Engineering the phosphoinositide-binding profile of a class I Pleckstrin homology domain

Pleckstrin homology (PH) domains are protein modules that bind with varying degrees of affinity and specificity membrane phosphoinositides. Previously we have shown that although the PH domains of the Ras GTPase-activating proteins GAP1(m) and GAP1(IP4BP) are 63% identical at the amino acid level they possess distinct phosphoinositide-binding profiles. The GAP1(m) PH domain binds phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5) P-3), whereas the domain from GAP1(IP4BP) binds PtdIns(3,4,5) P-3 and phosphatidylinositol 4,5-bisphosphate ( PtdIns(4,5) P-2) equally well. These phosphoinositide specificities are translated into distinct subcellular localizations. GAP1(m) is cytosolic and undergoes a rapid PtdIns(3,4,5) P-3-dependent association with the plasma membrane following growth factor stimulation. In contrast, GAP1(IP4BP) is constitutively associated, in a PtdIns(4,5) P-2-dependent manner, with the plasma membrane (Cozier, G. E., Lockyer, P. J., Reynolds, J. S., Kupzig, S., Bottomley, J. R., Millard, T., Banting, G., and Cullen, P. J. (2000) J. Biol. Chem. 275, 28261 - 28268). In the present study, we have used molecular modeling to identify residues in the GAP1(IP4BP) PH domain predicted to be required for high affinity binding to PtdIns( 4,5) P2. This has allowed the isolation of a mutant, GAP1(IP4BP)( K591T), which while retaining high affinity for PtdIns( 3,4,5) P-3 has a 6-fold reduction in its affinity for PtdIns( 4,5) P-2. Importantly, GAP1(IP4BP)-(K591T) is predominantly localized to the cytosol and undergoes a PtdIns( 3,4,5) P-3- dependent association with the plasma membrane following growth factor stimulation. We have therefore engineered the phosphoinositide-binding profile of the GAP1(IP4BP) PH domain, thereby emphasizing that subtle changes in PH domain structure can have a pronounced effect on phosphoinositide binding and the subcellular localization of GAP1(IP4BP).