The mechanism through which insulin binding to the extracellular domain of the insulin receptor activates the intrinsic tyrosine kinase in the intracellular domain of the protein is unknown. For the c-neu/erbB-2 (c-erbB-2) protooncogene, a single point mutation within the transmembrane (TM) domain converting Val-664 to Glu (erbB-2V-->E) results in elevated levels of tyrosine kinase activity and cellular transformation. We report the construction of a chimeric insulin receptor in which the TM domain of the receptor has been substituted with that encoded by erbB-2V-->E. When expressed in Chinese hamster ovary cells this chimeric receptor displays maximal levels of autophosphorylation and kinase activity in the absence of insulin. This activity results in an increase in the level of insulin-receptor substrate 1 phosphorylation but a down-regulation in insulin-receptor substrate 1 protein and desensitization to insulin stimulation of glycogen synthesis. By contrast, basal levels of DNA synthesis are elevated to levels approximately 60% of those observed in serum-stimulated cells. Over-expression of chimeric insulin receptors containing the c-erbB-2 TM domain or a single point mutation in the insulin receptor TM domain of Val-938-->Asp, on the other hand, shows none of these alterations. Thus, the TM domain encoded by erbB-2V-->E contains structural features that can confer ligand-independent activation in a heterologous protein. Constitutive activation of the insulin receptor results in a relative increase in basal levels of DNA synthesis, but an apparent resistance to the metabolic effects of insulin.

Insulin rapidly stimulates tyrosine kinase activity of its receptor resulting in phosphorylation of its cytosolic substrate, insulin receptor substrate-1 (IRS-1), which in turn associates with phosphatidylinositol 3-kinase (PI 3-kinase), thus activating the enzyme. Glucocorticoid treatment is known to produce insulin resistance, but the exact molecular mechanism is unknown. In the present study we have examined the levels and phosphorylation state of the insulin receptor and IRS-1, as well as the association/activation between IRS-1 and PI 3-kinase in the liver and muscle of rats treated with dexamethasone. After dexamethasone treatment (1 mg/kg per d for 5 d), there was no change in insulin receptor concentration in liver of rats as determined by immunoblotting with antibody to the COOH-terminus of the receptor. However, insulin stimulation of receptor autophosphorylation determined by immunoblotting with antiphosphotyrosine antibody was reduced by 46.7 +/- 9.1%. IRS-1 and PI 3-kinase protein levels increased in liver of dexamethasone-treated animals by 73 and 25%, respectively (P < 0.05). By contrast, IRS-1 phosphorylation was decreased by 31.3 +/- 10.9% (P < 0.05), and insulin stimulated PI 3-kinase activity in anti-IRS-1 immunoprecipitates was decreased by 79.5 +/- 11.2% (P < 0.02). In muscle, the changes were less dramatic, and often in opposite direction of those observed in liver. Thus, there was no significant change in insulin receptor level or phosphorylation after dexamethasone treatment. IRS-1 and PI 3-kinase levels were decreased to 38.6 and 65.6%, respectively (P < 0.01 and P < 0.05). IRS-1 phosphorylation showed no significant change in muscle, but insulin-stimulated IRS-1 associated PI 3-kinase was decreased by 41%. Thus, dexamethasone has differential effects on the proteins involved in the early steps in insulin action in liver and muscle. In both tissues, dexamethasone treatment results in a reduction in insulin-stimulated IRS-1-associated P I3-kinase, which may play a role in the pathogenesis of insulin resistance at the cellular level in these animals.

