Morris F. White

Mutation of the insulin receptor at tyrosine 960 inhibits signal transmission but does not affect its tyrosine kinase activity

Tyrosyl phosphorylation is implicated in the mechanism of insulin action. Mutation of the beta-subunit of the insulin receptor by substitution of tyrosyl residue 960 with phenylalanine had no effect on insulin-stimulated autophosphorylation or phosphotransferase activity of the purified receptor. However, unlike the normal receptor, this mutant was not biologically active in Chinese hamster ovary cells. Furthermore, insulin-stimulated tyrosyl phosphorylation of at least one endogenous substrate (pp185) was increased significantly in cells expressing the normal receptor but was barely detected in cells expressing the mutant. Therefore, beta-subunit autophosphorylation was not sufficient for the insulin response, and a region of the insulin receptor around Tyr-960 may facilitate phosphorylation of cellular substrates required for transmission of the insulin signal.

Contrasting Effects of IRS-1 Versus IRS-2 Gene Disruption on Carbohydrate and Lipid Metabolism in Vivo

To examine the impact of homozygous genetic disruption of insulin receptor substrate (IRS)-1 (IRS-1−/−) or IRS-2 (IRS-2−/−) on basal and insulin-stimulated carbohydrate and lipid metabolism in vivo, we infused 18-h fasted mice (wild-type (WT), IRS-1−/−, and IRS-2−/−) with [3-3H]glucose and [2H5]glycerol and assessed rates of glucose and glycerol turnover under basal (0–90 min) and hyperinsulinemic-euglycemic clamp (90–210 min; 5 mm glucose, and 5 milliunits of insulin·kg− 1·min− 1) conditions. Both IRS-1− /− and IRS-2− /− mice were insulin-resistant as reflected by markedly impaired insulin-stimulated whole-body glucose utilization compared with WT mice. Insulin resistance in the IRS-1− /− mice could be ascribed mainly to decreased insulin-stimulated peripheral glucose metabolism. In contrast, IRS-2− /− mice displayed multiple defects in insulin-mediated carbohydrate metabolism as reflected by (i) decreased peripheral glucose utilization, (ii) decreased suppression of endogenous glucose production, and (iii) decreased hepatic glycogen synthesis. Additionally, IRS-2− /− mice also showed marked insulin resistance in adipose tissue as reflected by reduced suppression of plasma free fatty acid concentrations and glycerol turnover during the hyperinsulinemic-euglycemic clamp. These data suggest important tissue-specific roles for IRS-1 and IRS-2 in mediating the effect of insulin on carbohydrate and lipid metabolismin vivo in mice. IRS-1 appears to have its major role in muscle, whereas IRS-2 appears to impact on liver, muscle, and adipose tissue.

Calmodulin Activates Phosphatidylinositol 3-Kinase

Calmodulin and phosphatidylinositol 3-kinase are vital components of a number of common intracellular events. Calmodulin, a ubiquitous Ca2+-dependent effector protein, regulates multiple processes in eukaryotic cells, including cytoskeletal organization, vesicular trafficking, and mitogenesis. Phosphatidylinositol 3-kinase participates in events downstream of the receptors for insulin and other growth factors. Here we demonstrate by coimmunoprecipitation and affinity chromatography that Ca2+/calmodulin associates with Src homology 2 domains in the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase, thereby significantly enhancing phosphatidylinositol 3-kinase activity in vitro and in intact cells. Furthermore, CGS9343B, a calmodulin antagonist, inhibited basal and Ca2+-stimulated phosphorylation of phosphatidylinositol in intact cells. These data demonstrate a novel mechanism for modulating phosphatidylinositol 3-kinase and provide a direct link between components of two fundamental signaling pathways.

