The role of insulin receptor kinase domain autophosphorylation in receptor-mediated activities

Analysis with insulin and anti-receptor antibodies

Peter A. Wilden, Kenneth Siddle, Eva Haring, Jonathan M. Backer, Morris F. White, C. Ronald Kahn

Research output: Contribution to journalArticle

91 Citations (Scopus)

Abstract

The role of specific tyrosine autophosphorylation sites in the human insulin receptor kinase domain (Tyr1158, Tyr1162, and Tyr1163) was analyzed using in vitro mutagenesis to replace tyrosine residues individually or in combination. Each of the three single-Phe, the three possible double-Phe a triple-Phe and a triple-Ser mutant receptors, stably expressed in Chinese hamster ovary cells, were compared with the wild-type receptor in their ability to mediate stimulation of receptor kinase activity, glycogen synthesis, and DNA synthesis by insulin or the human-specific anti-receptor monoclonal antibody 83-14. At a concentration of 0.1 nM insulin which produced approximately half-maximal responses with wild-type receptor, DNA synthesis and glycogen synthesis mediated by the three single-Phe mutants ranged from 52 to 88% and from 32 to 79% of the wild-type receptor, respectively. The corresponding figures for the double-Phe mutants averaged 15 and 6%, whereas the triple-mutants were unresponsive in both assays. The level of biological function approximately paralleled the insulin-stimulated tyrosine kinase activity in the intact cell as estimated by tyrosine phosphorylation of the insulin receptor and its endogenous substrate pp185/IRS-1. Interestingly, all mutants showed a marked decrease in insulin-stimulated receptor internalization. Antireceptor antibody stimulated receptor kinase activity and mimicked insulin action in these cells. In general, the impairment of the metabolic response was greater and impairment of the growth response was less when antibody was the stimulus. These experiments show that the level and specific sites of autophosphorylation are critical determinants of receptor function. The data are consistent with a requirement for the receptor tyrosine kinase either as an obligatory step or a modulator, in both metabolic and growth responses, and demonstrate the important role of the level of insulin receptor kinase domain autophosphorylation in regulating insulin sensitivity.

Original languageEnglish (US)
Pages (from-to)13719-13727
Number of pages9
JournalJournal of Biological Chemistry
Volume267
Issue number19
StatePublished - Jul 5 1992
Externally publishedYes

Fingerprint

Insulin Receptor
Anti-Idiotypic Antibodies
Phosphotransferases
Insulin
Tyrosine
Antibodies
Glycogen
Receptor Protein-Tyrosine Kinases
Growth
Cricetulus
Mutagenesis
Protein-Tyrosine Kinases
Insulin Resistance
Ovary
Phosphorylation
Monoclonal Antibodies
Modulators
Assays
DNA
Thermodynamic properties

ASJC Scopus subject areas

  • Biochemistry

Cite this

The role of insulin receptor kinase domain autophosphorylation in receptor-mediated activities : Analysis with insulin and anti-receptor antibodies. / Wilden, Peter A.; Siddle, Kenneth; Haring, Eva; Backer, Jonathan M.; White, Morris F.; Kahn, C. Ronald.

In: Journal of Biological Chemistry, Vol. 267, No. 19, 05.07.1992, p. 13719-13727.

Research output: Contribution to journalArticle

Wilden, Peter A. ; Siddle, Kenneth ; Haring, Eva ; Backer, Jonathan M. ; White, Morris F. ; Kahn, C. Ronald. / The role of insulin receptor kinase domain autophosphorylation in receptor-mediated activities : Analysis with insulin and anti-receptor antibodies. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 19. pp. 13719-13727.
@article{a38986ad248b45809b19e0d661c784ea,
title = "The role of insulin receptor kinase domain autophosphorylation in receptor-mediated activities: Analysis with insulin and anti-receptor antibodies",
abstract = "The role of specific tyrosine autophosphorylation sites in the human insulin receptor kinase domain (Tyr1158, Tyr1162, and Tyr1163) was analyzed using in vitro mutagenesis to replace tyrosine residues individually or in combination. Each of the three single-Phe, the three possible double-Phe a triple-Phe and a triple-Ser mutant receptors, stably expressed in Chinese hamster ovary cells, were compared with the wild-type receptor in their ability to mediate stimulation of receptor kinase activity, glycogen synthesis, and DNA synthesis by insulin or the human-specific anti-receptor monoclonal antibody 83-14. At a concentration of 0.1 nM insulin which produced approximately half-maximal responses with wild-type receptor, DNA synthesis and glycogen synthesis mediated by the three single-Phe mutants ranged from 52 to 88{\%} and from 32 to 79{\%} of the wild-type receptor, respectively. The corresponding figures for the double-Phe mutants averaged 15 and 6{\%}, whereas the triple-mutants were unresponsive in both assays. The level of biological function approximately paralleled the insulin-stimulated tyrosine kinase activity in the intact cell as estimated by tyrosine phosphorylation of the insulin receptor and its endogenous substrate pp185/IRS-1. Interestingly, all mutants showed a marked decrease in insulin-stimulated receptor internalization. Antireceptor antibody stimulated receptor kinase activity and mimicked insulin action in these cells. In general, the impairment of the metabolic response was greater and impairment of the growth response was less when antibody was the stimulus. These experiments show that the level and specific sites of autophosphorylation are critical determinants of receptor function. The data are consistent with a requirement for the receptor tyrosine kinase either as an obligatory step or a modulator, in both metabolic and growth responses, and demonstrate the important role of the level of insulin receptor kinase domain autophosphorylation in regulating insulin sensitivity.",
author = "Wilden, {Peter A.} and Kenneth Siddle and Eva Haring and Backer, {Jonathan M.} and White, {Morris F.} and Kahn, {C. Ronald}",
year = "1992",
month = "7",
day = "5",
language = "English (US)",
volume = "267",
pages = "13719--13727",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "19",

}

TY - JOUR

T1 - The role of insulin receptor kinase domain autophosphorylation in receptor-mediated activities

T2 - Analysis with insulin and anti-receptor antibodies

AU - Wilden, Peter A.

AU - Siddle, Kenneth

AU - Haring, Eva

AU - Backer, Jonathan M.

AU - White, Morris F.

AU - Kahn, C. Ronald

PY - 1992/7/5

Y1 - 1992/7/5

N2 - The role of specific tyrosine autophosphorylation sites in the human insulin receptor kinase domain (Tyr1158, Tyr1162, and Tyr1163) was analyzed using in vitro mutagenesis to replace tyrosine residues individually or in combination. Each of the three single-Phe, the three possible double-Phe a triple-Phe and a triple-Ser mutant receptors, stably expressed in Chinese hamster ovary cells, were compared with the wild-type receptor in their ability to mediate stimulation of receptor kinase activity, glycogen synthesis, and DNA synthesis by insulin or the human-specific anti-receptor monoclonal antibody 83-14. At a concentration of 0.1 nM insulin which produced approximately half-maximal responses with wild-type receptor, DNA synthesis and glycogen synthesis mediated by the three single-Phe mutants ranged from 52 to 88% and from 32 to 79% of the wild-type receptor, respectively. The corresponding figures for the double-Phe mutants averaged 15 and 6%, whereas the triple-mutants were unresponsive in both assays. The level of biological function approximately paralleled the insulin-stimulated tyrosine kinase activity in the intact cell as estimated by tyrosine phosphorylation of the insulin receptor and its endogenous substrate pp185/IRS-1. Interestingly, all mutants showed a marked decrease in insulin-stimulated receptor internalization. Antireceptor antibody stimulated receptor kinase activity and mimicked insulin action in these cells. In general, the impairment of the metabolic response was greater and impairment of the growth response was less when antibody was the stimulus. These experiments show that the level and specific sites of autophosphorylation are critical determinants of receptor function. The data are consistent with a requirement for the receptor tyrosine kinase either as an obligatory step or a modulator, in both metabolic and growth responses, and demonstrate the important role of the level of insulin receptor kinase domain autophosphorylation in regulating insulin sensitivity.

AB - The role of specific tyrosine autophosphorylation sites in the human insulin receptor kinase domain (Tyr1158, Tyr1162, and Tyr1163) was analyzed using in vitro mutagenesis to replace tyrosine residues individually or in combination. Each of the three single-Phe, the three possible double-Phe a triple-Phe and a triple-Ser mutant receptors, stably expressed in Chinese hamster ovary cells, were compared with the wild-type receptor in their ability to mediate stimulation of receptor kinase activity, glycogen synthesis, and DNA synthesis by insulin or the human-specific anti-receptor monoclonal antibody 83-14. At a concentration of 0.1 nM insulin which produced approximately half-maximal responses with wild-type receptor, DNA synthesis and glycogen synthesis mediated by the three single-Phe mutants ranged from 52 to 88% and from 32 to 79% of the wild-type receptor, respectively. The corresponding figures for the double-Phe mutants averaged 15 and 6%, whereas the triple-mutants were unresponsive in both assays. The level of biological function approximately paralleled the insulin-stimulated tyrosine kinase activity in the intact cell as estimated by tyrosine phosphorylation of the insulin receptor and its endogenous substrate pp185/IRS-1. Interestingly, all mutants showed a marked decrease in insulin-stimulated receptor internalization. Antireceptor antibody stimulated receptor kinase activity and mimicked insulin action in these cells. In general, the impairment of the metabolic response was greater and impairment of the growth response was less when antibody was the stimulus. These experiments show that the level and specific sites of autophosphorylation are critical determinants of receptor function. The data are consistent with a requirement for the receptor tyrosine kinase either as an obligatory step or a modulator, in both metabolic and growth responses, and demonstrate the important role of the level of insulin receptor kinase domain autophosphorylation in regulating insulin sensitivity.

UR - http://www.scopus.com/inward/record.url?scp=0026748806&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026748806&partnerID=8YFLogxK

M3 - Article

VL - 267

SP - 13719

EP - 13727

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 19

ER -