Insulin rapidly stimulates tyrosine phosphorylation of a protein of approximately 185 kD in most cell types. This protein, termed insulin receptor substrate-1 (IRS-1), has been implicated in insulin signal transmission based on studies with insulin receptor mutants. In the present study we have examined the levels of IRS-1 and the phosphorylation state of insulin receptor and IRS-1 in liver and muscle after insulin stimulation in vivo in two rat models of insulin resistance, i.e., insulinopenic diabetes and fasting, and a mouse model of non-insulin-dependent diabetes mellitus (ob/ob) by immunoblotting with anti-peptide antibodies to IRS-1 and anti-phosphotyrosine antibodies. As previously described, there was an increase in insulin binding and a parallel increase in insulin-stimulated receptor phosphorylation in muscle of fasting and streptozotocin-induced (STZ) diabetic rats. There was also a modest increase in overall receptor phosphorylation in liver in these two models, but when normalized for the increase in binding, receptor phosphorylation was decreased, in liver and muscle of STZ diabetes and in liver of 72 h fasted rats. In the hyperinsulinemic ob/ob mouse there was a decrease in insulin binding and receptor phosphorylation in both liver and muscle. The tyrosyl phosphorylation of IRS-1 after insulin stimulation reflected an amplification of the receptor phosphorylation in liver and muscle of hypoinsulinemic animals (fasting and STZ diabetes) with a twofold increase, and showed a significant reduction (approximately 50%) in liver and muscle of ob/ob mouse. By contrast, the levels of IRS-1 protein showed a tissue specific regulation with a decreased level in muscle and an increased level in liver in hypoinsulinemic states of insulin resistance, and decreased levels in liver in the hyperinsulinemic ob/ob mouse. These data indicate that: (a) IRS-1 protein levels are differentially regulated in liver and muscle; (b) insulin levels may play a role in this differential regulation of IRS-1; (c) IRS-1 phosphorylation depends more on insulin receptor kinase activity than IRS-1 protein levels; and (d) reduced IRS-1 phosphorylation in liver and muscle may play a role in insulin-resistant states, especially of the ob/ob mice.

The most widely distributed members of the family of insulin receptor substrate (IRS) proteins are IRS-1 and IRS-2. These proteins participate in insulin and insulin-like growth factor 1 signaling, as well as the actions of some cytokines, growth hormone, and prolactin. To more precisely define the specific role of IRS-1 in adipocyte biology, we established brown adipocyte cell lines from wild-type and IRS-1 knockout (KO) animals. Using differentiation protocols, both with and without insulin, preadipocyte cell lines derived from IRS-1 KO mice exhibited a marked decrease in differentiation and lipid accumulation (10 to 40%) compared to wild-type cells (90 to 100%). Furthermore, IRS-1 KO cells showed decreased expression of adipogenic marker proteins, such as peroxisome proliferator-activated receptor gamma (PPARgamma), CCAAT/enhancer-binding protein alpha (C/EBPalpha), fatty acid synthase, uncoupling protein-1, and glucose transporter 4. The differentiation deficit in the KO cells could be reversed almost completely by retrovirus-mediated reexpression of IRS-1, PPARgamma, or C/EBPalpha but not the thiazolidinedione troglitazone. Phosphatidylinositol 3-kinase (PI 3-kinase) assays performed at various stages of the differentiation process revealed a strong and transient activation in IRS-1, IRS-2, and phosphotyrosine-associated PI 3-kinase in the wild-type cells, whereas the IRS-1 KO cells showed impaired phosphotyrosine-associated PI 3-kinase activation, all of which was associated with IRS-2. Akt phosphorylation was reduced in parallel with the total PI 3-kinase activity. Inhibition of PI 3-kinase with LY294002 blocked differentiation of wild-type cells. Thus, IRS-1 appears to be an important mediator of brown adipocyte maturation. Furthermore, this signaling molecule appears to exert its unique role in the differentiation process via activation of PI 3-kinase and its downstream target, Akt, and is upstream of the effects of PPARgamma and C/EBPalpha.

Insulin receptor function was examined in cultured skin fibroblasts from three patients with leprechaunism (Ark-1, Minn-1, and Can-1), a rare syndrome of severe insulin resistance and neonatal growth retardation. All three patients cell lines demonstrated insulin binding less than 15% of control. This was primarily due to reduced affinity of the receptor in Can-1 and due to reduced number of receptors in the other two cell lines (Ark-1 and Minn-1). When expressed as a fraction of total insulin bound, the percentage of cell-associated insulin internalized and degraded did not differ between the patient cell lines and the controls. However, chloroquine, which inhibited degradation by 50% in the control cells, had no effect in the cells from the patients. When normalized to insulin binding, insulin receptor autophosphorylation was normal in cells from Can-1, but reduced in those of Ark-1 and Minn-1. In contrast, the receptor-associated tyrosine kinase activity toward exogenous substrates was decreased in all three patient cell lines. These results suggest that leprechaunism is a biochemically heterogenous disease associated with a variety of alterations in receptor function. Cells from Ark-1 and Minn-1 exhibit parallel alterations in receptor autophosphorylation and kinase activity. Cells from Can-1 demonstrate normal receptor autophosphorylation but reduced kinase activity, thus displaying a unique form of a mutant insulin receptor. Despite reduced kinase activity, all three cell lines exhibit normal rates of insulin internalization, but decreased lysosomal-mediated degradation. Our data imply that receptor autophosphorylation and kinase activity may be regulated separately and that kinase activity may be linked to insulin degradation, but not necessarily internalization.

