Antibodies to insulin receptor tyrosine kinase stimulate its activity towards exogenous substrates without inducing receptor autophosphorylation.
Anti-peptide antibodies directed against a highly-conserved sequence of the insulin receptor tyrosine kinase domain have been used to study the relationship between this specific region and kinase activation. Antibodies have been prepared by the injection into a rabbit of a synthetic peptide (P2) corresponding to residues 1110-1125 of the proreceptor. The peptide exhibits 88-95% sequence similarity with the corresponding sequence in the v-ros protein and in receptors for epidermal growth factor and for insulin-like growth factor 1. Two antibodies with different specificities could be separated from total antiserum obtained after immunization with P2. One antibody [anti-(P-Tyr)] cross-reacted with phosphotyrosine and immunoprecipitated solely autophosphorylated receptors. This antibody was shown to increase or decrease the receptor tyrosine kinase activity depending on its concentration. In all circumstances receptor autophosphorylation and substrate phosphorylation were modulated in a parallel fashion. The second antibody (anti-P2) failed to immunoprecipitate the insulin receptor, but was found to interact with both the peptide and the receptor by e.l.i.s.a. assay. Using a tyrosine co-polymer we found that anti-P2 activated the insulin receptor kinase leading to substrate phosphorylation at a level similar to that observed with insulin. This effect was additive to the hormonal effect. In contrast, receptor autophosphorylation was not modified by the anti-peptide. The differential effect of this anti-peptide further supports the idea that receptor autophosphorylation and kinase activity towards exogenous substrates might be independently regulated. Finally, our data suggest that conformational changes in the receptor tyrosine kinase domain may be sufficient for activation of its enzymic activity.
Institut National de la Santé et de la Recherche Médicale INSERM U 145, Faculté de Médecine, Nice, France.
V
V
Baron
N
N
Gautier
N
N
Rochet
R
R
Ballotti
B
B
Rossi
S
S
Saint-Pierre
E
E
Van Obberghen
J
J
Dolais-Kitabgi
D000818:Animals; D000906:Antibodies; D004789:Enzyme Activation; D010455:Peptides / Q000276:immunology; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D010455:Peptides; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin