We studied the inhibitory effect of non-phosphorylated and triphosphorylated synthetic peptides, corresponding to amino acids 1143-1155 of the insulin proreceptor (domain 1151) on autophosphorylation and kinase of the insulin receptor. Tyrosine-phosphorylated peptides were synthesized using the N-(9-fluorenylmethoxycarbonyl)-O-dibenzylphosphono-~-tyr0~~ne.The triphosphorylated peptide (1 1 51-P3) and the non-phosphorylated peptide (1 151-NP), respectively, inhibited insulin receptor autophosphorylation by 65% and 70%, in a dose-dependent and additive manner. When the receptor was pre-phosphorylated for 1 min with [y-32P]ATP, 1151-P3 decreased autophosphorylation to 60% of maximun, whereas 11 51-NP had no further effect. In both non-activated and preactivated receptors, 1 151-P3 inhibition of receptor autophosphorylation was prevented by adding 2 mM vanadate. Kinase activity towards exogenous substrate poly(Glu,, Tyr) was dose-dependently inhibited by both analogues. This effect was independent of the state of receptor phosphorylation or the addition of vanadate. Since 11 51 -P3 inhibited the exogenous kinase without altering receptor endogenous autophosphorylation after the addition of vanadate, we investigated 11 51-NP and 11 51-P3 competition for the phosphorylation of a resin-immobilized 1151 peptide. While 11 51 -NP (at 2 mM) was highly competitive, inhibiting phosphate incorporation by 70%, 11 51-P 3 caused a four-fold increase in the phosphorylation of 1151-NP -resin. The receptor underwent conformational changes during autophosphorylation and an antibody directed against a peptide corresponding to amino acids 1334-1330 of the proreceptor (1322Ab) was previously shown to immunoprecipitate specifically the non-phosphorylated reccptor forms. Nevertheless, the 1322Ab irnmunoprecipitated a fully autophosphorylated receptor in the presence of 1151-NP, but not of 1151-P3, thus suggesting a conformational change induced by the non-phosphorylated peptide.In conclusion, kinase inhibition was still observed after the addition of phosphate groups to three 11 51 -peptide tyrnsines, but the peptide effect on receptor autophosphorylation, phosphorylation of homologous 1151-NP -resin and conformational changes induced in the receptor was altered dramatically. These data may provide a basis for further understanding the role of tyrosine phosphorylation in insulin receptor kinase activation or regulation.The insulin receptor is a complex heterotetrameric glycoprotein of the plasma membrane and is composed of two distinct subunits [l -31. The 135-kDa CI subunit contains a high affinity insulin binding site and is externally oriented [4].The B subunit is a 95-kDa transmembrane protein which contains a tyrosine-specific protein kinase in the cytoplasmic domain [ 5 , 6 ] . Insulin binding to the CI subunit rapidly induces kinase activation of the p subunit, which undergoes auto- Abbreviutions. RP, reverse phase; HPCE, high-performance capillary electrophoresis; Fmoc, 9-fluorenylmethoxycarbonyl; Boc, t-butyloxycarbonyl; TyrO(P...