Abstract
Transgenic mice which overexpress kinase-deficient human insulin receptors in muscle were used to study the relationship between insulin receptor tyrosine kinase and the in vivo activation of several downstream signaling pathways. Intravenous insulin stimulated insulin receptor tyrosine kinase activity by 7-fold in control muscle versus < or = 1.5-fold in muscle from transgenic mice. Similarly, insulin failed to stimulate tyrosyl phosphorylation of receptor beta-subunits or insulin receptor substrate 1 (IRS-1) in transgenic muscle. Insulin substantially stimulated IRS-1-associated phosphatidylinositol (PI) 3-kinase in control versus absent stimulation in transgenic muscles. In contrast, insulin-like growth factor 1 modestly stimulated PI 3-kinase in both control and transgenic muscle. The effects of insulin to stimulate p42 mitogen-activated protein kinase and c-fos mRNA expression were also markedly impaired in transgenic muscle. Specific immunoprecipitation of human receptors followed by measurement of residual insulin receptors suggested the presence of hybrid mouse-human heterodimers. In contrast, negligible hybrid formation involving insulin-like growth factor 1 receptors was evident. We conclude that (i) transgenic expression of kinase-defective insulin receptors exerts dominant-negative effects at the level of receptor auto-phosphorylation and kinase activation; (ii) insulin receptor tyrosine kinase activity is required for in vivo insulin-stimulated IRS-1 phosphorylation, IRS-1-associated PI 3-kinase activation, phosphorylation of mitogen-activated protein kinase, and c-fos gene induction in skeletal muscle; (iii) hybrid receptor formation is likely to contribute to the in vivo dominant-negative effects of kinase-defective receptor expression.
Highlights
Insulin's diverse actions are initiated by binding to its specific transmembrane receptor
We investigated the consequences of impaired insulin receptor kinase activity for the ability of insulin administered in vivo to regulate molecules that participate in several important signaling pathways (IRS-I, PI 3-kinase, mitogen-activated protein (MAP) kinase, and c-fos mRNA) in muscle
Assessment of Potential Hybrid Receptor Formation-To explore potential mechanisms for the dominant-negative effects of mutant receptor expression, we investigated whether hybrid receptors composed of mouse-human insulin receptor halves or mouse IGF-1 receptor-human insulin receptor halves were present in muscle from transgenic mice
Summary
That most, if not all, of insulin's biological effects are mediated by the insulin receptor tyrosine kinase [1]. Recent studies have shown that insulin can activate both PI 3-kinase and the rasMAP kinase cascade in mice lacking IRS-I [15, 16] This IRSI-independent signaling may involve receptor-mediated phosphorylation of She [17] or a newly identified substrate referred to as IRS-2 [16]. Skeletal muscle from these transgenic mice displayed reduced insulin-stimulated insulin receptor tyrosine kinase activity [22] This animal model provides a unique opportunity to study the effects of altered insulin receptor tyrosine kinase in an important insulin target tissue. We investigated the consequences of impaired insulin receptor kinase activity for the ability of insulin administered in vivo to regulate molecules that participate in several important signaling pathways (IRS-I, PI 3-kinase, MAP kinase, and c-fos mRNA) in muscle. We provide evidence which supports the hypothesis that the mechanism(s) responsible for trans-dominant effects of kinase-deficient insulin receptor expression involves the formation of hybrid heterodimer complexes between endogenous murine receptors and overexpressed mutant human receptors
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