Proteolytic generation of constitutive tyrosine kinase activity of the human insulin receptor.
Structural modification induced by partial digestion with trypsin has been shown to stimulate the tyrosine kinase activity of the insulin receptor both in solution and in intact cells [Tamura, Fujita-Yamaguchi & Larner (1983) J. Biol. Chem. 258, 14749-14752; Goren, White & Kahn (1987) Biochemistry 26, 2374-2382; Leef & Larner (1987) J. Biol. Chem. 262, 14837-14842]. Furthermore, experiments involving deletion of sequences encoding the extracellular domain of the insulin receptor suggest that it may function as a protooncogene in fibroblasts [Wang et al., (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5725-5729]. To further understand the structural requirements that generate this activity, the major activated fragments generated in solution following trypsin digestion have been characterized here, one of which is shown to have a similar amino acid sequence to a transforming protein. Furthermore, treatment with trypsin of intact Chinese hamster ovary cells that overexpress the human insulin receptor stimulates both autophosphorylation of the receptor and 2-deoxyglucose uptake into the cells, but does not enhance receptor internalization. Unlike digestion in solution, no proteolysis or loss of activity of the activated insulin receptor beta-subunit could be detected using intact cells, even at high trypsin concentrations, despite the existence of extracellular sites that are readily cleaved by trypsin in the solubilized receptor. These studies provide further detail of a mechanism used during trypsinization of cells in culture which mimics activation of the insulin receptor and contributes to stimulation of growth.
Hsuan
J J
JJ
Ludwig Institute for Cancer Research, Middlesex Hospital/University College Branch, London U.K.
Downward
J
J
Clark
S
S
Waterfield
M D
MD
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D014357:Trypsin
D010446:Peptide Fragments; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D014357:Trypsin