antibodies was seen only within a narrow concentration range and monovalent antibody fragments were ineffective. Evidence was obtained by sucrose density-gradient centrifugation for the formation of antibody-receptor complexes which involved both inter- and intra-molecular cross-linking, although stimulation of autophosphorylation appeared to be preferentially associated with the latter. There was partial additivity between the effects of insulin and antibodies in stimulating autophosphorylation, although the sites of phosphorylation appeared identical on two-dimensional peptide maps. Antibodies for two further epitopes failed to activate receptor kinase, but inhibited its stimulation by insulin. The effects of antibodies on kinase activity paralleled their metabolic effects on adipocytes, except for one antibody which was potently insulin-like in its metabolic effects, but which antagonized insulin stimulation of kinase activity. It is concluded that antibodies activate the receptor by cross-linking subunits rather than by reacting at specific epitopes. The ability of some antibodies to activate receptor may depend on receptor environment as well as the disposition of epitopes.     

Formation of Hirano bodies induced by expression of an actin cross-linking protein with a gain-of-function mutation

Hirano bodies are paracrystalline actin filament-containing structures reported to be associated with a variety of neurodegenerative diseases. However, the biological function of Hirano bodies remains poorly understood, since nearly all prior studies of these structures were done with postmortem samples of tissue. In the present study, we generated a full-length form of a Dictyostelium 34-kDa actin cross-linking protein with point mutations in the first putative EF hand, termed 34-kDa ΔEF1. The 34-kDa ΔEF1 protein binds calcium normally but has activated actin binding that is unregulated by calcium. The expression of the 34-kDa ΔEF1 protein in Dictyostelium induces the formation of Hirano bodies, as assessed by both fluorescence microscopy and transmission electron microscopy. Dictyostelium cells bearing Hirano bodies grow normally, indicating that Hirano bodies are not associated with cell death and are not deleterious to cell growth. Moreover, the expression of the 34-kDa ΔEF1 protein rescues the phenotypes of cells lacking the 34-kDa protein and cells lacking both the 34-kDa protein and α-actinin. Finally, the expression of the 34-kDa ΔEF1 protein also initiates the formation of Hirano bodies in cultured mouse fibroblasts. These results show that the failure to regulate the activity and/or affinity of an actin cross-linking protein can provide a signal for the formation of Hirano bodies. More generally, the formation of Hirano bodies is a cellular response to or a consequence of aberrant function of the actin cytoskeleton.     

Insulin-mimetic effect of trypsin on the insulin receptor tyrosine kinase in intact adipocytes

It has previously been demonstrated that the insulin-mimetic agent trypsin stimulates autophosphorylation of purified insulin receptors and activates the insulin receptor tyrosine kinase in vitro. We now report the effects of trypsin on whole cell tyrosine kinase activation and insulin receptor autophosphorylation. Trypsin treatment of intact adipocytes produces a time-dependent stimulation of tyrosine kinase activity as measured in lectin extracts containing the insulin receptor, or specifically immunoprecipitated insulin receptor samples. Trypsin treatment of adipocytes also results in a loss of insulin binding capacity, and a linear correlation exists between loss of binding and stimulation of tyrosine kinase activity. Exposure of adipocytes to trypsin is known to result in a time- and dose-dependent activation of intracellular glycogen synthase. Examination of the time courses of stimulation of tyrosine kinase and glycogen synthase activation in our system indicates that the stimulation of tyrosine kinase activity by trypsin occurs with sufficient rapidity and magnitude to be consistent with a role of phosphorylation in the activation of glycogen synthase. Trypsin has further been demonstrated to stimulate autophosphorylation of the beta-subunit of the insulin receptor in intact adipocytes. Cells prelabeled with [32P]PO4 for 2 h were exposed to trypsin, and receptors were partially purified over wheat germ agglutinin-agarose columns. Receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the beta-subunit was identified by autoradiography. The protein was extracted and hydrolyzed, and the phosphoamino acids were separated by electrophoresis and quantitated. Two- and five-fold increases in phosphotyrosine were observed with 3 and 10 min of trypsin treatment, respectively. We conclude that trypsin-induced cleavage of the insulin receptor alpha-subunit is relevant to the ability of trypsin to activate the insulin receptor tyrosine kinase in intact adipocytes. We further conclude that autophosphorylation of the insulin receptor and activation of its tyrosine kinase by trypsin may be important to the insulin-mimetic anabolic effects of trypsin.     

