No abstract

The FYVE domain mediates the recruitment of proteins involved in membrane trafficking and cell signaling to phosphatidylinositol 3-phosphate (PtdIns(3)P)-containing membranes. To elucidate the mechanism by which the FYVE domain interacts with PtdIns(3)P-containing membranes, we measured the membrane binding of the FYVE domains of yeast Vps27p and Drosophila hepatocyte growth factor-regulated tyrosine kinase substrate and their mutants by surface plasmon resonance and monolayer penetration analyses. These measurements as well as electrostatic potential calculation show that PtdIns(3)P specifically induces the membrane penetration of the FYVE domains and increases their membrane residence time by decreasing the positive charge surrounding the hydrophobic tip of the domain and causing local conformational changes. Mutations of hydrophobic residues located close to the PtdIns(3)P-binding pocket or an Arg residue directly involved in PtdIns(3)P binding abrogated the penetration of the FYVE domains into the monolayer, the packing density of which is comparable with that of biological membranes and large unilamellar vesicles. Based on these results, we propose a mechanism of the membrane binding of the FYVE domain in which the domain first binds to the PtdIns(3)P-containing membrane by specific PtdIns(3)P binding and nonspecific electrostatic interactions, which is then followed by the PtdIns(3)P-induced partial membrane penetration of the domain.FYVE domains are small (70 -80 amino acids) cysteine-rich domains that bind two zinc ions (1, 2). They are named for the first letters of the first four proteins in which they were identified: Fablp, YOTB, Vac1p, and early endosomal antigen 1 (EEA1).1 A functional role for the FYVE domain was first recognized when it was found to facilitate the localization of EEA1 to endosomes (3). Thereafter, it has been demonstrated that the FYVE domain specifically binds phosphatidylinositol 3-phosphate (PtdIns(3)P) in vitro (4, 5). PtdIns(3)P is constitutively present in eukaryotic cells, and the majority of PtdIns(3)P in mammalian cells is produced by class III phosphoinositide 3-kinase (6). PtdIns(3)P is thought to be involved in vesicle trafficking as mutations and inhibition of phosphoinositide 3-kinase have caused mis-sorting of vacuolar proteins, changes in vacuole morphology, and defects in the endocytic pathway (7). As expected from proposed roles of PtdIns(3)P, it has been found in specific subcellular locales, including the cytoplasmic face of early endosomes and internal vesicles of multivesicular bodies (6). Consistent with its in vitro PtdIns(3)P specificity, the FYVE domain has been also shown to be localized to these membrane sites in vivo (8, 9). Recent structural analyses of several FYVE domains (10 -13), a crystallographic structure analysis of EEA1-FYVE in particular (13), have elucidated the mechanism by which FYVE domains specifically recognize the PtdIns(3)P molecule. However, less is known about the mechanism by which FYVE domains interact with and are targeted to P...

Phox (PX) domains are phosphoinositide (PI)-binding domains with broad PI specificity. Two cytosolic components of NADPH oxidase, p40(phox) and p47(phox), contain PX domains. The PX domain of p40(phox) specifically binds phosphatidylinositol 3-phosphate, whereas the PX domain of p47(phox) has two lipid binding sites, one specific for phosphatidylinositol 3,4-bisphosphate and the other with affinity for phosphatidic acid or phosphatidylserine. To delineate the mechanisms by which these PX domains interact with PI-containing membranes, we measured the membrane binding of these domains and respective mutants by surface plasmon resonance and monolayer techniques and also calculated the electrostatic potentials of the domains as a function of PI binding. Results indicate that membrane binding of both PX domains is initiated by nonspecific electrostatic interactions, which is followed by the membrane penetration of hydrophobic residues. The membrane penetration of the p40(phox) PX domain is induced by phosphatidylinositol 3-phosphate, whereas that of the p47(phox) PX domain is triggered by both phosphatidylinositol 3,4-bisphosphate and phosphatidic acid (or phosphatidylserine). Studies of enhanced green fluorescent protein-fused PX domains in HEK293 cells indicate that this specific membrane penetration is also important for subcellular localization of the two PX domains. Further studies on the full-length p40(phox) and p47(phox) proteins showed that an intramolecular interaction between the C-terminal Src homology 3 domain and the PX domain prevents the nonspecific monolayer penetration of p47(phox), whereas such an interaction is absent in p40(phox).

