Substrate Phosphorylation Catalyzed by the Insulin Receptor Tyrosine Kinase

Flores-Riveros, Jaime; Sibley, Eric; Kastelic, Tania; Lane, M. Daniel

doi:10.1016/s0021-9258(20)88221-0

Cited by 131 publications

(25 citation statements)

References 31 publications

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“…The mouse insulin receptor also reacted with antibody CT-l (results not shown). This is consistent with the known sequences of rodent and human receptors which are identical in the C-terminal 15 amino acids apart from the presence of valine (rat, mouse) rather than isoleucine (human) ten residues from the terminus (Ebina et al, 1985;Flores-Riveros et al, 1989;Goldstein & Dudley, 1990). Antibody CT-1 was approximately one-fifth as effective on a concentration basis as antibody (Fig.…”

Section: Resultssupporting

confidence: 87%

“…The mean density for negative colonies was 5.4 + 1.4 units. For comparison, the C-terminal sequences of the human insulin receptor (Ullrich et al, 1985;Ebina et al, 1985), the rat/mouse insulin receptor (Flores-Riveros et al, 1989;Goldstein & Dudley, 1990) and the human IGF-I receptor (Ullrich et al, 1986) are also shown.…”

Section: Rodent Insulin Receptormentioning

confidence: 99%

“…Sequences at the Cterminus are favoured in this context, as these are likely to be more accessible and less conformationally constrained than those in other parts of the molecule. In the case of the insulin receptor there is the additional advantage that the C-terminal sequence is conserved between species (Ullrich et al, 1985;Ebina et al, 1985;Flores-Riveros et al, 1989;Goldstein & Dudley, 1990). We set out to obtain antibodies for this sequence which would be useful in the precipitation, purification and assay of both human and rodent insulin receptors.…”

Section: Introductionmentioning

confidence: 99%

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A monoclonal anti-peptide antibody reacting with the insulin receptor β-subunit. Characterization of the antibody and its epitope and use in immunoaffinity purification of intact receptors

Ganderton

Stanley

Field

et al. 1992

Biochemical Journal

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A mouse monoclonal antibody (CT-1) was prepared against the C-terminal peptide sequence of the human insulin receptor beta-subunit (KKNGRILTLPRSNPS). The antibody reacted with native human and rat insulin receptors in solution, whether or not insulin was bound and whether or not the receptor had undergone prior tyrosine autophosphorylation. The antibody also reacted specifically with the receptor beta-subunit on blots of SDS/polyacrylamide gels. Preincubation of soluble receptors with antibody increased the binding of 125I-insulin approx. 2-fold. The antibody did not affect insulin-stimulated autophosphorylation, but increased the basal autophosphorylation rate approx. 2-fold. The amino acid residues contributing to the epitope for CT-1 were defined by construction and screening of an epitope library. Oligonucleotides containing 23 random bases were synthesized and ligated into the vector pCL627, and the corresponding peptide sequences expressed as fusion proteins in Escherichia coli were screened by colony blotting. Reactive peptides were identified by sequencing the oligonucleotide inserts in plasmids purified from positive colonies. Six different positive sequences were found after 900,000 colonies had been screened, and the consensus epitope was identified as GRVLTLPRS. Phosphorylation of the threonine residue within this sequence (corresponding to the known phosphorylation site Thr-1348 in the insulin receptor) decreased the affinity of antibody binding approx. 100-fold, as measured by competition in an e.l.i.s.a. Antibody CT-1 was used for immunoaffinity isolation of insulin receptor from detergent-solubilized human placental or rat liver microsomal membranes. Highly purified receptor was obtained in 60% yield by binding to CT-1-Sepharose immunoadsorbent and specific elution with a solution of peptide corresponding to the known epitope. This approach to purification under very mild conditions may in principle be used with any protein for which an antibody is available and for which a peptide epitope or 'mimotope' can be identified.

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Section: Resultssupporting

confidence: 87%

Section: Rodent Insulin Receptormentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A monoclonal anti-peptide antibody reacting with the insulin receptor β-subunit. Characterization of the antibody and its epitope and use in immunoaffinity purification of intact receptors

Ganderton

Stanley

Field

et al. 1992

Biochemical Journal

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“…(2) autophosphorylation in the central subdomain is associated with activation (Ellis et al, 1986;Flores Riveros et al, 1989; Herrera & Rosen, 1986; Murakami & Rosen, 1991;Tornqvist & Avruch, 1988;White et al, 1988;Zhang et al, 1991); and…”

