Abstract
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 micrometer 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.
Highlights
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
The polyphosphoinositides represent diverse membrane targeting sites for pleckstrin homology (PH) domains because they include PtdIns(3,4)P2 and PtdIns(3,4,5)P3, which appear upon cell stimulation by growth factors and other regulators of PI 3-kinases, as well as PtdIns(4,5)P2, which is present in cells constitutively at relatively high abundance (19)
Assuming a single binding site for the polyphosphoinositide on each PH domain, the highest apparent affinity for the labeled C8PtdIns(3,4,5)P3 was exhibited by the GRP1 PH domain
Summary
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. 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.
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