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...

The FYVE domain associates with phosphatidylinositol 3-phosphate [PtdIns(3)P] in membranes of early endosomes and penetrates bilayers. Here, we detail principles of membrane anchoring and show that the FYVE domain insertion into PtdIns(3)P-enriched membranes and membrane-mimetics is substantially increased in acidic conditions. The EEA1 FYVE domain binds to POPC/POPE/PtdIns(3)P vesicles with a Kd of 49 nM at pH 6.0, however associates ~24 fold weaker at pH 8.0. The decrease in the affinity is primarily due to much faster dissociation of the protein from the bilayers in basic media. Lowering the pH enhances the interaction of the Hrs, RUFY1, Vps27p and WDFY1 FYVE domains with PtdIns(3)P-containing membranes in vitro and in vivo, indicating that pH-dependency is a general function of the FYVE finger family. The PtdIns(3)P binding and membrane insertion of the FYVE domain is modulated by the two adjacent His residues of the R(R/K)HHCRXCG signature motif. Mutation of either His residue abolishes the pH-sensitivity. Both protonation of the His residues and nonspecific electrostatic contacts stabilize the FYVE domain in the lipid-bound form, promoting its penetration and increasing the membrane residence time.