Insulin receptor substrate-1 (IRS-1) is pivotal in mediating the actions of insulin and growth factors in most tissues of the body, but its role in insulin-producing beta islet cells is unclear. Freshly isolated islets from IRS-1 knockout mice and SV40-transformed IRS-1-deficient beta-cell lines exhibit marked insulin secretory defects in response to glucose and arginine. Furthermore, insulin expression is reduced by about 2-fold in the IRS-1-null islets and beta-cell lines, and this defect can be partially restored by transfecting the cells with IRS-1. These data provide evidence for an important role of IRS-1 in islet function and provide a novel functional link between the insulin signaling and insulin secretion pathways. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Insulin actions and receptors were studied in capillary endothelial cells cultured from diabetic BB rats and their nondiabetic colony mates. The endothelial cells from diabetic rats of 2 mo duration had persistent biological and biochemical defects in culture. Compared with normal rats, endothelial cells from diabetic rats grew 44% more slowly. Binding studies of insulin and insulin-like growth factor I (IGF-I) showed that cells from diabetic rats had 50% decrease of insulin receptor binding (nondiabetic: 4.6 +/- 0.7; diabetic: 2.6 +/- 0.4% per milligram protein, P less than 0.01), which was caused by a 50% decrease in the number of binding sites per milligram protein, whereas IGF-I binding was not changed. Insulin stimulation of 2-deoxy-glucose uptake and alpha-aminoisobutyric acid uptake were also severely impaired with a 80-90% decrease in maximal stimulation, in parallel with a 62% decrease in insulin-stimulated autophosphorylation (P less than 0.05). 125I-insulin cross-linking revealed an 140-kD alpha subunit of the insulin receptor similar to that in cells from nondiabetic rats, although bands at greater than 200 kD were also detected. The molecular weight of the insulin receptor beta subunit (by SDS-PAGE) was smaller in cells from diabetic than from normal rats (88-90 vs. 95 kD). Neuraminadase treatment of the partially purified insulin receptors decreased the molecular weight of the insulin receptors from nondiabetic rats to a greater degree than its diabetic counterpart. In contrast, Northern blot analysis of insulin receptor mRNAs using human cDNA probes revealed two species of 9.4 and 7.2 kb with no difference in mRNA abundance between cells from diabetic and nondiabetic rats. We conclude that the exposure of capillary endothelial cells to a diabetic milieu in vivo can cause specific and persistent changes in the insulin receptor and insulin action.

Mice made insulin receptor substrate 1 (IRS-1) deficient by targeted gene knockout exhibit growth retardation and abnormal glucose metabolism due to resistance to the actions of insulin-like growth factor 1 (IGF-1) and insulin (E. Araki et al., Nature 372:186-190, 1994; H. Tamemoto et al., Nature 372:182-186, 1994). Embryonic fibroblasts and 3T3 cell lines derived from IRS-1-deficient embryos exhibit no IGF-1-stimulated IRS-1 phosphorylation or IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity but exhibit normal phosphorylation of IRS-2 and Shc and normal IRS-2-associated PI 3-kinase activity. IRS-1 deficiency results in a 70 to 80% reduction in IGF-1-stimulated cell growth and parallel decreases in IGF-1-stimulated S-phase entry, PI 3-kinase activity, and induction of the immediate-early genes c-fos and egr-1 but unaltered activation of the mitogen-activated protein kinases ERK 1 and ERK 2. Expression of IRS-1 in IRS-1-deficient cells by retroviral gene transduction restores IGF-1-stimulated mitogenesis, PI 3-kinase activation, and c-fos and egr-1 induction in proportion to the level of reconstitution. Increasing the level of IRS-2 in these cells by using a retrovirus reconstitutes IGF-1 activation of PI 3-kinase and immediate-early gene expression to the same degree as expression of IRS-1; however, IRS-2 overexpression has only a minor effect on IGF-1 stimulation of cell cycle progression. These results indicate that IRS-1 is not necessary for activation of ERK 1 and ERK 2 and that activation of ERK 1 and ERK 2 is not sufficient for IGF-1-stimulated activation of c-fos and egr-1. These data also provide evidence that IRS-1 and IRS-2 are not functionally interchangeable signaling intermediates for stimulation of mitogenesis despite their highly conserved structure and many common functions such as activating PI 3-kinase and early gene expression.

