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)P(3)) and its corresponding inositol head-group inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). 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)P(3) 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)P(3), it also binds, with a comparable affinity, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) (K(d) values of 0.5 +/- 0.2 and 0.8 +/- 0.5 microm, respectively). Intriguingly, whereas this binding site overlaps with that for Ins(1,3,4,5)P(4), consistent with the constitutive plasma membrane association of GAP1(IP4BP) resulting from its PH domain-binding PtdIns(4,5)P(2), we show that in vivo depletion of PtdIns(4,5)P(2), but not PtdIns(3,4,5)P(3), results in dissociation of GAP1(IP4BP) from this membrane. Thus, the Ins(1,3,4,5)P(4)-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)P(4) rather than PtdIns(3,4,5)P(3).

Inositol 1,3,4,5-tetrakisphosphate (IP4), is a ubiquitous inositol phosphate that has been suggested to function as a second messenger. Recently, we purified and cloned a putative IP4 receptor, termed GAP1(IP4BP)[1], which is also a member of the GAP1 family of GTPase-activating proteins for the Ras family of GTPases. A homologue of GAP1(IP4BP), called GAP1(m), has been identified [2] and here we describe the cloning of a GAP1(m) cDNA from a human circulating-blood cDNA library. We found that a deletion mutant of GAP1(m), in which the putative phospholipid-binding domains (C2A and C2B) have been removed, binds to IP4 with a similar affinity and specificity to that of the corresponding GAP1(IP4BP) mutant. Expression studies of the proteins in either COS-7 or HeLa cells showed that, whereas GAP1(IP4BP) is located solely at the plasma membrane, GAP1(m) seems to have a distinct perinuclear localisation. By mutational analysis, we have shown that the contrast in subcellular distribution of these two closely related proteins may be a function of their respective pleckstrin homology (PH) domains. This difference in localisation has fundamental significance for our understanding of the second messenger functions of IP4.

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The universal role of 3-phosphorylated inositol lipids in cellular signal transduction has become apparent over the last few years. Of these lipids phosphatidylinositol 3,4,5-trisphosphate (PIP,) has aroused the most interest as a potential second messenger'. On aspect of our work is directed at identifying in vivo PIP' receptors in order to characterise the downstream pathways that become activated following PIP, production. Experimentally we have identified various proteins based initially on their ability to specifically bind in virro the inositol head group of PIP,: inositol 1,3,4,5-tetrakisphosphate (IP,). Using this approach we have characterised GAPl'P48P and GAPI? members of the GAP1 family of Ras GTPase-activating proteins', and the ARF exchange factor$, ARNO, GRPl and cytohesin-I, as high affinity isomerically specific IP, binding proteins; data that raises the issue of whether these proteins constitute PIP' binding proteins in v i~o '~~. Through the use of green fluorescent protein (GFP) technology coupled with live cell time-lapse confocal laser scanning microscopy we have shown that a GFP-ARNO chimera is rapidly (< 40 seconds) recruited from the cytosol to the plasma membrane of adipocytes following insulin stimulation'. This translocation not only requires the ability of insulin to generate plasma membrane PIP, but also the presence of the in vitro IP4 binding ARNO pleckstrin homology (PH) domain. Similar data has been obtained for GRPI4 and cytohesin-Is. We have also recently demonstrated that GAPI" is recruited to the plasma membrane of PC12 cells following EGF stimulation. As with ARNO, this recruitment requires PIP, generation and the GAPI" PH domain but interestingly does not appear to lead to an activation of the GAPl"' H-Ras GAP activity6. These results and others regarding inositol phosphate binding to PH domains and the effect of PIP, on ARF signalling will he discussed.A wide variety of growth factors, inflammatory stimuli and antigens use the phosphoinositide 30H-kinase (F' 13K) signalling pathway to regulate a range of complex cell responses. We still have an incomplete understanding however, of the molecular mechanisms by which the lipid products of agonist-stimulated PI3Ks (PtdIns(3,4)P* and PtdIns(3,4,5)P3) actually perform their regulatory roles. A number of approaches have been used to try and resolve this, including various assays for specific PtdIns(3,4,)Pz and RdIns(3,4,5)P3 binding proteins and also in vzfro assays where the effects of addition of these lipids have been measured on physiologically relevant cell responses. We are currently developing an in vifro assay in which activations of the GPy-sensitive plOllpllOy isoform of PDK abundant in leucocytes can regulate the rate of superoxide production by a collection of neutrophil proteins. The activation of the oxidase burst by G-protein coupled receptors (e.g. receptors for FMLP, PAF, histamine) on neutrophils has been identified previously as a PI3K regulated response (e.g. Arcaro & Wymann (1993) Biochem. J. 296, 297-301) ...

GAP1IP4BP is a Ras GTPase-activating protein (GAP) that in vitro is regulated by the cytosolic second messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. We have studied Ins(1,3,4,5)P4 binding to GAP1IP4BP, and shown that the inositol phosphate specificity and binding affinity are similar to Ins(1,3,4,5)P4 binding to Bruton's tyrosine kinase (Btk), evidence which suggests a similar mechanism for Ins(1,3,4,5)P4 binding. The crystal structure of the Btk pleckstrin homology (PH) domain in complex with Ins(1,3,4,5)P4 has shown that the binding site is located in a partially buried pocket between the β1/β2- and β3/β4-loops. Many of the residues involved in the binding are conserved in GAP1IP4BP. Therefore we generated a model of the PH domain of GAP1IP4BP in complex with Ins(1,3,4,5)P4 based on the Btk-Ins(1,3,4,5)P4 complex crystal structure. This model had the typical PH domain fold, with the proposed binding site modelling well on the Btk structure. The model has been verified by site-directed mutagenesis of various residues in and around the proposed binding site. These mutations have markedly reduced affinity for Ins(1,3,4,5)P4, indicating a specific and tight fit for the substrate. The model can also be used to explain the specificity of inositol phosphate binding.