Gap1(IP4BP) contains a novel group I pleckstrin homology domain that directs constitutive plasma membrane association

Abstract

The group I family of pleckstrin homology (PH) domains are characterized by their inherent ability to specifically bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and its corresponding inositol head-group inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). In vivo this interaction results in the regulated plasma membrane recruitment of cytosolic group I PH domain-containing proteins following agonist-stimulated PtdIns(3,4,5)P3 production. Among group I PH domain-containing proteins, the Ras GTPase-activating protein GAP1(IP4BP) is unique in being constitutively associated with the plasma membrane. Here we show that, although the GAP1(IP4BP) PH domain interacts with PtdIns(3,4,5)P3, it also binds, with a comparable affinity, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) (K(d) values of 0.5 ± 0.2 and 0.8 ± 0.5 μM, respectively). Intriguingly, whereas this binding site overlaps with that for Ins(1,3,4,5)P4, consistent with the constitutive plasma membrane association of GAP1(IP4BP) resulting from its PH domain-binding PtdIns(4,5)P2, we show that in vivo depletion of PtdIns(4,5)P2, but not PtdIns(3,4,5)P3, results in dissociation of GAP1(IP4BP) from this membrane. Thus, the Ins(1,3,4,5)P4-binding PH domain from GAP1(IP4BP) defines a novel class of group I PH domains that constitutively targets the protein to the plasma membrane and may allow GAP1(IP4BP) to be regulated in vivo by Ins(1,3,4,5)P4 rather than PtdIns(3,4,5)P3.