To test whether the tyrosine kinase activity of the insulin receptor is crucial for insulin action, we have constructed mutations of the human insulin receptor at Lys-1030, which is in the presumed ATP-binding region. By using oligonucleotide-directed mutagenesis, this lysine residue was replaced with either methionine, arginine, or alanine. Chinese hamster ovary cells were transfected by mutant cDNAs and the expressed insulin receptors were characterized. We show here that none of these mutants exhibited insulin-activated autophosphorylation and kinase activity in vitro. They also do not mediate insulin-and antibody-stimulated uptake of 2-deoxyglucose. The tyrosine kinase activity is thus required for a key physiological response of insulin.Insulin initiates its diverse biological effects by binding to its receptor, an integral membrane glycoprotein composed of two a (Mr = 135,000) and two ,B (Mr = 95,000) subunits linked by disulfide bonds (1, 2). An immediate consequence of insulin binding to the a subunit is the activation of an intrinsic tyrosine kinase activity located in the ,3 subunit (3, 4). Since tyrosine kinase activity is also found associated with receptors of several other hormones (5), and many of the effects of insulin are caused by changes in the phosphorylation state of various proteins (6), it has been suggested that some or all of the effects of insulin are mediated by the receptor's kinase activity. A variety of experiments supports this hypothesis (7)(8)(9)(10)(11)(12), including the finding that a long-term effect of insulin is blocked by a monoclonal antibody that inhibits the insulin receptor (IR) kinase (13). However, other studies utilizing polyclonal and monoclonal antireceptor antibodies show a stimulation of glucose uptake in adipocytes without stimulating the kinase activity of the receptor (14-17). Thus, the role the kinase activity plays in the responses to insulin is still uncertain.The isolation and sequencing of the human placental IR cDNA has now provided primary structural information about the receptor protein (18,19). The a subunit (735 amino acids) contains a cysteine-rich crosslinking domain and presumably the insulin-binding site; the /3 subunit (620 amino acids) contains the single transmembrane domain of the receptor and the tyrosine kinase domain with the presumed ATP-binding region and potential tyrosine phosphorylation sites (18, 19). Furthermore, the functional human IR has been expressed in Chinese hamster ovary (CHO) cells that are stably transformed with the human IR cDNA (20). This system permits the further analysis of the structure-function relationship of the receptor molecule and the role of the receptor kinase in eliciting the various physiological responses to insulin. In a previous study, Ellis et al. (21) have shown that removal of the C-terminal region of the IR renders the cytoplasmic domain unstable, and it is removed, presumably by processing. The resulting truncated receptor is capable of binding insulin normally but exhibits no kinase act...
The insulin receptor has an intrinsic tyrosine kinase activity that is essential for signal transduction. A mutant insulin receptor gene lacking almost the entire kinase domain has been identified in an individual with type A insulin resistance and acanthosis nigricans. Insulin binding to the erythrocytes or cultured fibroblasts from this individual was normal. However receptor autophosphorylation and tyrosine kinase activity toward an exogenous substrate were reduced in partially purified insulin receptors from the proband's lymphocytes that had been transformed by Epstein-Barr virus. The insulin resistance associated with this mutated gene was inherited by the proband from her mother as an apparently autosomal dominant trait. Thus a deletion in one allele of the insulin receptor gene may be at least partly responsible for some instances of insulin-resistant diabetes.
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