Tyrosine kinase-defective insulin receptors undergo decreased endocytosis but do not affect internalization of normal endogenous insulin receptors.
To characterize tyrosine kinase activity in signaling ligand/receptor internalization, metabolic labeling and surface radioligand binding were used to follow the processing of both normal and tyrosine kinase-deficient human insulin receptors. The mutant receptor (A/K1018) has an alanine substituted for lysine 1018 in the ATP-binding domain. Rat 1 fibroblasts, expressing either normal human insulin receptors (HIRc) or A/K1018 receptors, were assayed to determine the insulin receptor half-life as well as internalization and down-regulation. Our results show that insulin greatly reduces the half-life of normal insulin receptors (from 9.9 to 5.7 h). The A/K1018 receptor had a much longer half-life (24 h), which was not reduced by the presence of saturating insulin concentrations. The A/K1018 receptor does not undergo down-regulation after long term insulin exposure, while HIRc cells showed a 34% decrease in insulin receptor number. This down-regulation is accounted for by the accelerated turnover rate of normal receptors in the presence of insulin. To confirm that the kinase activity is necessary for normal endocytosis, we also show that ATP depletion in HIRc cells resulted in significant decreases in receptor internalization and that tyrosine kinase-defective receptors also fail to internalize in a different cell type (rat Fao hepatocytes). Lastly, the complement of normal rat insulin receptors in cells expressing the kinase-defective receptors endocytose normally. We conclude that the defect in endocytosis observed in kinase-defective receptors is intrinsic to this receptor and not due to a dominant inhibitory effect on cellular endocytotic machinery.
Grako
K A
KA
Department of Medicine, University of California-San Diego, La Jolla 92093.
Olefsky
J M
JM
McClain
D A
DA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism; D015536:Down-Regulation; D004705:Endocytosis; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection
D007328:Insulin; C008147:insulin, iodo-; D000255:Adenosine Triphosphate; D008715:Methionine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D003545:Cysteine