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dc.contributor.authorHERRERA, ROMAN
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 48-06, Section: B, page: 1577.
dc.description.abstractThe insulin receptor is a ligand-dependent protein tyrosine kinase whose activity plays a key role in the mechanism of insulin action. We have investigated the structure-function relationship of this receptor kinase activity as an approach to the study of the molecular mechanism of signal transduction.;Our results have shown that the receptor undergoes an intramolecular autophosphorylation reaction that is kinetically stimulated by the ligand, insulin. The phosphorylated receptor became an active, ligand-independent protein tyrosine kinase. Dephosphorylation of the receptor restored the kinase activity to its basal, inactive state.;In order to identify and correlate the functional domain of the receptor molecule with its catalytic activity, we have developed a battery of sequence-specific antibodies against the protein kinase domain of the receptor predicted by the DNA sequence of the proreceptor cDNA. Chemical cleavage at either tryptophan or methionine residues followed by immunoprecipitation with antipeptide antibodies was used to map the in vitro autophosphorylation sites of the {dollar}\beta{dollar} subunit of the insulin receptor. Two phosphorylated fragments were resolved. One, recognized by antibody elicited to the carboxyl terminus, includes amino acid residues 1328-1342 (P5). The other, recognized by antibody to P2, is located in a domain that includes tyrosine 1150. The rate of phosphorylation of this latter site correlates with the rate of activation of the insulin receptor kinase during in vitro autophosphorylation. This kinase activation is accompanied by a conformational change, detected by antibody directed to P2 domain. An antibody directed to a cytoplasmic domain near the membrane spanning region of the proreceptor, amino acids 952-967 (P4), inhibited both insulin-dependent autophosphorylation and exogenous substrate phosphorylation of the dephosphorylated receptor, but not the activity of the phosphorylated receptor.;We concluded that the insulin receptor kinase activity is regulated by its phosphorylation state and that immuno-detectable conformational changes of the receptor protein occur upon ligand-induced autophosphorylation. In addition, these antipeptide antibodies have been used to study the relationship between the human receptors for insulin and insulin-like growth factor (IGF-I). The results establish (1) that the IGF-I and insulin receptors share a specific amino acid sequences necessary for the expression of enzymatic activity, and (2) that the C terminus of the insulin receptor is not conserved in the IGF-I receptor.
dc.publisherProQuest Dissertations & Theses

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