Distinct polyphosphoinositide binding selectivities for pleckstrin homology domains of GRP1-like proteins based on diglycine versus triglycine motifs

GRP1 and the related proteins ARNO and cytohesin-1 are ARF exchange factors that contain a pleckstrin homology (PH) domain thought to target these pro teins 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 muM for GRP1, ARNO, and cytohesin-1, respe ctively. 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 ap parent affinity. Addition of a glycine to the Gly(274)-Gly(275) motif in GR P1 greatly increased its binding affinity for PtdIns(4,5)P-2 with little ef fect 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 reduc ed 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 recru ited to ruffles in the cell surface in response to insulin, as were full-le ngth 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 opp osed to the triglycine in ARNO and cytohesin-1, directs its remarkable PtdI ns(3,4,5)P-3 binding selectivity.