We identified a potential phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P 3 ) binding pleckstrin homology domain in the data bases and have cloned and expressed its full coding sequence (LL5␤). The protein bound PtdIns(3,4,5)P 3 selectively in vitro. Strikingly, a substantial proportion of LL5␤ became associated with an unidentified intracellular vesicle population in the context of low PtdIns(3,4,5)P 3 levels produced by the addition of wortmannin or LY294002. In addition, expression of platelet-derived growth factor-receptor mutants unable to activate type 1A phosphoinositide 3-kinase (PI3K) or serum starvation in porcine aortic endothelial cells lead to redistribution of LL5␤ to this vesicle population. Importantly, pleckstrin homology domain mutants of LL5␤ that could not bind PtdIns(3,4,5)P 3 were constitutively localized to this vesicle population. At increased PtdIns(3,4,5)P 3 levels, LL5␤ was redirected to a predominantly cytoplasmic distribution, presumably through a PI3K-dependent block on its targeting to the vesicular compartment. Furthermore, at high, hormone-stimulated PtdIns-(3,4,5)P 3 levels, it became significantly plasma-membrane localized. The distribution of LL5␤ is thus dramatically and uniquely sensitive to low levels of PtdIns(3,4,5)P 3 indicating it can act as a sensor of both low and hormone-stimulated levels of PtdIns(3,4,5)P 3 . In addition, LL5␤ bound to the cytoskeletal adaptor, ␥-filamin, tightly and in a PI3K-independent fashion, both in vitro and in vivo. This interaction could co-localize heterologously expressed ␥-filamin with GFP-LL5␤ in the unidentified vesicles.Phosphoinositide 3-kinases (PI3Ks) 1 3-phosphorylate phosphoinositides. There are three classes of PI3Ks. The type I enzymes seem to act as PtdIns(4,5)P 2 3-kinases in vivo; they can be activated by a variety of close-to-receptor transduction events and drive accumulation of PtdIns(3,4,5)P 3 in the inner leaflet of the plasma membrane. This PtdIns(3,4,5)P 3 serves as signal recruiting proteins from the cytosol that possess modules, typically PH domains, capable of binding its head group (1).There are a variety of reagents that can be used to inhibit PI3K activity. Most widely used are wortmannin (2) and LY294002 (3); both of which potently inhibit nearly all classes of PI3K and hence cannot generally be used to implicate a particular PI3K in a process. More specific are receptor tyrosine kinase Tyr 3 Phe mutants. A number of receptor tyrosine kinases (relevant here, the PDGF ␤-receptor) are capable of binding type IA PI3Ks at specific tyrosine residues that become phosphorylated following ligand binding (4). Mutation of these tyrosines to phenylalanine blocks type IA PI3K binding and activation but does not affect association of other effectors (1, 5). Stable, clonal cell lines have been created overexpressing wild-type PDGF-␤ receptors or (Y740F/Y751F) PDGF-␤ receptors (6) that have allowed the impact of selectivity blocking type IA PI3K activation to be assessed in vivo (7).There is now a substantial famil...

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