GRP1 Pleckstrin Homology Domain:  Activation Parameters and Novel Search Mechanism for Rare Target Lipid

Abstract

Pleckstrin homology (PH) domains play a central role in a wide array of signaling pathways by binding second messenger lipids of the phosphatidylinositol phosphate (PIP) lipid family. A given type of PIP lipid is formed in a specific cellular membrane where it is generally a minor component of the bulk lipid mixture. For example, the signaling lipid PI(3,4,5)P 3 (or PIP 3 ) is generated primarily in the inner leaflet of the plasma membrane where it is believed to never exceed 0.02% of the bulk lipid. The present study focuses on the PH domain of the general receptor for phosphoinositides, isoform 1 (GRP1), which regulates the actin cytoskeleton in response to PIP 3 signals at the plasma membrane surface. The study systematically analyzes both the equilibrium and kinetic features of GRP1-PH domain binding to its PIP lipid target on a bilayer surface. Equilibrium binding measurements utilizing protein-to-membrane fluorescence resonance energy transfer (FRET) to detect GRP1-PH domain docking to membrane-bound PIP lipids confirm specific binding to PIP 3 . A novel FRET competitive binding measurement developed to quantitate docking affinity yields a K D of 50 ± 10 nM for GRP1-PH domain binding to membrane-bound PIP 3 in a physiological lipid mixture approximating the composition of the plasma membrane inner leaflet. This observed K D lies in a suitable range for regulation by physiological PIP 3 signals. Interestingly, the affinity of the interaction decreases at least 12-fold when the background anionic lipids phosphatidylserine (PS) and phosphatidylinositol (PI) are removed from the lipid mixture. Stopped-flow kinetic studies using protein-to-membrane FRET to monitor association and dissociation time courses reveal that this affinity decrease arises from a corresponding decrease in the on-rate for GRP1-PH domain docking with little or no change in the off-rate for domain dissociation from membrane-bound PIP 3 . Overall, these findings indicate that the PH domain interacts not only with its target lipid, but also with other features of the membrane surface. The results are consistent with a previously undescribed type of two-step search mechanism for lipid binding domains in which weak, nonspecific electrostatic interactions between the PH domain and background anionic lipids facilitate searching of the membrane surface for PIP 3 headgroups, thereby speeding the high-affinity, specific docking of the domain to its rare target lipid.Many significant events in cell signaling take place at membrane surfaces, particularly at the surface of the plasma membrane where receptors, channels, and signaling complexes make critical decisions to turn specific pathways on or off. An important class of membranebound second messengers that often play key roles in such decisions are the phosphatidylinositol phosphate lipids (PIP 1 lipids). Three of the most well characterized PIP signaling lipids in the plasma membrane are PI(3,4,5)P 3 , PI(3,4)P 2 , and PI(4,5)P 2 . These † Support provided by NIH Grant GM R01-63235 (to J.J...

Determining selectivity of phosphoinositide-binding domains

“…Two methods that have been employed for this are fluorescence resonance energy transfer (FRET) and isothermal titration calorimetry (ITC) [35,36]. Corbin et al [37] have followed vesicle association of the Grp1 PH domain by monitoring FRET between tryptophans in the PH domain and dansylated lipid incorporated into the vesicles. A similar FRET approach has also been employed for assessing the binding of Texas Red labeled MARCKS peptide to vesicles containing fluorescent PtdIns(4,5)P 2 [38].…”

Structural Basis for the Golgi Association by the Pleckstrin Homology Domain of the Ceramide Trafficking Protein (CERT)

“…However, the contributions of those nonspecific interactions are less prominent, as compared with that of the CERT PH domain (32,33). For example, in the case of the GRP1 PH domain, the K D to PtdIns(3,4,5)P 3 -embedded liposomes is 50 Ϯ 10 nM, and the affinity was reduced by only 12-fold when the acidic lipid components were eliminated from the liposome (34).…”

Molecular mechanism of membrane targeting by the GRP1 PH domain*

“…The b1-b2, b3-b4, and b6-b7 loops form a PI binding pocket, and their length and primary sequence define the PH domain specificity. Like other PH modules, the GRP1 PH domain is electrostatically polarized and has a strong positive electrostatic potential surrounding the binding site, which may contribute to both the specific PtdIns(3,4,5)P 3 binding and the weak nonspecific electrostatic interactions with other acidic lipids in membranes (14,18,19). Although the majority of PH domains exhibit broad selectivity and associate with several PIs, recruiting their host proteins to different intracellular membranes, PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 remain the most common targets for this family of lipid binding effectors.…”