Insulin Receptor Substrate-2 Is Not Necessary for Insulin- and Exercise-stimulated Glucose Transport in Skeletal Muscle

Insulin receptor substrate-2-deficient (IRS2−/−) mice develop type 2 diabetes. The purpose of this study was to determine whether there is a defect in basal, insulin-, and exercise-stimulated glucose transport in the skeletal muscle of these animals. IRS2−/− and wild-type (WT) mice (male, 8–10 weeks) exercised on a treadmill for 1 h or remained sedentary. 2-Deoxyglucose (2DG) uptake was measured in isolated soleus muscles incubated in vitro in the presence or absence of insulin. Resting blood glucose concentration in IRS2−/−mice (10.3 mm) was higher than WT animals (4.1 mm), but there was a wide range among the IRS2−/− mice (3–25 mm). Therefore, IRS2−/− mice were divided into two subgroups based on blood glucose concentrations (IRS2−/−L < 7.2 mm, IRS2−/−H > 7.2 mm). Only IRS2−/−H had lower basal, exercise-, and submaximally insulin-stimulated 2DG uptake, while maximal insulin-stimulated 2DG uptake was similar among the three groups. The ED50 for insulin to stimulate 2DG uptake above basal in IRS2−/−H was higher than WT and IRS2−/−L mice, suggesting insulin resistance in the skeletal muscle from the IRS2−/− mice with high blood glucose concentrations. Furthermore, resting blood glucose concentrations from all groups were negatively correlated to submaximally insulin-stimulated 2DG uptake (r 2 = 0.33, p < 0.01). Muscle GLUT4 content was significantly lower in IRS2−/−H mice compared with WT and IRS2−/−L mice. These results demonstrate that the IRS2 protein in muscle is not necessary for insulin- or exercise-stimulated glucose transport, suggesting that the onset of diabetes in the IRS2−/− mice is not due to a defect in skeletal muscle glucose transport; hyperglycemia may cause insulin resistance in the muscle of IRS2−/− mice.

Human Skeletal Muscle Insulin Receptor Substrate-1: Characterization of the cDNA, Gene, and Chromosomal Localization

Insulin receptor substrate-1 is a major substrate of insulin receptor Tyr kinase. We have now cloned the IRS-1 cDNA from human skeletal muscle, one of the most important target tissues of insulin action, localized and cloned the human IRS-1 gene, and studied the expression of the protein in Chinese hamster ovary cells. Human IRS-1 cDNA encodes a 1242 amino acid sequence that is 88% identical with rat liver IRS-1. The 14 potential Tyr phosphorylation sites include 6 Tyr-Met-X-Met motifs and 3 Tyr-X-X-Met motifs that are completely conserved in human IRS-1. Human IRS-1 has >50 possible Ser/Thr phosphorylation sites and one potential ATP-binding site close to the NH2-terminal. The human IRS-1 gene contains the entire 5ʹ-untranslated region and protein coding region in a single exon and was localized on chromosome 2 q36–37 by in situ hybridization. By Northern blot analysis, IRS-1 mRNA is rare and consists of two species of 6.9 and 6 kilobase. By using quantitative polymerase chain reaction after reverse transcription of total RNA from human fetal tissues, IRS-1 mRNA could be identified in all tissues. When human IRS-1 cDNA was expressed in Chinese hamster ovary cells, the protein migrated between 170,000–180,000 Mr in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was rapidly Tyr phosphorylated upon insulin stimulation. Thus, IRS-1 is widely expressed and highly conserved across species and tissues. Compared with rat protein, human IRS-1 contains more potential Ser/Thr phosphorylation sites and only one nucleotide binding site. The entire protein coding sequence is contained within a single exon.

The insulin receptor and its substrate: molecular determinants of early events in insulin action.

Publisher Summary This chapter describes insulin receptor and its substrate. It discusses the two earliest molecular events, binding and activation of the insulin receptor kinase and phosphorylation and subsequent signal transduction by the insulin receptor substrate IRS-1. Defining these events has helped add a whole new dimension to the understanding of insulin action. At the same time, studies defining the molecular events at the end of the insulin action cascade, such as activation of glucose transport and regulation of gene expression, have also begun to clarify the specific components required for these signaling events. Although a black box remains between the early and late events in insulin action, it is becoming smaller.

