MOLECULAR RECOGNITION AT THE PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE-BINDING SITE - STUDIES USING THE PERMUTED ISOMERS OF PHOSPHATIDYLINOSITOL TRISPHOSPHATE

Permuted isomers of L-alpha-phosphatidyl-D-myo-inositol trisphosphate (PtdInsP(3)), including PtdIns(3,4,5)P-3, PtdIns(3,4,6)P-3, PtdIns(3,5 ,6)P-3, and PtdIns(4,5,6)P-3, have been synthesized as part of our eff ort to understand the underlying principles governing ligand selection for Ptdlns(3,4,5)P-3-specific binding proteins. These PtdInsP(3) isom ers are examined by using two PtdIns(3,4,5)P-3-dependent functional as says: binding to the C-terminal SH2 domain of the p85 regulatory subun it of PI 3-kinase and platelet aggregation. Our data show that all the se isomers bind to the SH2 domain with comparable affinity despite var iation in the regioisomeric distribution of phosphate functions. Moreo ver, all these phospholipids are capable of triggering platelet aggreg ation with the relative potency of PtdIns(3,4,5)P-3 > PtdIns(3,5,6)P-3 > PtdIns(4,5,6)P-3 > PtdIns(3,4,6)P-3. Evidence suggests that these P tdInsP(3)'s facilitate cell aggregation by activating Ca2+ influx acro ss the plasma membrane. In contrast, other inositol lipids examined in cluding PtdIns(3,4)P-2, PtdIns(4,5)P-2, PtdIns(3)P, and PtdIns(4)P are ineffective in eliciting the aggregation even at much higher concentr ations. Taken together, the present data suggest that the charge densi ty on the phosphorylated inositol ring represents a key factor in dete rmining the phosphoinositide binding specificity of target proteins. I t is conceivable that the interaction with the PtdIns(3,4,5)P-3-bindin g motif requires the participation of all three phosphates on the head group of PtdIns(3,4,5)P-3. Consequently, other membrane phosphoinositi des (e.g., the bis- and monophosphates) become thermodynamically unfav orable for the binding to these PtdIns(3,4,5)P-3 targets.