INTERACTION OF THE MOLECULAR-WEIGHT 85K REGULATORY SUBUNIT OF THE PHOSPHATIDYLINOSITOL 3-KINASE WITH THE INSULIN-RECEPTOR AND THE INSULIN-LIKE GROWTH-FACTOR-I (IGF-I) RECEPTOR - COMPARATIVE-STUDY USING THE YEAST 2-HYBRID SYSTEM

Activation of the phosphatidyl inositol 3-kinase (PI3-kinase) is one o f the immediate events in signaling by the insulin receptor (IR) and t he insulin-like growth factor-I receptor (IGF-IR). The IR and IGF-IR a re two closely related tyrosine kinases, which are activated on bindin g of their respective ligands. Previous studies have proposed that the two receptors interact directly with the SH2 domains of the M(r) 85K regulatory subunit (p85) of PI 3-kinase via phosphorylated Y(1322)THM and Y(1316)AHM sequences located in the carboxyl-terminal domain of th e IR and IGF-IR, respectively. In this study we have used the yeast tw o-hybrid system to compare the interaction of the cytoplasmic domains of the IR and the IGF-IR with p85. We found that the IR is more effici ent in interacting with the p85 than is the IGF-IR. For both receptors , deletion of the region containing the Y(1322)THM sequence in the IR and the Y(l316)AHM-similar sequence in the IGF-IR decreases their abil ity to interact with p85. However, these mutated receptors still inter acted with the full-length p85, suggesting that other regions in the r eceptors might be involved in the interaction. Furthermore, mutations of the three major autophosphorylation sites indicate that interaction s with p85 are dependent on the receptor tyrosine kinase activity. Fin ally, we asked whether the two SH2 domains of p85 (n-SH2 and c-SH2) ar e involved in the same fashion in their association with the two recep tors. Interestingly, we observed that the carboxyl-terminal domain of the IGF-IR associates only with the p85 c-SH2 domain, whereas the corr esponding domain of the IR interacts with both the n-SH2 and the c-SH2 domains. In combination, both SH2 domains (n/c-SH2) contribute to the maximal interaction observed with the full-length p85. Although the p recise impact on signaling resulting from these differences in the int eraction of p85 with the IR vs. the IGF-IR remains to be determined, i t is tempting to propose that they contribute, at least in part, to th e specificity of the biological responses induced by insulin us. IGF-I .