GRP1 and the related proteins ARNO and cytohesin-1 are ARF exchange factors that contain a pleckstrin homology (PH) domain thought to target these proteins to cell membranes through binding polyphosphoinositides. Here we show the PH domains of all three proteins exhibit relatively high affinity for dioctanoyl phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P 3 ), with K D values of 0.05, 1.6 and 1.0 M for GRP1, ARNO, and cytohesin-1, respectively. However, the GRP1 PH domain was unique among these proteins in its striking selectivity for PtdIns(3,4,5)P 3 versus phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P 2 ), for which it exhibits about 650-fold lower apparent affinity. Addition of a glycine to the Gly 274 -Gly 275 motif in GRP1 greatly increased its binding affinity for PtdIns(4,5)P 2 with little effect on its binding to PtdIns(3,4,5)P 3 , while deletion of a single glycine in the corresponding triglycine motif of the ARNO PH domain markedly reduced its binding affinity for PtdIns(4,5)P 2 but not for PtdIns(3,4,5)P 3 . In intact cells, the hemagglutinin epitope-tagged PH domain of GRP1 was recruited to ruffles in the cell surface in response to insulin, as were full-length GRP1 and cytohesin-1, but the PH domain of cytohesin-1 was not. These data indicate that the unique diglycine motif in the GRP1 PH domain, as opposed to the triglycine in ARNO and cytohesin-1, directs its remarkable PtdIns(3,4,5)P 3 binding selectivity.Cell signaling processes are often initiated by the recruitment of protein complexes to the cytoplasmic face of the plasma membrane, where they act to elicit signaling events. Specialized regions or domains within such signaling proteins function as adapters in the recruitment process, linking these proteins to chemical motifs generated by receptor activation. For example, specific membrane-bound protein phosphotyrosine sites appear in response to activation of transmembrane receptor tyrosine kinases by growth factors and other stimuli, attracting Src homology (SH) 1 2 domains within proteins that bind these sites (1). The proteins that contain SH2 domains are enzymes, regulator proteins, or simply adapters themselves that connect other proteins to the localized protein phosphotyrosines. This paradigm has been extended to a large number of protein domains and their respective ligands, and provides for an effective means of mobilizing cellular signaling machines (2). A particularly interesting membrane localization domain that has been identified in over 100 proteins is the PH domain, which spans approximately 120 residues and contains an invariant tryptophan in its COOH-terminal region (3). Several PH domain structures have been solved by NMR and by x-ray crystallography, giving rise to the concept that their overall protein fold is formed from seven  sheets with connecting loops that form a ligand binding scaffold (4 -10). The PH domain fold is similar to that of several other ligand binding protein domains that differ substantially in amino acid sequence, and has therefore been ...