Generation of Oligomeric Insulin Receptor Forms by Intramolecular Sulfhydryl-Disulfide Exchange. Involvement of Masked Sulfhydryl Groups

Krämer, Helmut; Deger, Arno; Koch, R; Rapp, Reinhard; Hinz, Marianne; Weber, Ulrich

doi:10.1515/bchm3.1987.368.1.471

Cited by 12 publications

(1 citation statement)

References 23 publications

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“…The lysate was incubated with an excess of Ultralink-immobilized protein G (Pierce) and autoantibodies to the IR that were obtained from patient B-19 with clinical type B insulin-resistance syndrome (15). In contrast to other experiments described here, proteins were separated under non-reducing conditions by omitting dithiothreitol and adding 10 mM NEM (16) to the lysate in addition to the Laemmli sample buffer. Samples were boiled 3 min before resolving using SDS-PAGE.…”

Section: Expression In Mammalian Cellsmentioning

confidence: 99%

Characterization of Drosophila Insulin Receptor Substrate

Poltilove¹,

Jacobs²,

Haft³

et al. 2000

Journal of Biological Chemistry

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Insulin receptor substrate (IRS) proteins are phosphorylated by multiple tyrosine kinases, including the insulin receptor. Phosphorylated IRS proteins bind to SH2 domain-containing proteins, thereby triggering downstream signaling pathways. The Drosophila insulin receptor (dIR) C-terminal extension contains potential binding sites for signaling molecules, suggesting that dIR might not require an IRS protein to accomplish its signaling functions. However, we obtained a cDNA encoding Drosophila IRS (dIRS), and we demonstrated expression of dIRS in a Drosophila cell line. Like mammalian IRS proteins, the N-terminal portion of dIRS contains a pleckstrin homology domain and a phosphotyrosine binding domain that binds to phosphotyrosine residues in both human and Drosophila insulin receptors. When coexpressed with dIRS in COS-7 cells, a chimeric receptor (the extracellular domain of human IR fused to the cytoplasmic domain of dIR) mediated insulin-stimulated tyrosine phosphorylation of dIRS. Mutating the juxtamembrane NPXY motif markedly reduced the ability of the receptor to phosphorylate dIRS. In contrast, the NPXY motifs in the C-terminal extension of dIR were required for stable association with dIRS. Coimmunoprecipitationexperimentsdemonstratedinsulindependent binding of dIRS to phosphatidylinositol 3-kinase and SHP2. However, we did not detect interactions with Grb2, SHC, or phospholipase C-␥. Taken together with published genetic studies, these biochemical data support the hypothesis that dIRS functions directly downstream from the insulin receptor in Drosophila.

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Section: Expression In Mammalian Cellsmentioning

confidence: 99%

Characterization of Drosophila Insulin Receptor Substrate

Poltilove¹,

Jacobs²,

Haft³

et al. 2000

Journal of Biological Chemistry

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Truncation of the ectodomain of the human insulin receptor results in secretion of a soluble insulin binding protein from transfected CHO cells

Ellis

Sissom

Levitan

1988

J of Molecular Recognition

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The insulin receptor is an integral transmembrane glycoprotein comprised of two alpha-(approximately 135 kDa) and two beta-(approximately 95 kDa) subunits, which is synthesized as a single polypeptide chain precursor (alpha beta). The primary sequence of the human insulin receptor (hIR) protein, deduced from the nucleotide sequence of cloned human placental mRNAs, predicts two large domains (929 and 403 residues) on either side of a single membrane spanning domain (23 residues); each of these major domains has a distinct function (insulin binding and protein/tyrosine kinase activity, respectively). To experimentally test this deduced topology, and to explore the potential for independent domain function by the hIR extracellular domain, we have constructed an expression plasmid encoding an hIR deletion mutant which is truncated 8 residues from the beginning of the predicted transmembrane domain (i.e., 921 residues). This domain of the hIR is in fact processed into alpha- and truncated beta-subunits and secreted with high efficiency from transfected CHO cell lines which express this mutant hIR, and the protein accumulates as an (alpha beta)2 dimer in the medium. This molecule is recognized by a battery of 13 monoclonal antibodies to epitopes on the IR extracellular domain, four of which block insulin binding and two of which require the native conformation of the IR for recognition. Further, this domain binds insulin with an apparent dissociation constant comparable to that of the wild-type hIR. However, the secreted dimer displays a linear Scatchard plot, while that of the wild-type membrane-associated hIR is curvilinear.(ABSTRACT TRUNCATED AT 250 WORDS)

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