title: Molecular mechanism of membrane targeting by the GRP1 PH domain.
abstract: 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)P(3)]. GRP1's pleckstrin homology (PH) domain recognizes PtdIns(3,4,5)P(3) 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)P(3) binding, regulated by pH and facilitated by electrostatic interactions with other anionic lipids. The specific recognition of PtdIns(3,4,5)P(3) triggers insertion of the GRP1 PH domain into membranes. An acidic environment enhances PtdIns(3,4,5)P(3) 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)P(3) 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)P(3) and significant membrane penetration, as illustrated by mutagenesis data. The binding affinity of the GRP1 PH domain for PtdIns(3,4,5)P(3)-containing vesicles is further amplified (by approximately 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.
authors.affiliation: Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, CO, USA.
authors.forename: Ju
authors.initials: J
authors.lastname: He
authors.forename: Rachel M
authors.initials: RM
authors.lastname: Haney
authors.forename: Mohsin
authors.initials: M
authors.lastname: Vora
authors.forename: Vladislav V
authors.initials: VV
authors.lastname: Verkhusha
authors.forename: Robert V
authors.initials: RV
authors.lastname: Stahelin
authors.forename: Tatiana G
authors.initials: TG
authors.lastname: Kutateladze
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology*
chemical_list: D008051:Lipid Bilayers; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; D018160:Receptors, Cytoplasmic and Nuclear; C060974:phosphatidylinositol 3,4,5-triphosphate; C040985:phosphatidylinositol receptors; D006639:Histidine