Title

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

UMMS Affiliation

Program in Molecular Medicine and Department of Biochemistry and Molecular Biology

Date

7-27-2000

Document Type

Article

Subjects

Amino Acid Sequence; Animals; Binding Sites; Binding, Competitive; CHO Cells; Cell Adhesion Molecules; Cricetinae; GTPase-Activating Proteins; Guanine Nucleotide Exchange Factors; Kinetics; Molecular Sequence Data; Peptide Fragments; Phosphatidylinositol Phosphates; Receptors, Cytoplasmic and Nuclear; Recombinant Fusion Proteins; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Transfection; src Homology Domains

Disciplines

Life Sciences | Medicine and Health Sciences

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.

Rights and Permissions

Citation: J Biol Chem. 2000 Oct 20;275(42):32816-21. Link to article on publisher's site

DOI of Published Version

10.1074/jbc.M002435200

Related Resources

Link to Article in PubMed

Journal Title

The Journal of biological chemistry

PubMed ID

10913124