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 ]. GRP1ʼs 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 ?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.-He, J., R. M. Haney, M. Vora, V. V. Verkhusha, R. V. Stahelin, and T. G. Kutateladze. The signaling lipid phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] is produced in plasma membranes in response to stimulation of cell surface receptors by growth factors and hormones (1). Class I phosphoinositide (PI) 3-kinases phosphorylate the inositol headgroup of the relatively abundant phosphatidylinositol 4,5-bisphosphate [Ptdns(4,5)P 2 ], transiently elevating the level of PtdIns (3,4,5)P 3 from undetectable to nearly 10% of the PtdIns (4,5)P 2 level (2-4). The concentration of PtdIns(3,4,5)P 3 is tightly regulated by the activity of PI 5-and 3-phosphatases, such as SHIP1/2 and PTEN, which dephosphorylate the inositol ring generating PtdIns(3,4)P 2 and PtdIns (4,5)P 2 (5, 6). Despite the transitory accumulation and low concentrations in the plasma membrane, PtdIns (3,4,5)P 3 is implicated in fundamental biological processes including growth, proliferation, migration, and survival of cells (1, 7). The PtdIns(3,4,5)P 3 -mediated signals are primarily recognized and transduced by pleckstrin homology (PH) domain-containing proteins that bind strongly and in some cases specifically to PtdIns(3,4,5)P 3 . Mutations in the PH domains that disrupt or promote PtdIns (3,4,5)P 3 binding cause various signaling disarrays leading to sever...

Structural and functional characterization of the two phosphoinositide binding sites of PROPPINs, a β-propeller protein family

“…Docking of PtdIns(3,5)P 2 was only successful when H178 and H249 of KlHsv2 were protonated. This is reminiscent of the histidine switch observed for PtdIns3P binding of the EEA1-FYVE domain (47) and PtdIns(3,4,5)P 3 binding of the GRP1 PH domain (48). We thus speculate that PtdIns(3,5)P 2 binding might also depend on pH.…”

Lipid binding domains: more than simple lipid effectors

“…The Kutateladze laboratory identified perhaps the most compelling feature among FYVE domains in their ability to target endosomes at acidic pH due to His protonation (19). This opened the door for them to demonstrate that other domains such as PH (20) and ENTH (21) can target membranes in a pH-dependent manner due to His protonation.…”

Molecular Basis of Phosphatidylinositol 4-Phosphate and ARF1 GTPase Recognition by the FAPP1 Pleckstrin Homology (PH) Domain

“…Monolayer Measurements-The insertion of FAPP1 PH into a phospholipid monolayer was investigated by measuring the change in surface pressure () of the invariable surface area upon the addition of the protein (23,30). POPC/POPE (80: 20) or POPC/POPE/PI4P (75:20:5) monolayers were spread onto the subphase composed of 10 mM HEPES, 160 mM KCl (pH 7.4) until the desired initial surface pressure ( 0 ) was reached.…”

“…The amino acid sequential assignments of the 15 N/ 13 C-labeled FAPP1 PH domain were obtained by collecting and analyzing a set of triple-resonance experiments, including HNCACB, CBCA(CO)NH, HNCO, C(CO)NH, and H(CCO)NH as described in Ref. 23. Spectra were processed with NMRPipe (24) and analyzed using CCPN (25), nmrDraw, and in-house software programs.…”