Interaction of p85 subunit of PI 3-kinase with insulin and IGF-1 receptors analysed by using the two-hybrid system

Interaction of the p85 subunit of PI 3-kinase with the insulin receptor (IR) and the IGF-1 receptor (IGF-1R) was investigated using the two-hybrid system by assessing for his3 and lacZ activation in S. cerevisiae. The experiments were performed with the cytoplasmic β domain (wild 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 indicated that p85, N + C-SH2 and C-SH2 interact with both IRβ and IGF-1Rβ, whereas N-SH2 and SH3 + N-SH2 interact only with IRβ. Interaction of p85 and N + C-SH2 with IRβ(ΔC-43) or IGF-1Rβ(ΔC-43) in which the C-terminal 43 amino acids (including the YXXM motif) were deleted, persisted. The internal binding site thus revealed was not altered by further mutating Y 960/F for IR or Y 950/F for IGF-1R. Activation of lacZ upon interaction of p85 with IRβ(ΔC-43) was 4-fold less as compared to IRβ. This activation with p85 and IGF-1Rβ was 4-fold less as compared to IRβ and was somewhat increased (2-fold) for IGE-1Rβ(ΔC-43). Thus, the C-terminal domain in IGF-1R appears to exert a negative control on binding of p85 thereby providing a possible regulatory mechanism for direct activation of the PI 3-kinase pathway.