INTERACTION OF P85 SUBUNIT OF PI-3-KINASE WITH INSULIN AND IGF-1 RECEPTORS ANALYZED BY USING THE 2-HYBRID SYSTEM

Interaction of the p85 subunit of PI 3-kinase with the insulin recepto r (IR) and the IGF-1 receptor (IGF-1R) was investigated using the two- hybrid system by assessing for his3 and lacZ activation in S. cerevisi ae. The experiments were performed with the cytoplasmic beta domain (w ild type or mutated) of IR and IGF-1R and p85 or its subdomains (N + C -SH2, N-SH2, C-SH2, SH3 + N-SH2). The results of his3 activation indic ated that p85, N + C-SH2 and C-SH2 interact with both IR beta and IGF- 1R beta, whereas N-SH2 and SH3 + N-SH2 interact only with IR beta. Int eraction of p85 and N + C-SH2 with IR beta(Delta C-43) or IGF-1R beta( Delta C-43) in which the C-terminal 43 amino acids (including the YXXM motif) were deleted, persisted, The internal binding site thus reveal ed was not altered by further mutating Y-960/F for IR or Y-950/F for I GF-1R. Activation of lacZ upon interaction of p85 with IR beta(Delta C -43) was 4-fold less as compared to IR beta. This activation with p85 and IGF-1R beta was 4-fold less as compared to IR beta and was somewha t increased (2-fold) for IGF-1R beta(Delta C-43). Thus, the C-terminal domain in IGF-1R appears to exert a negative control on binding of p8 5 thereby providing a possible regulatory mechanism for direct activat ion of the PI 3-kinase pathway.