title: Changes in insulin-receptor structure associated with trypsin-induced activation of the receptor tyrosine kinase.
abstract: The tyrosine kinase of the insulin receptor can be activated by trypsin treatment. The concomitant abolition of insulin binding has been postulated to result from proteolytic destruction of the receptor. A discrepancy between the decrease in insulin binding and receptor immunoreactivity after trypsin treatment led us to investigate more closely the structure of the trypsin-treated receptor. After trypsin treatment of the CHOT cell line, which over-expresses transfected human insulin receptors, insulin binding was significantly decreased, but reactivity with five alpha-subunit monoclonal antibodies was either unaffected or only moderately decreased, indicating that the alpha-subunit was substantially intact. Examination of receptor structure after trypsin treatment, receptor autophosphorylation and gel electrophoresis revealed a single band at 110 kDa in non-reduced gels, comprising a small fragment (21 kDa) of the alpha-subunit linked to the beta-subunit by class II disulphides. When the receptor was radio-labelled with 125I, two additional alpha-subunit bands of 142 kDa and 81 kDa (composed of identical reduced bands) were observed on non-reduced gels, which contained disulphide-linked (class I) fragments. All fragments could be precipitated by antibodies to both alpha- and beta-subunits. However, only antibodies directed towards the N-terminus of the receptor could immunoblot trypsin-treated fragments. Thus activation of the receptor tyrosine kinase by trypsin occurs after cleavage, but not loss of the alpha-subunit. This finding has implications for the mechanism of transmembrane activation of the receptor kinase by insulin.
authors.affiliation: Burnet Clinical Research Unit, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.
authors.forename: S
authors.initials: S
authors.lastname: Clark
authors.forename: G
authors.initials: G
authors.lastname: Eckardt
authors.forename: K
authors.initials: K
authors.lastname: Siddle
authors.forename: L C
authors.initials: LC
authors.lastname: Harrison
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008970:Molecular Weight; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology*
chemical_list: D000911:Antibodies, Monoclonal; D004220:Disulfides; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D014357:Trypsin; D004229:Dithiothreitol