Background: Propofol binding to GABA A R sites of uncertain location potentiates receptor function and produces anesthesia in vivo. Results: A photoreactive propofol analog identifies propofol-binding sites in ␣1␤3 GABA A Rs. Conclusion: Propofol binds to each class of intersubunit sites in the GABA A R transmembrane domain. Significance: This study demonstrates that propofol binds to the same sites in a GABA A R as etomidate and barbiturates.

The mechanism by which inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5, 6)P4) regulates chloride (Cl-) secretion was evaluated in the colonic epithelial cell line T84 using whole cell voltage clamp techniques. Our studies focused on the calcium-dependent chloride conductance (gClCa) that was activated either by mobilizing intracellular calcium (Cai) stores with thapsigargin or by introduction of the autonomous, autophosphorylated calmodulin-dependent protein kinase II (CaMKII) into the cell via the patch pipette. Basal concentrations of Ins(3,4,5,6)P4 (1 microM) present in the pipette solution had no significant effect on Cl- current; however, as the concentration of the polyphosphate was increased there was a corresponding reduction in anion current, with near complete inhibition at 8-10 microM Ins(3,4,5,6)P4. Corresponding levels are found in cells after sustained receptor-dependent activation of phospholipase C. The Ins(3,4,5, 6)P4-induced inhibition of gClCa was isomer specific; neither Ins(1, 3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, nor Ins(1,3,4,5,6)P5 induced current inhibition at concentrations of up to 100 microM. Annexin IV also plays an inhibitory role in modulating gClCa in T84 cells. When 2 microM annexin IV was present in the pipette solution, a concentration that by itself has no effect on gClCa, the potency of Ins(3,4,5,6)P4 was approximately doubled. The combination of Ins(3,4,5,6)P4 and annexin IV did not alter the in vitro activity of CaMKII. These data demonstrate that Ins(3,4,5,6)P4 is an additional cellular signal that participates in the control of salt and fluid secretion, pH balance, osmoregulation, and other physiological activities that depend upon gClCa activation. Ins(3,4,5,6)P4 metabolism and action should also be taken into account when designing treatment strategies for cystic fibrosis.

Studies of cellular signal transduction mechanisms involving receptor-mediated generation of inositol phosphates and phosphorylated phosphatidylinositols require easy access to these naturally occurring products. Although numerous synthetic methods have been developed during the past decade, most of these methods suffer from excessive length and lack of generality. In this work we describe the comprehensive and uniform synthesis of all naturally occurring phosphatidylinositols such as phosphatidylinositol, phosphatidylinositol 3-phosphate, 4-phosphate, 5-phosphate, 3,4-bisphosphate, 3,5-bisphosphate, 4,5-bisphosphate, and 3,4,5-trisphosphate, featuring both saturated and unsaturated fatty acid chains.

