Characterization of skeletal muscle insulin receptor

Dubler, Robert E.; Whipple, Judith H.; Larner, Joseph

doi:10.1016/0003-9861(85)90611-3

Cited by 11 publications

(2 citation statements)

References 31 publications

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“…Tyrosine phosphorylation was detected with antiphosphotyrosine-horseradish peroxidase conjugates (PY-20). To determine the effects of gangliosides on IR phosphorylation, IR was partially purified from skeletal muscle of C57BL/6 mice at 4°C by a modification of the methods of Dubler et al (21). Briefly, the particulate fractions of homogenates, prepared in 50 mM HEPES, pH 7.5, 8% sucrose, 10 mM EDTA, 10 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 M leupeptin, and 1 M pepstatin A, were solubilized with 2% Triton X-100 and further purified by sequential column chromatography on Sephacryl S-200 and wheat germ agglutinin-agarose.…”

Section: Methodsmentioning

confidence: 99%

Overexpression of Plasma Membrane-associated Sialidase Attenuates Insulin Signaling in Transgenic Mice

Sasaki

Hata

Suzuki

et al. 2003

Journal of Biological Chemistry

108

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Plasma membrane-associated sialidase is a key enzyme for ganglioside hydrolysis, thereby playing crucial roles in regulation of cell surface functions. Here we demonstrate that mice overexpressing the human ortholog (NEU3) develop diabetic phenotype by 18 -22 weeks associated with hyperinsulinemia, islet hyperplasia, and increased ␤-cell mass. As compared with the wild type, insulin-stimulated phosphorylation of the insulin receptor (IR) and insulin receptor substrate I was significantly reduced, and activities of phosphatidylinositol 3-kinase and glycogen synthase were low in transgenic muscle. IR phosphorylation was already attenuated in the younger mice before manifestation of hyperglycemia. Transient transfection of NEU3 into 3T3-L1 adipocytes and L6 myocytes caused a significant decrease in IR signaling. In response to insulin, NEU3 was found to undergo tyrosine phosphorylation and subsequent association with the Grb2 protein, thus being activated and causing negative regulation of insulin signaling. In fact, accumulation of GM1 and GM2, the possible sialidase products in transgenic tissues, caused inhibition of IR phosphorylation in vitro, and blocking of association with Grb2 resulted in reversion of impaired insulin signaling in L6 cells. The data indicate that NEU3 indeed participates in the control of insulin signaling, probably via modulation of gangliosides and interaction with Grb2, and that the mice can serve as a valuable model for human insulin-resistant diabetes.Gangliosides are a family of sialic acid-containing glycosphingolipids present in the cell surface membranes. Several lines of evidence suggest their important functional roles in regulating a wide range of biological processes including cell growth, cell differentiation, and transmembrane signaling (1-3). Most of the observations on ganglioside function, however, have been performed so far by using bacterial sialidases and exogenous gangliosides to mimic in vivo expression. To obtain further insights into their physiological significance and regulation mechanism, it is necessary to focus on endogenous sialidases responsible for ganglioside hydrolysis inside the cells. Three types of mammalian sialidase have been cloned and established to have low identity with each other in their primary structure. They differ in enzymatic properties and subcellular localization, being mainly found in lysosomes (Neu1), cytosol (Neu2), and plasma membranes (Neu3). Other than the contribution of lysosomal sialidase to glycoconjugate catabolism in lysosomes, their cellular roles are not well understood. The fact that plasma membrane sialidase is unique in specifically hydrolyzing gangliosides (4 -10) and in its subcellular localization in plasma membranes, where levels of other glycosidases are very low, suggests participation in cell surface events through modulation of gangliosides. To obtain evidence of this, we previously cloned sialidase cDNAs of mammalian origin (8, 9, 11, 12), and we have recently investigated effects of overexpression in transgenic...

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Section: Methodsmentioning

confidence: 99%

Overexpression of Plasma Membrane-associated Sialidase Attenuates Insulin Signaling in Transgenic Mice

Sasaki

Hata

Suzuki

et al. 2003

Journal of Biological Chemistry

108

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“…Of these, skeletal muscle represents the largest target, making up -2 5 -4 5 % of body weight in men (2). Although insulin receptors from this tissue have been identified and characterized (3,4), relatively few studies have been performed on the receptor kinase of skeletal muscle, largely due to technical difficulties in isolating the receptor.…”

mentioning

confidence: 99%

Calmodulin as Substrate for Insulin-Receptor Kinase: Phosphorylation by Receptors From Rat Skeletal Muscle

Sacks

McDonald²

1989

Diabetes

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Calmodulin is a substrate for insulin-receptor kinase obtained from rat adipocytes and hepatocytes and human placenta. In this study, we demonstrate that insulin stimulates the phosphorylation of calmodulin via insulin receptors partially purified from rat skeletal muscle. Phosphorylation of calmodulin was maximal in the presence of Mg2+ and insulin and the absence of Ca2+. Free-Ca2+ concentrations greater than 0.1 microM progressively inhibited phosphorylation with almost total inhibition at 200 microM Ca2+. Insulin-stimulated phosphorylation of calmodulin was dose dependent and saturable with half-maximal effect obtained at approximately 5 x 10(-10) M insulin. There was an absolute requirement for certain basic proteins, e.g., polylysine or protamine sulfate, to obtain phosphate incorporation into calmodulin. Polylysine stimulated the phosphorylation of calmodulin independently of insulin, but this was increased up to sixfold by the addition of insulin. Phosphate incorporation into calmodulin increased with increasing concentration of the substrate up to a saturating concentration of 2.4 microM. The Km for calmodulin was approximately 0.2 microM. Up to 0.15 mol of phosphate was incorporated per mole of calmodulin with tyrosine the predominant amino acid phosphorylated. The observations that calmodulin is phosphorylated by insulin-receptor kinase from all three classic target organs for insulin confirm that calmodulin is a general substrate for this kinase and suggest that Ca2+ and calmodulin may be components of the insulin-signaling mechanism.

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Hormonal control of muscle growth

1987

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In muscle of whole animals, pituitary growth hormone, the thyroid hormones, and insulin are major growth-promoting hormones, and the glucocorticoids have significant catabolic actions. At the cellular level the primary anabolic hormones for cultured myoblasts are the somatomedins (insulin-like growth factors) and fibroblast growth factor. In these cells physiological concentrations of growth hormone, thyroid hormones, and insulin have no growth-promoting effect; some of the reported actions of insulin probably result from cross-reaction with the somatomedin receptor. Results with purified proteins do not support the view that mitogens block myoblast differentiation; transforming growth factor-beta and interferon are nonmitogenic proteins that inhibit differentiation, insulin-like growth factors are mitogens that stimulate differentiation, and fibroblast growth factor is the only purified mitogen that inhibits differentiation. At least six serum-free media have now been devised for the growth of various kinds of muscle cells under closely defined conditions.

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Characterization of skeletal muscle insulin receptor

Cited by 11 publications

References 31 publications

Overexpression of Plasma Membrane-associated Sialidase Attenuates Insulin Signaling in Transgenic Mice

Overexpression of Plasma Membrane-associated Sialidase Attenuates Insulin Signaling in Transgenic Mice

Calmodulin as Substrate for Insulin-Receptor Kinase: Phosphorylation by Receptors From Rat Skeletal Muscle

Hormonal control of muscle growth

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