To investigate the roles of insulin receptor substrate 3 (IRS-3) and IRS-4 in the insulin-like growth factor 1 (IGF-1) signaling cascade, we introduced these proteins into 3T3 embryonic fibroblast cell lines prepared from wild-type (WT) and IRS-1 knockout (KO) mice by using a retroviral system. Following transduction of IRS-3 or IRS-4, the cells showed a significant decrease in IRS-2 mRNA and protein levels without any change in the IRS-1 protein level. In these cell lines, IGF-1 caused the rapid tyrosine phosphorylation of all four IRS proteins. However, IRS-3- or IRS-4-expressing cells also showed a marked decrease in IRS-1 and IRS-2 phosphorylation compared to the host cells. This decrease was accounted for in part by a decrease in the level of IRS-2 protein but occurred with no significant change in the IRS-1 protein level. IRS-3- or IRS-4-overexpressing cells showed an increase in basal phosphatidylinositol 3-kinase activity and basal Akt phosphorylation, while the IGF-1-stimulated levels correlated well with total tyrosine phosphorylation level of all IRS proteins in each cell line. IRS-3 expression in WT cells also caused an increase in IGF-1-induced mitogen-activated protein kinase phosphorylation and egr-1 expression ( approximately 1.8- and approximately 2.4-fold with respect to WT). In the IRS-1 KO cells, the impaired mitogenic response to IGF-1 was reconstituted with IRS-1 to supranormal levels and was returned to almost normal by IRS-2 or IRS-3 but was not improved by overexpression of IRS-4. These data suggest that IRS-3 and IRS-4 may act as negative regulators of the IGF-1 signaling pathway by suppressing the function of other IRS proteins at several steps.

We have studied the function of a mutant insulin receptor (IR) molecule in which Tyr-1146, one of the first autophosphorylation sites in the beta subunit, was replaced with phenylalanine (IRF1146). Autophosphorylation of the partially purified IRF1146 was reduced 60-70% when compared to the wild-type IR but was still stimulated by insulin. The phosphotransferase activity of the dephospho form of both the IR and IRF1146 toward exogenous substrates was stimulated 3- to 4-fold by insulin. However, the wild-type IR was activated 12-fold by autophosphorylation, whereas the IRF1146 was activated only 2-fold. When the IRF1146 was expressed in Chinese hamster ovary (CHO) cells, insulin binding was normal, whereas autophosphorylation was reduced 80% when compared to cells expressing the wild-type IR. Endogenous substrates of the insulin receptor kinase were not detected during insulin stimulation of CHO cells expressing the IRF1146. Moreover, the IRF1146 did not internalize insulin rapidly or stimulate DNA synthesis in the presence of insulin. In contrast, both the IR and IRF1146 stimulated glycogen synthase equally in CHO cells. These data suggest that activation of the IR tyrosine kinase can be resolved into two components: the first is dependent on insulin binding and the second is dependent on the subsequent insulin-stimulated autophosphorylation cascade. Thus, at least two signal transduction pathways diverging from the IR are implicated in the mechanism of insulin action.

Insulin stimulates tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), which in turn binds to and activates phosphatidylinositol 3-kinase (PI 3-kinase). In the present study, we have examined these processes in animal models of insulin-resistant and insulin-deficient diabetes mellitus. After in vivo insulin stimulation, there was a 60-80% decrease in IRS-1 phosphorylation in liver and muscle of the ob/ob mouse. There was no insulin stimulation of PI 3-kinase (85 kD subunit) association with IRS-1, and IRS-1-associated PI 3-kinase activity was reduced 90%. Insulin-stimulated total PI 3-kinase activity was also absent in both tissues of the ob/ob mouse. By contrast, in the streptozotocin diabetic rat, IRS-1 phosphorylation increased 50% in muscle, IRS-1-associated PI 3-kinase activity was increased two- to threefold in liver and muscle, and there was a 50% increase in the p85 associated with IRS-1 after insulin stimulation in muscle. In conclusion, (a) IRS-1-associated PI 3-kinase activity is differentially regulated in hyperinsulinemic and hypoinsulinemic diabetic states; (b) PI 3-kinase activation closely correlates with IRS-1 phosphorylation; and (c) reduced PI 3-kinase activity may play a role in the pathophysiology of insulin resistant diabetic states, such as that seen in the ob/ob mouse.

