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Molecular Mechanism of Membrane Targeting by the GRP1 PH Domain

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Molecular Mechanism of Membrane Targeting by the GRP1 PH Domain Supplemental Material can be found at: http://www.jlr.org/cgi/content/full/M800150-JLR200/DC1 Molecular mechanism of membrane targeting by the GRP1 PH domain † † † Ju He,* Rachel M. Haney, ,§ Mohsin Vora, Vladislav V. Verkhusha,** Robert V. Stahelin, ,§ and Tatiana G. Kutateladze1,* Department of Pharmacology,* University of Colorado Health Sciences Center, Aurora, CO; † Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, South Bend, IN; Department of Chemistry and Biochemistry and The Walther Center for Cancer Research,§ University of Notre Dame, South Bend, IN; and Department of Anatomy and Structural Biology,** Downloaded from Albert Einstein College of Medicine, Bronx, NY Abstract The general receptor for phosphoinositides iso- Supplementary key words general receptor for phosphoinositides iso- • • • form 1 (GRP1) is recruited to the plasma membrane in re- form 1 pleckstrin homology domain phosphoinositide phosphati- dylinositol 3,4,5-trisphosphate sponse to activation of phosphoinositide 3-kinases and www.jlr.org accumulation of phosphatidylinositol 3,4,5-trisphosphate ʼ [PtdIns(3,4,5)P3]. GRP1 s pleckstrin homology (PH) do- main recognizes PtdIns(3,4,5)P3 with high specificity and af- The signaling lipid phosphatidylinositol 3,4,5-trisphos- finity, however, the precise mechanism of its association phate [PtdIns(3,4,5)P3] is produced in plasma membranes at Albert Einstein College of Medicine Library on July 14, 2008 with membranes remains unclear. Here, we detail the mo- in response to stimulation of cell surface receptors by lecular basis of membrane anchoring by the GRP1 PH do- growth factors and hormones (1). Class I phosphoinositide main. Our data reveal a multivalent membrane docking (PI) 3-kinases phosphorylate the inositol headgroup of the involving PtdIns(3,4,5)P binding, regulated by pH and fa- 3 relatively abundant phosphatidylinositol 4,5-bisphosphate cilitated by electrostatic interactions with other anionic lip- [Ptdns(4,5)P2], transiently elevating the level of PtdIns ids. The specific recognition of PtdIns(3,4,5)P3 triggers insertion of the GRP1 PH domain into membranes. An (3,4,5)P3 from undetectable to nearly 10% of the PtdIns – acidic environment enhances PtdIns(3,4,5)P3 binding and (4,5)P2 level (2 4). The concentration of PtdIns(3,4,5)P3 is increases membrane penetration as demonstrated by tightly regulated by the activity of PI 5- and 3-phosphatases, NMR and monolayer surface tension and surface plasmon such as SHIP1/2 and PTEN, which dephosphorylate resonance experiments. The GRP1 PH domain displays a the inositol ring generating PtdIns(3,4)P2 and PtdIns 28 nM affinity for POPC/1-palmitoyl-2-oleoyl-sn-glycero-3- (4,5)P2 (5, 6). Despite the transitory accumulation and phosphoethanolamine/PtdIns(3,4,5)P3 vesicles at pH 6.0, low concentrations in the plasma membrane, PtdIns but binds 22-fold weaker at pH 8.0. The pH sensitivity is attributed in part to the His355 residue, protonation of (3,4,5)P3 is implicated in fundamental biological processes which is required for the robust interaction with PtdIns including growth, proliferation, migration, and survival of (3,4,5)P3 and significant membrane penetration, as illus- cells (1, 7). The PtdIns(3,4,5)P3-mediated signals are pri- trated by mutagenesis data. The binding affinity of the marily recognized and transduced by pleckstrin homol- GRP1 PH domain for PtdIns(3,4,5)P3-containing vesicles ogy (PH) domain-containing proteins that bind strongly ? is further amplified (by 6-fold) by nonspecific electrostatic and in some cases specifically to PtdIns(3,4,5)P3. Muta- interactions with phosphatidylserine/phosphatidylinositol. tions in the PH domains that disrupt or promote PtdIns Together, our results provide new insight into the multiva- (3,4,5)P binding cause various signaling disarrays lead- lent mechanism of the membrane targeting and regulation 3 ing to severe immune system disorders and cancers of the GRP1 PH domain.—He, J., R. M. Haney, M. Vora, V. V. Verkhusha, R. V. Stahelin, and T. G. Kutateladze. Molecular mechanism of membrane targeting by the GRP1 PH domain. J. Lipid Res. 2008. 49: 1807–1815. Abbreviations: GRP1, general receptor for phosphoinositides iso- form 1; HSQC, heteronuclear single quantum coherence; IP4, inositol 1,3,4,5-tetrakisphosphate; PH, pleckstrin homology; PI, phosphoinositide; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; PS, phos- This research was supported by an Indiana University Biomedical Research phatidylserine; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; grant; American Cancer Society Grant IRG-84-002-22 (R.V.S.); and National PtdIns(4,5)P , phosphatidylinositol 4,5-bisphosphate; SPR, surface plas- Institutes of Health Grants GM-070358, GM-073913 (V.V.V.), and GM-071424 2 and CA-95144 (T.G.K.). mon resonance. 