Molecular Recognition at the Phosphatidylinositol 3,4,5-Trisphosphate-Binding Site. Studies Using the Permuted Isomers of Phosphatidylinositol Trisphosphate

Permuted isomers of L-α-phosphatidyl-D-myo-inositol trisphosphate (PtdInsP₃), including PtdIns(3,4,5)P₃, PtdIns(3,4,6)P₃, PtdIns(3,5,6)P₃, and PtdIns(4,5,6)P₃, have been synthesized as part of our effort to understand the underlying principles governing ligand selection for PtdIns(3,4,5)P₃-specific binding proteins. These PtdInsP₃ isomers are examined by using two PtdIns(3,4,5)P₃-dependent functional assays: binding to the C-terminal SH2 domain of the p85 regulatory subunit of PI 3-kinase and platelet aggregation. Our data show that all these isomers bind to the SH2 domain with comparable affinity despite variation in the regioisomeric distribution of phosphate functions. Moreover, all these phospholipids are capable of triggering platelet aggregation with the relative potency of PtdIns(3,4,5)P₃ > PtdIns(3,5,6)P₃ > PtdIns(4,5,6)P₃ > PtdIns(3,4,6)P₃. Evidence suggests that these PtdInsP₃'s facilitate cell aggregation by activating Ca²⁺ influx across the plasma membrane. In contrast, other inositol lipids examined including PtdIns(3,4)P2, PtdIns(4,5)P₂, PtdIns(3)P, and PtdIns(4)P are ineffective in eliciting the aggregation even at much higher concentrations. Taken together, the present data suggest that the charge density on the phosphorylated inositol ring represents a key factor in determining the phosphoinositide binding specificity of target proteins. It is conceivable that the interaction with the PtdIns(3,4,5)P₃-binding motif requires the participation of all three phosphates on the headgroup of PtdIns(3,4,5)P₃. Consequently, other membrane phosphoinositides (e.g., the bis- and monophosphates) become thermodynamically unfavorable for the binding to these PtdIns(3,4,5)P₃ targets.