Distinct polyphosphoinositide binding selectivities for pleckstrin homology domains of GRP1-like proteins based on diglycine versus triglycine motifs
UMass Chan Affiliations
Program in Molecular Medicine and Department of Biochemistry and Molecular BiologyDocument Type
Journal ArticlePublication Date
2000-07-27Keywords
Amino Acid SequenceAnimals
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
Life Sciences
Medicine and Health Sciences
Metadata
Show full item recordAbstract
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.Source
J Biol Chem. 2000 Oct 20;275(42):32816-21. Link to article on publisher's siteDOI
10.1074/jbc.M002435200Permanent Link to this Item
http://hdl.handle.net/20.500.14038/42400PubMed ID
10913124Related Resources
Link to Article in PubMedae974a485f413a2113503eed53cd6c53
10.1074/jbc.M002435200