Section: Discussionmentioning

confidence: 99%

“…reviews by Ellis et al (1991) and Olefsky, (1990)]. There are three subdomains that undergo autophosphorylation, including seven discrete tyrosine residues of the /3-subunit (Ellis et al, 1986;Flores Riveros et al, 1989; Herrera & Rosen, 1986; Murakami & Rosen, 1991; Tornqvist & Avruch, 1988; White et al, 1988), two of which are first identified in the following paper in this issue (Kohanski, 1993). The isolated cytoplasmic kinase domain of the insulin receptor has been expressed using insect cell cultures and the baculovirus expression system (Cobb et al, 1989;Villalba et al, 1989).…”

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confidence: 99%

Insulin receptor autophosphorylation. I. Autophosphorylation kinetics of the native receptor and its cytoplasmic kinase domain

Ra¹

1993

Biochemistry

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Kinetic analysis of autophosphorylation was done using a non-Michaelis-Menten kinetic model. This model describes autophosphorylation in terms of a fast reaction phase, a slow reaction phase, and a partition function for the two phases. Kinetic parameters determined by this new approach show that insulin stimulates autophosphorylation by promoting (1) a 10-fold increase in the rate constant for the fast phase of the reaction and (2) a 2-fold increase in the partition function favoring the fast phase. Insulin did not significantly affect the binding constant for ATP in this fast phase. Kinetic parameters obtained for the cytoplasmic kinase domain were similar to those obtained for the native insulin receptor in the absence of insulin. The insulin receptor has three subdomains encompassing its seven autophosphorylation sites. The juxtamembrane sites react primarily in the slow kinetic phase, favored by the absence of stimulation and low ATP concentrations. The carboxy-terminal and central autophosphorylation subdomains react primarily in the fast kinetic phase, favored by raising the ATP concentration and/or the presence of insulin. These observations demonstrate that: (1) both ATP and insulin regulate reaction in each autophosphorylation subdomain, (2) insulin stimulation occurs predominantly in the central and carboxy-terminal regions, and (3) autophosphorylation observed with the cytoplasmic kinase domain was similar to native insulin receptor in the absence of insulin. These findings are consistent with conclusions based on the kinetic analysis of autophosphorylation.

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Antidiabetic agent pioglitazone increases insulin receptors on 3T3‐L1 adipocytes

Swanson

Bleasdale

1995

Drug Development Research

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Pioglitazone and other antidiabetic thiazolidinediones appear to overcome insulin resistance by affecting an unidentified early event in insulin action. Because attempts to determine changes in insulin receptors in tissues from thiazolidinedione-treated diabetic animals are complicated by drug-induced reduction of hyperinsulinemia and consequent receptor up-regulation, 3T3-LI cells were utilized here as an in vitro model system. After the excess insulin required for optimal differentiation of 3T3-LI adipocytes was removed from the culture medium, there was an increase in specific binding of insulin to the cell surface that was further augmented 25 and 47% in cells treated with pioglitazone for 12 and 24 h, respectively (EC,, about 0.6 pM, maximal at 5-25 FM). Pioglitazone increased the number of insulin receptors without changing binding affinity. Specific IGF-I binding was not affected by pioglitazone in the 3T3-LI cells. Glycerol 3-phosphate dehydrogenase activity, a marker of adipocyte differentiation, was also increased in a dose-dependent manner by pioglitazone (maximal at 10-25 pM). Tunicamycin, which inhibits the N-linked glycosylation of newly synthesized insulin receptors that is required for their translocation to the cell surface, decreased specific insulin binding by about 50% after 24 h, and this decrease remained unchanged when pioglitazone was simultaneously present. Pioglitazone increased not only cell surface insulin receptors, but also total cellular insulin receptors (quantitated either by specific insulin binding or by immunoblotting) and insulin receptor mRNA. In addition, among several structurally related thiazolidinediones there was a positive correlation between in vivo antihyperglycemic activity and their ability to increase insulin binding in vitro, suggesting a possible common molecular mechanism for these actions. o 1995 WiIey-Liss, Inc.

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Substrate Phosphorylation Catalyzed by the Insulin Receptor Tyrosine Kinase

Cited by 131 publications

References 31 publications

A monoclonal anti-peptide antibody reacting with the insulin receptor β-subunit. Characterization of the antibody and its epitope and use in immunoaffinity purification of intact receptors

A monoclonal anti-peptide antibody reacting with the insulin receptor β-subunit. Characterization of the antibody and its epitope and use in immunoaffinity purification of intact receptors

Insulin receptor autophosphorylation. I. Autophosphorylation kinetics of the native receptor and its cytoplasmic kinase domain

Antidiabetic agent pioglitazone increases insulin receptors on 3T3‐L1 adipocytes

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