Anti-(insulin receptor) monoclonal antibody-stimulated tyrosine phosphorylation in cells transfected with human insulin receptor cDNA.
The effects of insulin and anti-(insulin receptor) monoclonal antibodies on tyrosine phosphorylation were investigated in fibroblasts transfected with human insulin receptor cDNA (NIH 3T3HIR3.5 cells) using anti-phosphotyrosine immunoblotting. Insulin increased levels of tyrosine phosphorylation in two major proteins of molecular mass 97 kDa (pp97, assumed to be the insulin receptor beta-subunit) and 185 kDa (pp185). Insulin-mimetic anti-receptor antibodies also stimulated tyrosine phosphorylation of both pp97 and pp185. The observation of antibody-stimulated pp97 phosphorylation, as detected by immunoblotting, is in contrast with previous data which failed to show receptor autophosphorylation in NIH 3T3HIR3.5 cells labelled with [32P]P1. The effect of insulin on pp97 was maximal within 1 min, but the response to antibody was apparent only after a lag of 1-2 min and rose steadily over 20 min. The absolute level of antibody-stimulated phosphorylation of both pp97 and pp185 after 20 min was only about 20% of the maximum level induced by equivalent concentrations of insulin, even at concentrations of antibody sufficient for full occupancy of receptors. Another insulin-mimetic agent, wheat-germ agglutinin, stimulated receptor autophosphorylation with kinetics similar to those produced by the antibody. It is suggested that the relatively slow responses to both agents may be a function of the dependence on receptor cross-linking. These data are consistent with a role for the insulin receptor tyrosine kinase activity in the mechanism of action of insulin-mimetic anti-receptor antibodies.
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, U.K.
N P
NP
Brindle
J M
JM
Tavare
M
M
Dickens
J
J
Whittaker
K
K
Siddle
D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D002460:Cell Line; D004247:DNA / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D010766:Phosphorylation; D019000:Phosphotyrosine; D011506:Proteins / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D013268:Stimulation, Chemical; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism*; D014909:Wheat Germ Agglutinins / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D007328:Insulin; D011506:Proteins; D014909:Wheat Germ Agglutinins; D019000:Phosphotyrosine; D014443:Tyrosine; D004247:DNA; D011972:Receptor, Insulin