The incidence of genetic disease and the impact on man of an altered mutation rate

In order to adequately assess the genetic risks to man of an altered mutation rate, it is necessary to know the naturally occurring frequency of mutation-maintained genetic ill-health and the burden that such defects impose. The relevant data that are available are largely inadequate to determine the incidence of genetic disease that is maintained by mutation, and measures of various aspects of the social and personal burdens due to hereditary ill-health are almost wholly lacking. It is suggested that the creation of individual and family histories, using large scale automatic record linkage and existing files of vital and ill-health records, may be a useful approach to these kinds of problems. Using such linked individual health histories, new data are presented that relate to measures of the burden due to childhood dominant and recessive diseases and congenital malformations.     

A naturally occurring mutation of insulin receptor alanine 1134 impairs tyrosine kinase function and is associated with dominantly inherited insulin resistance

We have identified a previously undescribed genetic variant of the insulin receptor (Ala1134----Thr1134) in a family with the Type A syndrome of insulin resistance. Using the polymerase chain reaction to amplify insulin receptor cDNA and genomic DNA (exon 19), this mutation was detected in 1/2 alleles in the proband, her two affected sisters, and her affected father. Two normal alleles were present in the unaffected mother. No additional structural changes were encoded by the remainder of the proband's receptor cDNA. The Ala1134 mutant receptor was expressed in Chinese hamster ovary cells. The expressed mutant receptors were processed normally and displayed normal affinity of insulin binding but were markedly deficient in insulin-stimulated autophosphorylation. The mutant receptor was unable to catalyze the phosphorylation of the endogenous substrate, pp185, and insulin-stimulated kinase activity toward an exogenous substrate in vitro also was markedly impaired. Ala1134 is a highly conserved residue located in a consensus sequence found in most tyrosine kinases. It is likely that this previously uncharacterized residue and/or the immediate region surrounding it are important for normal kinase function in other members of this receptor family. This study also demonstrates that severe insulin resistance with dominant inheritance may be caused by a missense mutation in one allele of the insulin receptor gene.     

YMXM motifs and signaling by an insulin receptor substrate 1 molecule without tyrosine phosphorylation sites.

Tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) by the activated receptors for insulin, IGF-1, and various cytokines creates binding sites for signaling proteins with Src homology 2 domains (SH2 proteins). Determining the role of specific SH2 proteins during insulin signaling has been difficult because IRS-1 possesses as many as 18 potential tyrosine phosphorylation sites, several of which contain redundant motifs. Using 32D cells, which contain no endogenous IRS proteins, we compared the signaling ability of an IRS-1 molecule in which 18 potential tyrosine phosphorylation sites were replaced by phenylalanine (IRS-1(F18)) with two derivative molecules which retained three YMXM motifs (IRS-1(3YMXM)) or the two COOH-terminal SHP2-Fyn binding sites (IRS-1(YCT)). During insulin stimulation, IRS-1(F18) failed to undergo tyrosine phosphorylation or mediate activation of the phosphotidylinositol (PI) 3'-kinase or p70(s6k); IRS-1(YCT) was tyrosine phosphorylated but also failed to mediate these signaling events. Neither IRS-1(3YMXM) nor IRS-1(YCT) mediated activation of mitogen-activated protein kinases. IRS-1(F18) and IRS-1(YCT) partially mediated similar levels of insulin-stimulated mitogenesis at high insulin concentrations, however, suggesting that IRS-1 contains phosphotyrosine-independent elements which effect mitogenic signals, and that the sites in IRS-l(YCT) do not augment this signal. IRS-1(3YMXM) mediated the maximal mitogenic response to insulin, although the response to insulin was more sensitive with wild-type IRS-1. By contrast, the association of IRS-1(3YMXM) with PI 3'-kinase was more sensitive to insulin than the association with IRS-1. Thus, the binding of SH2 proteins (such as PI 3'-kinase) by YMXM motifs in IRS-1 is an important element in the mitogenic response, but other elements are essential for full mitogenic sensitivity.