Intracellular insulin signaling involves a series of alternative and complementary pathways created by the multiple substrates of the insulin receptor (IRS) and the various isoforms of SH2 domain signaling molecules that can interact with these substrates. In this study, we have evaluated the roles of IRS-1 and IRS-2 in signaling to the phosphatidylinositol (PI) 3-kinase pathway in the ob/ob mouse, a model of the insulin resistance of obesity and non-insulin-dependent diabetes mellitus. We find that the levels of expression of both IRS-1 and IRS-2 are decreased approximately 50% in muscle, whereas in liver the decrease is significantly greater for IRS-2 (72%) than for IRS-1 (29%). This results in differential decreases in IRS-1 and IRS-2 phosphorylation, docking of the p85alpha regulatory subunit of PI 3-kinase, and activation of this enzyme in these two insulin target tissues. In ob/ob liver there is also a change in expression of the alternatively spliced isoforms of the regulatory subunits for PI 3-kinase that was detected at the protein and mRNA level. This resulted in a 45% decrease in the p85alpha form of PI 3-kinase, a ninefold increase in the AS53/p55alpha, and a twofold increase in p50alpha isoforms. Thus, there are multiple alterations in the early steps of insulin signaling in the ob/ob mouse, with differential regulation of IRS-1 and IRS-2, various PI 3-kinase regulatory isoforms, and a lack of compensation for the decrease in insulin signaling by any of the known alternative pathways at these levels.

To investigate potential interactions between angiotensin II (AII) and the insulin signaling system in the vasculature, insulin and AII regulation of insulin receptor substrate-1 (IRS-1) phosphorylation and phosphatidylinositol (PI) 3-kinase activation were examined in rat aortic smooth muscle cells. Pretreatment of cells with AII inhibited insulin-stimulated PI 3-kinase activity associated with IRS-1 by 60%. While AII did not impair insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR) beta-subunit, it decreased insulin-stimulated tyrosine phosphorylation of IRS-1 by 50%. AII inhibited the insulin-stimulated association between IRS-1 and the p85 subunit of PI 3-kinase by 30-50% in a dose-dependent manner. This inhibitory effect of AII on IRS-1/PI 3-kinase association was blocked by the AII receptor antagonist saralasin, but not by AT1 antagonist losartan or AT2 antagonist PD123319. AII increased the serine phosphorylation of both the IR beta-subunit and IRS-1. In vitro binding experiments showed that autophosphorylation increased IR binding to IRS-1 from control cells by 2.5-fold versus 1.2-fold for IRS-1 from AII-stimulated cells, suggesting that AII stimulation reduces IRS-1's ability to associate with activated IR. In addition, AII increased p85 serine phosphorylation, inhibited the total pool of p85 associated PI 3-kinase activity, and decreased levels of the p50/p55 regulatory subunit of PI 3-kinase. These results suggest that activation of the renin-angiotensin system may lead to insulin resistance in the vasculature.

Insulin resistance is a feature of many common disorders including obesity and type 2 diabetes mellitus. In these disorders, the beta-cells compensate for the insulin resistance for long periods of time with an increase in secretory capacity, an increase in beta-cell mass, or both. To determine whether the beta-cell response might relate to a circulating growth factor, we have transplanted normal islets under the kidney capsule of normoglycemic insulin-resistant mice with two different models of insulin resistance: lean mice that have a double heterozygous deletion of the insulin receptor and insulin receptor substrate-1 (DH) or the obese, hyperglycemic ob/ob mice. In the grafts transplanted into both hosts, there was a marked increase in beta-cell mitotic activity and islet mass that was comparable with that observed in the endogenous pancreas. By contrast, islets of the DH mouse transplanted into normal mice showed reduced mitotic index. These data suggest the insulin resistance is associated with a circulating islet cell growth factor that is independent of glucose and obesity.