The mechanism of phosphatidylinositol-specific phospholipase C (PI-PLC) has been suggested to resemble that of ribonuclease A. The goal of this work is to rigorously evaluate the mechanism of PI-PLC from Bacillus thuringiensis by examining the functional and structural roles of His-32 and His-82, along with the two nearby residues Asp-274 and Asp-33 (which form a hydrogen bond with His-32 and His-82, respectively), using site-directed mutagenesis. In all, twelve mutants were constructed, which, except D274E, showed little structural perturbation on the basis of 1D NMR and 2D NOESY analyses. The H32A, H32N, H32Q, H82A, H82N, H82Q, H82D, and D274A mutants showed a 10 4 -10 5 -fold decrease in specific activity toward phosphatidylinositol; the D274N, D33A, and D33N mutants retained 0.1-1% activity, whereas the D274E mutant retained 13% activity. Steady-state kinetic analysis of mutants using (2R)-1,2-dipalmitoyloxypropane-3-(thiophospho-1D-myo-inositol) (DPsPI) as a substrate generally agreed well with the specific activity toward phosphatidylinositol. The results suggest a mechanism in which His-32 functions as a general base to abstract the proton from 2-OH and facilitates the attack of the deprotonated 2-oxygen on the phosphorus atom. This general base function is augmented by the carboxylate group of Asp-274 which forms a diad with His-32. The H82A and D33A mutants showed an unusually high activity with substrates featuring low pK a leaving groups, such as DPsPI and p-nitrophenyl inositol phosphate (NPIPs). These results suggest that His-82 functions as the general acid with assistance from Asp-33, facilitating the departure of the leaving group by protonation of the glycerol O3 oxygen. The Brønsted coefficients obtained for the WT and the D33N mutant indicate a high degree of proton transfer to the leaving group and further underscore the "helper" function of Asp-33. The complete mechanism also includes activation of the phosphate group toward nucleophilic attack by a hydrogen bond between Arg-69 and a nonbridging oxygen atom. The overall mechanism can be described as "complex" general acid-general base since three elements are required for efficient catalysis.Phosphatidylinositol-specific phospholipase C (PI-PLC) 1 plays a key role in receptor-mediated transformations of inositol phospholipids (1-4). The products of mammalian PI-PLC play important biological roles because they serve as a source of intracellular signaling molecules, diacylglycerol and inositol 1,4,5-trisphosphate. Diacylglycerol activates protein kinase C (5), and inositol 1,4,5-trisphosphate is involved in releasing intracellular calcium stores (6). The released calcium then activates many types of cellular processes. Glycosylphosphatidylinositol-specific phospholipase C, a subclass of PI-PLC, cleaves the spacer arm of glycosylphosphatidylinositol-anchored proteins to release extracellular enzymatic activities of these proteins (7). As shown in Figure 1, the reaction of bacterial PI-PLC consists of two steps: fast cleavage of PI int...

Mammalian phospholipases D (PLD), which catalyze the hydrolysis of phosphatidylcholine to phosphatidic acid (PA), have been implicated in various cell signaling and vesicle trafficking processes. Mammalian PLD1 contains two different membrane-targeting domains, pleckstrin homology and Phox homology (PX) domains, but the precise roles of these domains in the membrane binding and activation of PLD1 are still unclear. To elucidate the role of the PX domain in PLD1 activation, we constructed a structural model of the PX domain by homology modeling and measured the membrane binding of this domain and selected mutants by surface plasmon resonance analysis. The PLD1 PX domain was found to have high phosphoinositide specificity, i.e. phosphatidylinositol 3,4,5-trisphosphate (PtdIns-(3,4,5)P 3 ) > > phosphatidylinositol 3-phosphate > phosphatidylinositol 5-phosphate > > other phosphoinositides. The PtdIns(3,4,5)P 3 binding was facilitated by the cationic residues (Lys 119 , Lys 121 , and Arg 179 ) in the putative binding pocket. Consistent with the model structure that suggests the presence of a second lipid-binding pocket, vesicle binding studies indicated that the PLD1 PX domain could also bind with moderate affinity to PA, phosphatidylserine, and other anionic lipids, which were mediated by a cluster of cationic residues in the secondary binding site. Simultaneous occupancy of both binding pockets synergistically increases membrane affinity of the PX domain. Electrostatic potential calculations suggest that a highly positive potential near the secondary binding site may facilitate the initial adsorption of the domain to the anionic membrane, which is followed by the binding of PtdIns(3,4,5)P 3 to its binding pocket. Collectively, our results suggest that the interaction of the PLD1 PX domain with PtdIns(3,4,5)P 3 and/or PA (or phosphatidylserine) may be an important factor in the spatiotemporal regulation and activation of PLD1. Mammalian phospholipase D (PLD)1 catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid (PA) and choline (1, 2). PA may act as a lipid mediator for various proteins involved in cell signaling and vesicle trafficking (3, 4) and may also regulate the physical property of the cellular membranes (5, 6). Two isoforms of mammalian PLDs, PLD1 and PLD2, have been implicated in numerous cellular processes, including vesicle trafficking, cytoskeletal rearrangement, and proliferation (1,3,4,7,8). PLDs are activated in many cell types in response to growth factors, hormones, and neurotransmitters (9). It has been reported that PLD activities are regulated through interactions with a wide variety of molecules, including small GTP-binding proteins, such as ADPribosylation factor (Arf), Rho, Rac, and Cdc42, and protein kinase C isoforms (10 -16).In most mammalian cells, PLD activities have been found associated with the membrane fraction but PLDs show complex membrane localization patterns depending on cell types. While PLD2 is mainly found at the plasma membrane (17), PLD1 shows dynam...