Differential effect of pp120 on insulin endocytosis by two variant insulin receptor isoforms

Calzi, Sergio Li; Choice, Curtis V.; Najjar, Sonia M.

doi:10.1152/ajpendo.1997.273.4.e801

Cited by 17 publications

(28 citation statements)

References 30 publications

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“…Nonetheless, they persist in the fusion pellet independently of the phosphorylation state of pp120, suggesting that they may not be significant components of the internalization complex. DISCUSSION We have reported previously that co-expression of fulllength, not truncated, pp120 in NIH 3T3 cells increased receptor-mediated insulin endocytosis and degradation compared with cells expressing insulin receptors alone (11,12). In the current studies, we observed that pp120 regulated receptormediated insulin endocytosis by taking part in a complex of proteins required for insulin endocytosis.…”

Section: Fig 2 Indirect Immunofluorescence Pp120 Does Not Undergo supporting

confidence: 62%

“…Moreover, receptor-mediated insulin endocytosis and degradation were 2-3-fold higher in NIH 3T3 cells co-expressing fulllength pp120 than in cells expressing insulin receptors alone. More recently, we have reported that full-length pp120 stimulated insulin endocytosis via high affinity (receptor-A) rather than low affinity (receptor-B) insulin receptors, despite being equally phosphorylated by both isoforms (12). This suggests that pp120-induced increase in insulin endocytosis and degradation does not depend on its phosphorylation state.…”

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confidence: 86%

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Insulin Stimulates pp120 Endocytosis in Cells Co-expressing Insulin Receptors

Choice¹,

Howard²,

Poy³

et al. 1998

Journal of Biological Chemistry

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pp120, a substrate of the insulin receptor tyrosine kinase, is a plasma membrane glycoprotein that is expressed in the hepatocyte as two spliced isoforms differing by the presence (full-length) or absence (truncated) of most of the intracellular domain including all phosphorylation sites. Co-expression of full-length pp120, but not its phosphorylation-defective isoforms, increased receptor-mediated insulin endocytosis and degradation in NIH 3T3 fibroblasts. We, herein, examined whether internalization of pp120 is required to mediate its effect on insulin endocytosis. The amount of fulllength pp120 expressed at the cell surface membrane, as measured by biotin labeling, markedly decreased in response to insulin only when insulin receptors were coexpressed. In contrast, when phosphorylation-defective pp120 mutants were co-expressed, the amount of pp120 expressed at the cell surface did not decrease in response to insulin. Indirect immunofluorescence analysis revealed that upon insulin treatment of cells co-expressing insulin receptors, full-length, but not truncated, pp120 co-localized with ␣-adaptin in the adaptor protein complex that anchors endocytosed proteins to clathrincoated pits. This suggests that full-length pp120 is part of a complex of proteins required for receptor-mediated insulin endocytosis and that formation of this complex is regulated by insulin-induced pp120 phosphorylation by the receptor tyrosine kinase. In vitro GST binding assays and co-immunoprecipitation experiments in intact cells further revealed that pp120 did not bind directly to the insulin receptor and that its association with the receptor may be mediated by other cellular proteins.Insulin binding to the extracellular ␣-subunits of its receptor triggers activation of the tyrosine kinase in the intracellular domain of the ␤-subunits. Activation of the tyrosine kinase causes phosphorylation of the receptor and of endogenous substrates, including pp120, a plasma membrane glycoprotein of M r ϳ120,000 that is expressed in the liver as two spliced variants differing by the inclusion (full-length) or exclusion (truncated) of a 61 amino acid (aa) 1 segment in the C terminus of its cytoplasmic domain (1). The truncated isoform lacks all phosphorylation sites. Site-directed mutagenesis studies in NIH 3T3 mouse skin fibroblasts revealed that full-length pp120 is constitutively phosphorylated by cAMP-dependent serine kinase on Ser 503 and that this phosphorylation is required for its phosphorylation on Tyr 488 residue by the insulin receptor tyrosine kinase (2). Tyr 513 , the other tyrosine residue in the cytoplasmic domain of pp120, is not involved in insulinmediated pp120 phosphorylation (2).Binding of insulin to its receptor triggers the entry of the insulin-receptor complex into clathrin-coated pits, which pinch off the surface membrane to form clathrin-coated vesicles (3). The clathrin coat is formed upon receptor activation by the sequestration of the cytoplasmic adaptor proteins 2 (AP2) to the inner surface of the membrane where they bin...

