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Papers In Press, published online ahead of print August 1, 2008
J. Lipid Res., doi:10.1194/jlr.M800150-JLR200

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Journal of Lipid Research, Vol. 49, 1807-1815, August 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

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. Kutateladze^1,*

^* 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
^** Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY

^* This research was supported by an Indiana University Biomedical Research grant; American Cancer Society Grant IRG-84-002-22 (R.V.S.); and National Institutes of Health Grants GM-070358, GM-073913 (V.V.V.), and GM-071424 and CA-95144 (T.G.K.).

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of one figure.

Published, JLR Papers in Press, May 9, 2008.

¹ To whom correspondence should be addressed. e-mail: Tatiana.Kutateladze{at}UCHSC.edu

The general receptor for phosphoinositides isoform 1 (GRP1) is recruited to the plasma membrane in response to activation of phosphoinositide 3-kinases and accumulation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P₃]. GRP1's pleckstrin homology (PH) domain recognizes PtdIns(3,4,5)P₃ with high specificity and affinity, however, the precise mechanism of its association with membranes remains unclear. Here, we detail the molecular basis of membrane anchoring by the GRP1 PH domain. Our data reveal a multivalent membrane docking involving PtdIns(3,4,5)P₃ binding, regulated by pH and facilitated by electrostatic interactions with other anionic lipids. The specific recognition of PtdIns(3,4,5)P₃ triggers insertion of the GRP1 PH domain into membranes. An acidic environment enhances PtdIns(3,4,5)P₃ binding and increases membrane penetration as demonstrated by NMR and monolayer surface tension and surface plasmon resonance experiments. The GRP1 PH domain displays a 28 nM affinity for POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/PtdIns(3,4,5)P₃ vesicles at pH 6.0, but binds 22-fold weaker at pH 8.0. The pH sensitivity is attributed in part to the His355 residue, protonation of which is required for the robust interaction with PtdIns(3,4,5)P₃ and significant membrane penetration, as illustrated by mutagenesis data. The binding affinity of the GRP1 PH domain for PtdIns(3,4,5)P₃-containing vesicles is further amplified (by 6-fold) by nonspecific electrostatic interactions with phosphatidylserine/phosphatidylinositol. Together, our results provide new insight into the multivalent mechanism of the membrane targeting and regulation of the GRP1 PH domain.

Supplementary key words general receptor for phosphoinositides isoform 1 • pleckstrin homology domain • phosphoinositide • phosphatidylinositol 3,4,5-trisphosphate

Abbreviations: GRP1, general receptor for phosphoinositides isoform 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, phosphatidylserine; PtdIns(3,4,5)P₃, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(4,5)P₂, phosphatidylinositol 4,5-bisphosphate; SPR, surface plasmon resonance

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