Transmembrane signaling by the human insulin receptor kinase. Relationship between intramolecular beta subunit trans- and cis-autophosphorylation and substrate kinase activation.
To examine the role of intramolecular beta subunit trans- and cis-autophosphorylation in signal transduction, the vaccinia virus/bacteriophage T7 expression system was used to generate insulin holoreceptors composed of a kinase-defective half-receptor precursor (alpha beta A/K or alpha beta A/K.delta CT) and a kinase-active half-receptor precursor (alpha beta delta CT or alpha beta WT). In the alpha beta A/K-alpha beta delta CT hybrid insulin receptor, insulin stimulated a 20-fold increase in intramolecular beta subunit trans-phosphorylation, whereas cis-phosphorylation increased only 3-fold over the basal state. Similarly, in the alpha beta WT-alpha beta A/K.delta CT hybrid insulin receptor, insulin stimulated trans-phosphorylation approximately 30-fold and cis-phosphorylation only 3-fold over the basal state. Although cis-phosphorylation of the kinase-functional alpha beta half-receptor was observed within these hybrid receptor species, this was not sufficient to stimulate exogenous substrate kinase activity. These data demonstrate that insulin primarily activates an intramolecular beta subunit trans-phosphorylation reaction within the insulin holoreceptor and suggest that this reaction is necessary for activation of the holoreceptor. Furthermore, our results suggest a molecular basis for the dominant-negative phenotype observed in insulin-resistant patients possessing one kinase-defective insulin receptor allele.
Frattali
A L
AL
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242.
Treadway
J L
JL
Pessin
J E
JE
D001483:Base Sequence; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism* / Q000648:ultrastructure; D011972:Receptor, Insulin / Q000378:metabolism* / Q000648:ultrastructure; D011994:Recombinant Proteins; D015398:Signal Transduction; D013329:Structure-Activity Relationship
D007328:Insulin; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin