Submitted on March 24, 2008
Revised on May 7, 2008
Accepted on May 8, 2008
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
Pharmacology, University of Colorado Health Sciences Center, Aurora, CO
Corresponding Author: Tatiana.Kutateladze{at}uchsc.edu
The general receptor for phosphoinositides isoform 1 (GRP1) is recruited to the plasma membrane in response to activation of PI 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 describe 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, monolayer surface tension and surface plasmon resonance experiments. The GRP1 PH domain displays a 28 nM affinity for POPC/POPE/PtdIns(3,4,5)P3 vesicles at pH 6.0, however 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 PS/PI. Together our results provide new insight into the multivalent mechanism of the membrane targeting and regulation of the GRP1 PH domain.
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