Leptin is a 16-kDa hormone secreted by adipocytes and plays an important role in control of feeding behavior and energy expenditure. In obesity, circulating levels of leptin and insulin are high because of the presence of increased body fat mass and insulin resistance. Recent reports have suggested that leptin can act through some of the components of the insulin signaling cascade, such as insulin receptor substrates (IRS-1 and IRS-2), phosphatidylinositol 3-kinase (PI 3-kinase), and mitogen-activated protein kinase, and can modify insulin-induced changes in gene expression in vitro and in vivo. Well differentiated hepatoma cells (Fao) possess both the long and short forms of the leptin receptor and respond to leptin with a stimulation of c-fos gene expression. In Fao cells, leptin alone had no effects on the insulin signaling pathway, but leptin pretreatment transiently enhanced insulin-induced tyrosine phosphorylation and PI 3-kinase binding to IRS-1, while producing an inhibition of tyrosine phosphorylation and PI 3-kinase binding to IRS-2. Leptin alone also induced serine phosphorylation of Akt and glycogen synthase kinase 3 but to a lesser extent than insulin, and the combination of these hormones was not additive. These results suggest complex interactions between the leptin and insulin signaling pathways that can potentially lead to differential modification of the metabolic and mitotic effects of insulin exerted through IRS-1 and IRS-2 and the downstream kinases that they activate.

We have studied the effects of oral administration of vanadate, an insulinometic agent and a potent inhibitor of phosphotyrosyl protein phosphatase (PTPase) in vitro, on blood glucose and PTPase action, in two hyperinsulinemic rodent models of non-insulin-dependent diabetes mellitus (NIDDM). Oral administration of vanadate (0.25 mg/ml in the drinking water) to ob/ob mice for 3 wk lowered blood glucose level from 236 +/- 4 to 143 +/- 2 mg/dl without effect on body weight. Administration of vanadate to db/db mice produced a similar effect. Electron microscopic examination revealed no signs of hepatotoxicity after 47 d of treatment. There was a slight reduction in insulin receptor autophosphorylation when tested by immunoblotting with antiphosphotyrosine antibody after in vivo stimulation, and the phosphorylation of the endogenous substrate of the insulin receptor, pp185, was markedly decreased in the ob/ob mice. Both cytosolic and particulate PTPase activities in liver of ob/ob mice measured by dephosphorylation of a 32P-labeled peptide corresponding to the major site of insulin receptor autophosphorylation were decreased by approximately 50% (P less than 0.01). In db/db diabetic mice, PTPase activity in the cytosolic fraction was decreased to 53% of control values (P less than 0.02) with no significant difference in the particulate PTPase activity. Treatment with vanadate did not alter hepatic PTPase activity as assayed in vitro, or receptor and substrate phosphorylation as assayed in vivo, in ob/ob mice despite its substantial effect on blood glucose. These data indicate that vanadate is an effective oral hypoglycemic treatment in NIDDM states and suggest that its major effects occurs distal to the insulin receptor tyrosine kinase.

The principal substrate for the insulin and insulin-like growth factor-1 (IGF-1) receptors is the cytoplasmic protein insulin-receptor substrate-1 (IRS-1/pp185). After tyrosine phosphorylation at several sites, IRS-1 binds to and activates phosphatidylinositol-3'-OH kinase (PI(3)K) and several other proteins containing SH2 (Src-homology 2) domains. To elucidate the role of IRS-1 in insulin/IGF-1 action, we created IRS-1-deficient mice by targeted gene mutation. These mice had no IRS-1 and showed no evidence of IRS-1 phosphorylation or IRS-1-associated PI(3)K activity. They also had a 50 per cent reduction in intrauterine growth, impaired glucose tolerance, and a decrease in insulin/IGF-1-stimulated glucose uptake in vivo and in vitro. The residual insulin/IGF-1 action correlated with the appearance of a new tyrosine-phosphorylated protein (IRS-2) which binds to PI(3)K, but is slightly larger than and immunologically distinct from IRS-1. Our results provide evidence for IRS-1-dependent and IRS-1-independent pathways of insulin/IGF-1 signalling and for the existence of an alternative substrate of these receptor kinases.

