Abstract
We studied a patient with severe insulin resistance and a remarkable decrease in the in vivo autophosphorylation of the insulin receptor. Using a polymerase chain reaction-single strand conformation polymorphism method and direct sequencing, we identified a heterozygous mutation substituting Gln for Arg1131 in the putative "catalytic loop" of the tyrosine kinase domain of the insulin receptor gene. The Gln1131 mutant receptor was expressed by transfection in Chinese hamster ovary cells and compared with cells expressing the wild-type insulin receptor. Both mutant and wild-type receptors were expressed on the cell surface and displayed similar insulin-binding affinity. The Gln1131 mutation impaired the activity of the receptor tyrosine kinase and inhibited the ability of insulin to phosphorylate the endogenous substrate insulin receptor substrate-I. In addition, the Gln1131 mutant receptor exhibited diminished tyrosine-phosphorylated phosphatidylinositol 3-kinase and myelin basic protein kinase activities compared with the wild-type cells. It also demonstrated a defective mediation of the insulin signal stimulating 2-deoxy-D-glucose transport and thymidine incorporation, resistance to endocytosis, and insulin-induced down-regulation. Unlike a previously described mutation in the putative catalytic loop of the receptor that substituted Glu for Ala1135, the Gln1131 mutation retained proteolytic cleavage of the proreceptor into separate subunits. Our results demonstrate that a naturally occurring mutation (R1131Q) in the putative catalytic loop of the insulin receptor results in severe impairment of the tyrosine kinase function in our patient. In addition, our results indicate that Arg1131 is important for receptor-mediated insulin action in vivo and suggest that the amino acids constituting the catalytic loop of protein kinases may possess different modes in order to retain kinase function.
Highlights
A NEWLY IDENTIFIED MUTATION IN THE CATALYTIC LOOP OF THE TYROSINE KINASE DOMAIN OF THE HUMAN INSULIN RECEF'TOR*
The cloning and sequencingof insulin receptor cDNA in the phism method and direct sequencing, we identified a last few years have made ipt ossible to identify over 40 mutaheterozygous mutation substitutingGln forArg"" in the tions in the human insulirneceptor gene [8,9].Recent studies putative "catalytic loop" of the tyrosine kinase domain revealed that the kinase domain in thecytoplasmic portion of of the insulin receptor gene
Consistent with the factthat mutations that alter the tyrosine kinase domain have been identified in insulin receptor genes of patients with genetic formsof ited the ability of insulin to phosphorylate the endog- insulin resistance (10, ll),studies using in vitro site-directed enous substrate insulin receptor substrate-I
Summary
A NEWLY IDENTIFIED MUTATION IN THE CATALYTIC LOOP OF THE TYROSINE KINASE DOMAIN OF THE HUMAN INSULIN RECEF'TOR*. Both mutant and wild-type receptors were expressed on the cell surface and displayed similar insulin-binding affinity. Our results demonstrate that a naturally occurring mutation (R1131Q) in the putative catalytic loop of the insulin receptor results in severe impairment of the tyrosine kinase function in our patient.
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