Insulin-stimulated oocyte maturation requires insulin receptor substrate 1 and interaction with the SH2 domains of phosphatidylinositol 3-kinase

Xenopus oocytes from unprimed frogs possess insulin-like growth factor I (IGF-I) receptors but lack insulin and IGF-I receptor substrate 1 (IRS-1), the endogenous substrate of this kinase, and fail to show downstream responses to hormonal stimulation. Microinjection of recombinant IRS-1 protein enhances insulin-stimulated phosphatidylinositol (PtdIns) 3-kinase activity and restores the germinal vesicle breakdown response. Activation of PtdIns 3-kinase results from formation of a complex between phosphorylated IRS-1 and the p85 subunit of PtdIns 3-kinase. Microinjection of a phosphonopeptide containing a pYMXM motif with high affinity for the src homology 2 (SH2) domain of PtdIns 3-kinase p85 inhibits IRS-1 association with and activation of the PtdIns 3-kinase. Formation of the IRS-1-PtdIns 3-kinase complex and insulin-stimulated PtdIns 3-kinase activation are also inhibited by microinjection of a glutathione S-transferase fusion protein containing the SH2 domain of p85. This effect occurs in a concentration-dependent fashion and results in a parallel loss of hormone-stimulated oocyte maturation. These inhibitory effects are specific and are not mimicked by glutathione S-transferase fusion proteins expressing the SH2 domains of ras-GAP or phospholipase C gamma. Moreover, injection of the SH2 domains of p85, ras-GAP, and phospholipase C gamma do not interfere with progesterone-induced oocyte maturation. These data demonstrate that phosphorylation of IRS-1 plays an essential role in IGF-I and insulin signaling in oocyte maturation and that this effect occurs through interactions of the phosphorylated YMXM/YXXM motifs of IRS-1 with SH2 domains of PtdIns 3-kinase or some related molecules.

7 The Insulin Receptor and Tyrosine Phosphorylation

Publisher Summary This chapter discusses the structure and function of the insulin receptor to establish the hypothesis that tyrosine kinases play a key role in cellular regulation and transmission of the insulin signal. The insulin receptor is composed of two functional domains: (1) an extracellular regulatory subunit that binds insulin and (2) a transmembrane subunit that contains tyrosine kinase activity. The tyrosine kinase is intrinsic to the insulin receptor and is present in all cells studied so far that contain the receptors. One of the earliest detectable responses to insulin binding in an intact cell is activation of this kinase and tyrosine phosphorylation of intracellular domains on the β-subunit. From studies with the purified insulin receptor, autophosphorylation activates the phosphotransferase in the β-subunit that catalyzes tyrosine phosphorylation of other proteins on tyrosine residues. The search for substrates in the intact cell has been difficult because of their very low concentration; however, phosphotyrosine antibodies have provided a very sensitive tool to begin identifying the cellular substrates for the insulin receptor and other tyrosine kinases.

The Insulin Receptor Protein Kinase

Phosphorylation reactions catalyzed by protein kinases rapidly and reversibly modify the activity of cellular enzymes. Considerable evidence indicates that phosphorylation and dephosphorylation of proteins play an important role in the action of insulin and other hormones.(1–4) Receptors for several growth factors and neurotransmitters(5–14) are also phosphorylated, and in some cases this phosphorylation is increased after binding of the hormone. In addition, protein kinases are associated with certain transforming retroviruses that have profound effects on cellular growth and metabolism.(14–20) These findings suggest that phosphorylation may be an early step in the transmembrane signal process. In 1981, we began to study the possibility that the insulin receptor undergoes a phosphorylation reaction.(21) In this chapter, we review briefly the structure of the insulin receptor kinase, describe the kinetic characteristics of the receptor kinase measured in vitro, compare and contrast the phosphorylation of the receptor in broken and intact cells, and finally describe the phosphorylation of the receptor in certain insulin-resistant states.

Structure and function of tyrosine kinase receptors.

Over the past ten years, several growth factor receptors have been shown to be ligand-regulated tyrosine kinases. Tyrosine kinase activity is essential for signal transmission, suggesting that phosphorylation cascades may play an important role. Considerable effort has gone into understanding the structure and function of tyrosine kinase receptors in order to define their mechanisms of signal transmission. However, the protein substrates of the receptor kinases have proven to be difficult to isolate and clone. This review focuses on the receptors for insulin, epidermal growth factor, and platelet-derived growth factor. They are all tyrosine kinases, but emerging evidence suggests that they utilize multiple separate signal transduction pathways. Work carried out during the next several years should yield considerable insight into the complexity of the components which interact with these tyrosine kinase receptors to regulate cellular growth and metabolism.