Abstract
The intracellular domain of the insulin receptor possesses activity as a tyrosine-specific protein kinase which is stimulated by insulin binding to the extracellular domain of the receptor. We have identified a patient with a genetic form of insulin resistance who is heterozygous for a mutation substituting Glu for Ala1135 in the putative "catalytic loop" of the tyrosine kinase domain of the receptor. In this investigation, the Glu1135 mutant receptor was expressed by transfection of mutant cDNA into NIH-3T3 cells. Like previously described mutations in the tyrosine kinase domain, the Glu1135 mutation impairs receptor tyrosine kinase activity and inhibits the ability of insulin to stimulate thymidine incorporation and receptor endocytosis. These data support the hypothesis that the receptor tyrosine kinase activity plays a necessary role in the ability of the receptor to mediate insulin action in vitro and in vivo. However, unlike previously described mutations in the intracellular domain of the receptor, the Glu1135 mutation impairs proteolytic cleavage of the proreceptor into separate subunits and impairs the transport of the receptor to the cell surface. These latter defects provide an explanation for the decrease in the number of receptors on the cell surface observed in the patient's circulating monocytes despite the fact that the mutant receptor is resistant to endocytosis and insulin-induced down-regulation.
Highlights
Which is stimulated by insulin binding to the extracel- dependent proteinkinase, amino acid residue Ala1135is located lular domain of thereceptor
Unlike previously de- patient had previously been noted to have a marked decrease scribed mutations in the intracellular domain of the in the number of insulin receptors on the cell surface (Kahn receptor,the G ~ u "m~ut~ationimpairsproteolytic et al, 1976;Bar et al, 1980).Inasmuch as theG ~ u m" u~ta~tion cleavage of the proreceptor into separate subunits ancdauses a defect in receptor-mediated endocytosis i n vitro, it is impairs the transpoorftthe receptor to ctheell surface. unlikely thatthe decrease inthe number of cell surface
The supernatants were immunoprecipitatedwith site-specific anti-receptor antiserum rAb-50 (1:200) or with normal rabbit serum, from GCGA"to GAG"" (Fig. 1).The presenceof the mutation was confirmed by two techniques: sequencing the antisense strand of amplified genomic DNA and allele-specific oligonucleotidehybridization of amplifiedgenomicDNA and and the immunoprecipitateswere analyzed by NaDodS04-polyacryl- cDNA.o investigate the inheritancoef the amide gel electrophoresis followed by fluorography in the presencoef Glul13S mutation, we amplified exon 19of the insulinreceptor
Summary
Vol 268, No 11, Issue of April 15,pp. 8060-8069,1993 Printed in U.S.A. Substitution of Glutamic Acid for Alanine1135 in the Putative "Catalytic Loop" of the Tyrosine KinaseDomain of the Human Insulin Receptor. In most previously described patients (Cama et al, 1991, 1992; Yamamoto-Honda etal., 1990; Moller et al, 1990a,1990b, 1991),mutations in the tyrosine kinase domain of the receptor are associated with a normal number of receptors on the cell cytosis. On the basis twice with ice-cold PBS' to remove unbound insulin, the cells were of binding assays and immunoblot analysis, the partially purified solubilized in 1N NaOH for 2 h, and cell-associated radioactivity was extracts from the transfected cells were diluted to approximately quantitated in a y counter (Kadowaki et aL, 1990a, 1990d). Receptor Tyrosine Kinase Actiuity-Partially purified insulin receptors (50 pl) were incubated in the presence or absence of insulin l The abbreviations used are: PBS,phospha~-buffered saline; overnight a t 4 "C in 60 pl of phosphorylation reaction mixture con- DMEM, Dulbecco's modified Eagle's medium. At each time point,cells were washed with ice-cold PBS andsolubilized in buffer containingoctyl glucoside
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