GAP1IP4BP contains a novel Group I pleckstrin homology domain that directs constitutive plasma membrane association.

The Group I family of pleckstrin homology (PH) domains are characterised 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. Amongst Group I PH domain-containing proteins, the Ras GTPase-activating protein GAP1IP4BP is unique in being constitutively associated with the plasma membrane. Here we show that although the GAP1IP4BP PH domain interacts with PtdIns(3,4,5)P3 it also binds, with a comparable affinity, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) (Kd’s of 0.5 ± 0.2 and 0.8 ± 0.5 m M respectively). Intriguingly, whereas this binding site overlaps with that for Ins(1,3,4,5)P4, consistent with the constitutive plasma membrane association of GAP1IP4BP 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 GAP1IP4BP from this membrane. Thus the Ins(1,3,4,5)P4-binding PH domain from GAP1IP4BP defines a novel class of Group I PH domains that constitutively targets the protein to the plasma membrane and may allow GAP1IP4BP to be regulated in vivo by Ins(1,3,4,5)P4 rather than PtdIns(3,4,5)P3.


Background
Pleckstrin homology (PH) domains are protein modules of approximately 120 amino acids that were initially identified as regions of weak sequence homology repeated in pleckstrin (1,2). Subsequently PH domains have been identified in more than 100 different proteins that despite possessing low sequence similarities (10-20%) have been shown, or predicted to have, very similar overall topologies (3).
Many of these PH domain-containing proteins are involved in intracellular signalling, where the majority require membrane association for their function. Recent work has highlighted a pivotal role for PH domains in this membrane targeting (4,5).

The GAP1 IP4BP PH/Btk domain is the sole requirement for plasma membrane association.
To

Mutations within the PH/Btk domain that inhibit Ins(1,3,4,5)P 4 -binding result in GAP1 IP4BP dissociating from the plasma membrane.
In order to determine the correlation between Ins(1,3,4,5)P 4 -binding to GAP1 IP4BP and the ability to associate with the plasma membrane, the subcellular localisation of the GAP1 IP4BP mutants was determined. As seen in Figure 2, GAP1 IP4BP mutants with dramatically reduced abilities to bind Ins(1,3,4,5)P 4 , -K 585 →R, -A 587 →F, -N 597 →D, -F 598 →Q, -R 601 →K and -R 601 →C, no longer associate with the plasma membrane but were instead primarily cytosolic. This contrasted with GAP1 IP4BP -K 614 →E which, consistent with its ability to bind Ins(1,3,4,5)P 4 , retained a predominant plasma membrane localisation (Fig. 2). However, detailed image analysis revealed a detectable increase in cytosolic fluorescence compared to wild type (Fig. 3). Thus there does appear to be a strong correlation between the ability of the PH/Btk domain to bind phosphorylated forms of inositol and an ability to associate with the plasma membrane. Such a relationship suggests a potential molecular explanation for the plasma membrane localisation of GAP1 IP4BP ; namely that the PH/Btk domain may directly bind phosphoinositides present within the inner plasma membrane leaflet.

In unstimulated cells GAP1 IP4BP is not localised to the plasma membrane via an ability to bind basal PtdIns(3,4,5)P 3 .
To address whether the plasma membrane association of GAP1 IP4BP may result from binding to a low resting level of PtdIns(3,4,5)P 3 , we transiently  (Fig. 6). Again GFP-GAP1 IP4BP remained predominantly localised to the plasma membrane. Together these data emphasise that the plasma membrane association of GAP1 IP4BP is unlikely to result from its PH/Btk domain-binding resting levels of PtdIns(3,4,5)P 3 .

PtdIns(4,5)P 2 .
If the plasma membrane association of GAP1 IP4BP is a consequence of its PH/Btk domain-binding PtdIns(4,5)P 2 , then manipulation of the concentration of this lipid should result in the dissociation of GAP1 IP4BP from the plasma membrane. PLC and results in a subsequent decrease in plasma membrane PtdIns(4,5)P 2 levels (35). As shown in Figure 7, addition of ionomycin caused the dissociation of GFP-GAP1 IP4BP from the plasma membrane and a simultaneous appearance of GFP fluorescence in the cytosol (Fig. 7A). This dissociation was rapid and paralleled control experiments using the well characterised in vivo PtdIns(4,5)P 2 -binding PH domain from PLC-δ 1 (Fig. 7B (35,36)). To test the role of PLC in the ionomycininduced GAP1 IP4BP dissociation we incubated GFP-GAP1 IP4BP expressing HeLa cells with the relatively specific PLC inhibitor U73122 (37). Before ionomycin addition, the localisation of GFP-GAP1 IP4BP was unaltered and upon ionomycin addition U73122-treated cells failed to show the plasma membrane dissociation observed in control cells (Fig. 7C).
As a second, independent, approach we made use of the observation that high concentrations of wortmannin (1 ¼M) induce a gradual fall in the levels of plasma membrane PtdIns(4,5)P 2 due to an inhibition of type III PtdIns 4-kinase (38,39).

Discussion
In The similar affinity of GAP1 IP4BP for PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 suggests, given that plasma membrane PtdIns(4,5)P 2 is more abundant than PtdIns(3,4,5)P 3 even after agonist stimulation (34), that the constitutive plasma membrane association of GAP1 IP4BP may occur as a consequence of its PH/Btk domain-binding plasma membrane PtdIns(4,5)P 2 . If such a mechanism were correct then one would expect that since GAP1 m is not constitutively associated with the plasma membrane it should have a lower affinity for PtdIns(4,5)P 2 . As stated above, this is indeed the case. Furthermore, any mechanism that induces a significant depletion of plasma membrane PtdIns(4,5)P 2 should result in the dissociation of GAP1 IP4BP . Experimentally we have presented direct evidence that reducing PtdIns(4,5)P 2 content within the inner leaflet of the plasma membrane, does indeed result in the plasma membrane dissociation of GAP1 IP4BP . Together therefore these data strongly suggest that, in an unstimulated cell, the plasma membrane association of GAP1 IP4BP results from its PH/Btk domain binding PtdIns(4,5)P 2 .
The conclusion that GAP1 IP4BP constitutes a receptor for Ins(1,3,4,5)P 4 rather than PtdIns(3,4,5)P 3 has a significant baring on the molecular events that may Future experiments will need to address the downstream consequences of these interactions on GAP1 IP4BP regulated Ras signalling.