Abstract
pp120/HA4 is a hepatocyte membrane glycoprotein phosphorylated by the insulin receptor tyrosine kinase. In this study, we have investigated the role of pp120/HA4 in insulin action. Transfection of antisense pp120/HA4 cDNA in H35 hepatoma cells resulted in inhibition of pp120/HA4 expression and was associated with a 2-3-fold decrease in the rate of insulin internalization. Furthermore, insulin internalization in NIH 3T3 fibroblasts co-transfected with insulin receptors and pp120/HA4 was increased 2-fold compared with cells expressing insulin receptors alone. In contrast, no effect on internalization was observed in cells overexpressing a naturally occurring splice variant of pp120/HA4 that lacks the phosphorylation sites in the intracellular domain. Insulin internalization was also unaffected in cells expressing three site-directed mutants of pp120/HA4 in which the sites of phosphorylation by the insulin receptor kinase had been removed (Y488F, Y488F/Y513F, and S503A). Our data suggest that pp120/HA4 is part of a complex of proteins required for receptor-mediated internalization of insulin. It is possible that this function is regulated by insulin-induced phosphorylation of the intracellular domain of pp120/HA4.
Highlights
From the ‡Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, and the §Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43699-0008
We have investigated the role of pp120/ HA4, a substrate of the insulin receptor kinase that is predominantly expressed in liver, in the internalization process. pp120/HA4 is a transmembrane glycoprotein that is phosphorylated by the insulin receptor tyrosine kinase in intact cells (17–20) and in cell-free systems (21)
We report that insulin-induced receptor internalization is inhibited 2–3-fold in H35 hepatoma cells transfected with an antisense pp120/HA4 cDNA
Summary
Vol 270, No 41, Issue of October 13, pp. 24073–24077, 1995 Printed in U.S.A. Pietro Formisano‡, Sonia M. Insulin internalization was unaffected in cells expressing three site-directed mutants of pp120/HA4 in which the sites of phosphorylation by the insulin receptor kinase had been removed (Y488F, Y488F/Y513F, and S503A). We have investigated the role of pp120/ HA4, a substrate of the insulin receptor kinase that is predominantly expressed in liver, in the internalization process. Internalization rates were unaffected in NIH 3T3 cells expressing the short isoform of pp120/HA4 or by expression of mutant pp120/HA4 molecules in which the potential phosphorylation sites of the intracellular domain (Tyr488 and Ser503) had been replaced by site-directed mutagenesis with nonphosphorylatable amino acids
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