Leprechaunism is a rare genetic disorder characterized by severe growth retardation and insulin resistance. Maximal epidermal growth factor (EGF) binding was reduced in fibroblasts from three unrelated patients with leprechaunism (Ark-1, Can-1, and Minn-1) compared with control (0.8-2.2%/mg protein vs. 5.5%/mg protein). This was due to a decrease in receptor affinity in Ark-1 and Can-1 and a decrease in receptor number in Minn-1. In all cell lines, EGF-stimulated receptor autophosphorylation was also decreased to 18-60% of control, whereas EGF internalization and degradation was normal. Sphingosine (40 microM), a protein kinase C inhibitor, increased EGF receptor affinity twofold in control cells and six- to nine-fold in cells of leprechaunism. However, sphingosine did not enhance EGF-stimulated receptor autophosphorylation in either the controls or the patients' cells. By contrast, only one of the three cell lines of patients with the type A syndrome demonstrated a decrease in EGF binding and all demonstrated normal or near normal EGF-stimulated receptor autophosphorylation. These data indicate that in patients with leprechaunism, there are functional abnormalities of the EGF receptor, as well as of the insulin receptor, that may contribute to the severity of the syndrome. These data also suggest a role for the insulin receptor in maintaining normal EGF receptor function in these cells.

Interaction between a peptide hormone and extracellular domains of its receptor is a crucial step for initiation of hormone action. We have developed a modification of the yeast two-hybrid system to study this interaction and have used it to characterize the interaction of insulin-like growth factor 1 (IGF-1) with its receptor by using GAL4 transcriptional regulation with a beta-galactosidase assay as readout. In this system, IGF-1 and proIGF-1 bound to the cysteine-rich domain, extracellular domain, or entire IGF-1 proreceptor. This interaction was specific. Thus, proinsulin showed no significant interaction with the IGF-1 receptor, while a chimeric proinsulin containing the C-peptide of IGF-1 had an intermediate interaction, consistent with its affinity for the IGF-1 receptor. Over 2000 IGF-1 mutants were generated by PCR and screened for interaction with the color assay. About 40% showed a strong interaction, 20% showed an intermediate interaction, and 40% give little or no signal. Of 50 mutants that were sequenced, several (Leu-5 --> His, Glu-9 --> Val, Arg-37 --> Gly, and Met-59 --> Leu) appeared to enhance receptor association, others resulted in weaker receptor interaction (Tyr-31 --> Phe and Ile-43 --> Phe), and two gave no detectable signal (Leu-14 --> Arg and Glu-46 --> Ala). Using PCR-based mutagenesis with proinsulin, we also identified a gain of function mutant (proinsulin Leu-17 --> Pro) that allowed for a strong IGF-1-receptor interaction. These data demonstrate that the specificity of the interaction between a hormone and its receptor can be characterized with high efficiency in the two-hybrid system and that novel hormone analogues may be found by this method.

Phosphotyrosine phosphatase (PTPase) activity in rat liver was measured using a phosphopeptide substrate containing sequence identity to the major site of insulin receptor autophosphorylation. PTPase activity was detected in both cytosolic and particulate fractions of rat liver and produced linear dephosphorylation over a 15-min time course. In rats made insulin-deficient diabetic by streptozotocin treatment (STZ), cytosolic PTPase activity increased to 180% of the control values after 2 d of diabetes and remained elevated at 30 d (P less than 0.02). Gel filtration on Sephadex-75 revealed a single peak of activity in the cytosol in both control and diabetic animals and confirmed the increased levels. In BB diabetic rats, another model of insulin deficiency, the PTPase activity in the cytosolic fraction was increased to approximately 230% of control values. PTPase activity in the particulate fraction of liver was also increased by 30 and 80% after 2 and 8 d of STZ diabetes, respectively. However, this increase was not sustained and after 30 d of STZ diabetes, PTPase activity associated with the particulate fraction in the BB diabetic rat was reduced to approximately 70% of the control levels. Treatment of STZ diabetic rats with subcutaneous insulin or vanadate in their drinking water for 3 d reduced tyrosine PTPase activity in the particulate, but not in the cytosolic fraction. This was associated with a change in blood glucose toward normal. These data indicate insulin deficient diabetes is accompanied by significant changes in hepatic PTPase activity. Since tyrosine phosphorylation plays a central role in the cellular action of insulin receptor, an increase in PTPase activity may be an important factor in the altered insulin action associated with these diabetic states.