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Section: Fig 2 Indirect Immunofluorescence Pp120 Does Not Undergo supporting

confidence: 62%

mentioning

confidence: 86%

Insulin Stimulates pp120 Endocytosis in Cells Co-expressing Insulin Receptors

Choice¹,

Howard²,

Poy³

et al. 1998

Journal of Biological Chemistry

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“…Confluent cells were lysed in 1% Triton X-100 lysis buffer supplemented with protease inhibitors (7) prior to analysis by SDS-PAGE and immunoblotting with α-CC1, as described previously (21). Screening for expression of EGFRs was achieved by measurement of EGF binding in intact cells, as described previously (7,26).…”

Section: Methodsmentioning

confidence: 99%

“…It exerts this effect by undergoing internalization as part of the receptor-mediated insulin endocytosis complex and increasing its cellular uptake via clathrin-coated vesicles (25)(26)(27)(28). Transgenic mice with liver-specific overexpression of the phosphorylation-defective S503A CEACAM1-4L mutant (L-SACC1) develop impaired insulin clearance, hyperinsulinemia, insulin resistance, and increased visceral obesity (9).…”

Section: Introductionmentioning

confidence: 99%

CEACAM1 modulates epidermal growth factor receptor–mediated cell proliferation

Abou-Rjaily¹,

Lee²,

May³

et al. 2004

J. Clin. Invest.

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Phosphorylation of the cell adhesion protein CEACAM1 increases insulin sensitivity and decreases insulindependent mitogenesis in vivo. Here we show that CEACAM1 is a substrate of the EGFR and that upon being phosphorylated, CEACAM1 reduces EGFR-mediated growth of transfected Cos-7 and MCF-7 cells in response to EGF. Using transgenic mice overexpressing a phosphorylation-defective CEACAM1 mutant in liver (L-SACC1), we show that the effect of CEACAM1 on EGF-dependent cell proliferation is mediated by its ability to bind to and sequester Shc, thus uncoupling EGFR signaling from the ras/MAPK pathway. In L-SACC1 mice, we also show that impaired CEACAM1 phosphorylation leads to ligand-independent increase of EGFR-mediated cell proliferation. This appears to be secondary to visceral obesity and the metabolic syndrome, with increased levels of output of free fatty acids and heparin-binding EGF-like growth factor from the adipose tissue of the mice. Thus, L-SACC1 mice provide a model for the mechanistic link between increased cell proliferation in states of impaired metabolism and visceral obesity.

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“…Insulin Binding and Internalization-Primary hepatocytes, isolated as described previously (5), were grown for 24 h prior to [ 125 I]insulin (10 pM) binding at 4°C (30). Unbound insulin was removed, and cells were incubated at 37°C for 0 -60 min before incubating in 0.1% bovine serum albumin/phosphate-buffered saline (pH 3.5) for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Differential effect of pp120 on insulin endocytosis by two variant insulin receptor isoforms

Cited by 17 publications

References 30 publications

Insulin Stimulates pp120 Endocytosis in Cells Co-expressing Insulin Receptors

Insulin Stimulates pp120 Endocytosis in Cells Co-expressing Insulin Receptors

CEACAM1 modulates epidermal growth factor receptor–mediated cell proliferation

Interaction between Altered Insulin and Lipid Metabolism in CEACAM1-inactive Transgenic Mice

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