1 To whom correspondence should be addressed. Manuscript received 24 March 2008 and in revised form 7 May 2008. e-mail: [email protected] Published, JLR Papers in Press, May 9, 2008. The online version of this article (available at http://www.jlr.org) DOI 10.1194/jlr.M800150-JLR200 contains supplementary data in the form of one figure. Copyright © 2008 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org Journal of Lipid Research Volume 49, 2008 1807 (8, 9). Among the best characterized PtdIns(3,4,5)P3- Despite the critical role in GRP1 function, the precise recognizing protein effectors are Brutonʼs tyrosine ki- mechanism by which the PH domain associates with nase, ADP-ribosylation factor (Arf)-nucleotide binding site PtdIns(3,4,5)P3-enriched membranes remains unclear. opener (ARNO), Cytohesin-1, and general receptor for Because PtdIns(3,4,5)P3 is present at a very low level even phosphoinositides isoform 1 (GRP1) (10–12). GRP1 is a in stimulated cells, elucidation of the overall membrane member of the GTP/GDP exchange factors family that targeting is essential for our understanding of how the catalyzes guanine nucleotide exchange of Arf proteins es- GRP1 PH domain distinguishes PtdIns(3,4,5)P3 and selec- sential for actin rearrangement, endo- and exocytosis, tively binds this relatively rare lipid but does not recognize membrane budding, and trafficking. Whereas the enzy- other, more-abundant PIs. Here we present the molecular matic activity of GRP1 is attributed to the central Sec7 basis of membrane docking, penetration, and pH regula- homology domain, its membrane anchoring relies exclu- tion of the GRP1 PH domain based on structural and sively on the carboxy-terminal PH domain (13). quantitative analysis of its interactions with lipids and The PH domains comprise one of the largest families membrane-mimicking monolayers and bilayers. Our re- of signaling modules and are the most thoroughly charac- sults, derived from lipid binding measurements using terized among PI binding domains (14, 15). The PH do- NMR, surface plasmon resonance (SPR), liposome bind- main was identified within a set of human proteins in ing assays, and monolayer surface tension combined with 1993 and named after the two homologous regions of mutagenesis data, provide novel insights into the mecha- Downloaded from pleckstrin, the major protein kinase C substrate of platelets nism of membrane recruitment and anchoring by GRP1. (16, 17). Since then, it has been found in several hundred eukaryotic proteins involved in intracellular signaling, membrane trafficking, cytoskeletal dynamics, and lipid MATERIALS AND METHODS modifications. The PH domain contains ?120 residues that are folded in a highly conserved three-dimensional Subcloning, expression, and purification of GRP1 www.jlr.org structure despite little sequence similarity between the fam- PH domain ily members. The fold consists of a seven-stranded b-barrel, The DNA fragment encoding residues 261–385 of the PH capped by an amphipathic a helix at one edge, whereas domain of human GRP1 was cloned into the pRSET A vector at Albert Einstein College of Medicine Library on July 14, 2008 15 15 13 the opposite edge is framed by three variable loops. The (Invitrogen). The unlabeled, N-labeled, and N/ C-labeled b b b b b b proteins were expressed in Escherichia coli Rosetta in Luria- 1- 2, 3- 4, and 6- 7 loops form a PI binding pocket, 15 Bertani or minimal media supplemented with NH4Cl and and their length and primary sequence define the PH do- 13 C6-glucose (Cambridge Isotope). Bacteria were harvested by main specificity. Like other PH modules, the GRP1 PH do- centrifugation after induction with isopropyl-1-thio-b-D-galacto- main is electrostatically polarized and has a strong positive pyranoside (0.1 mM) and lysed by French Press. The 63 His- electrostatic potential surrounding the binding site, which fusion proteins were purified on a Talon-resin column (Clontech may contribute to both the specific PtdIns(3,4,5)P3 bind- Laboratories, Inc.). The His tag was cleaved with EKMax (Invitro- ing and the weak nonspecific electrostatic interactions with gen). The proteins were further purified by ion exchange chro- other acidic lipids in membranes (14, 18, 19). Although matography on a HiTrap SP HP column (Amersham) in Bis-Tris the majority of PH domains exhibit broad selectivity and buffer, pH 6.5, and concentrated in Millipore concentrators. associate with several PIs, recruiting their host proteins PCR mutagenesis of GRP1 PH to different intracellular membranes, PtdIns(4,5)P2 and PtdIns(3,4,5)P remain the most common targets for this Site-directed mutagenesis of the GRP1 PH domain was per- 3 formed using a QuikChange kit (Stratagene). The sequence of family of lipid binding effectors. the H355A construct was confirmed by DNA sequencing. The details of how the PH domain recognizes the pat- tern of sequential 3-, 4-, and 5-phosphate groups in PtdIns NMR spectroscopy and sequence-specific assignments (3,4,5)P is provided by the crystal structure of the GRP1 3 Multidimensional homo- and heteronuclear NMR spectra were PH domain in complex with inositol 1,3,4,5-tetrakisphos- recorded at 25°C on Varian INOVA 500, 600, and 800 MHz phate (IP4), an isolated headgroup of PtdIns(3,4,5)P3 (20, spectrometers.
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