Transmembrane signals initiated by a broad range of extracellular stimuli converge on nodes that regulate phospholipase C (PLC)-dependent inositol lipid hydrolysis for signal propagation. We describe how heterotrimeric guanine nucleotide-binding proteins (G proteins) activate PLC-βs and in turn are deactivated by these downstream effectors. The 2.7-angstrom structure of PLC-β3 bound to activated Gα q reveals a conserved module found within PLC-βs and other effectors optimized for rapid engagement of activated G proteins. The active site of PLC-β3 in the complex is occluded by an intramolecular plug that is likely removed upon G protein-dependent anchoring and orientation of the lipase at membrane surfaces. A second domain of PLC-β3 subsequently accelerates guanosine triphosphate hydrolysis by Gα q , causing the complex to dissociate and terminate signal propagation. Mutations within this domain dramatically delay signal termination in vitro and in vivo. Consequently, this work suggests a dynamic catch-and-release mechanism used to sharpen spatiotemporal signals mediated by diverse sensory inputs.Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] to the second messengers inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] and diacylglycerol in an essential step for the physiological action of many hormones, neurotransmitters, growth factors, and other extracellular stimuli (1-3). These cascades use

Specific recognition of phosphoinositides is crucial for protein sorting and membrane trafficking. Protein transport to the yeast vacuole depends on the Vam7 t-SNARE and its phox homology (PX) domain. Here, we show that the PX domain of Vam7 targets to vacuoles in vivo in a manner dependent on phosphatidylinositol 3-phosphate generation. A novel phosphatidylinositol-3-phosphate-binding motif and an exposed loop that interacts with the lipid bilayer are identified by nuclear magnetic resonance spectroscopy. Conservation of key structural and binding site residues across the diverse PX family indicates a shared fold and phosphoinositide recognition function.

Regulators of G-protein signaling (RGS) proteins enhance the intrinsic GTPase activity of G protein alpha (Galpha) subunits and are vital for proper signaling kinetics downstream of G protein-coupled receptors (GPCRs). R7 subfamily RGS proteins specifically and obligately dimerize with the atypical G protein beta5 (Gbeta5) subunit through an internal G protein gamma (Ggamma)-subunit-like (GGL) domain. Here we present the 1.95-A crystal structure of the Gbeta5-RGS9 complex, which is essential for normal visual and neuronal signal transduction. This structure reveals a canonical RGS domain that is functionally integrated within a molecular complex that is poised for integration of multiple steps during G-protein activation and deactivation.

Targeting of a wide variety of proteins to membranes involves specific recognition of phospholipid head groups and insertion into lipid bilayers. For example, proteins that contain FYVE domains are recruited to endosomes through interaction with phosphatidylinositol 3-phosphate (PtdIns(3)P). However, the structural mechanism of membrane docking and insertion by this domain remains unclear. Here, the depth and angle of micelle insertion and the lipid binding properties of the FYVE domain of early endosome antigen 1 are estimated by NMR spectroscopy. Spin label probes incorporated into micelles identify a hydrophobic protuberance that inserts into the micelle core and is surrounded by interfacially active polar residues. A novel proxyl PtdIns(3)P derivative is developed to map the position of the phosphoinositide acyl chains, which are found to align with the membrane insertion element. Dual engagement of the FYVE domain with PtdIns(3)P and dodecylphosphocholine micelles yields a 6-fold enhancement of affinity. The additional interaction of phosphatidylserine with a conserved basic site of the protein further amplifies the micelle binding affinity and dramatically alters the angle of insertion. Thus, the FYVE domain is targeted to endosomes through the synergistic action of stereospecific PtdIns(3)P head group ligation, hydrophobic insertion and electrostatic interactions with acidic phospholipids.Cellular processes including signal transduction, vesicular trafficking, and cytoskeletal rearrangement require selective recruitment of proteins to membrane surfaces. Well established mechanisms for localizing cytosolic proteins to membranes include electrostatic interactions through a basic peptide sequence, anchoring by covalently attached acyl chains, and association with the cytoplasmic domains of transmembrane proteins (reviewed in Refs. 1 and 2). (4), PDZ (5), and PTB (6) domains. Although the majority of these domains can interact with several PIs, the FYVE domain is remarkably selective for PtdIns(3)P (7-9). In addition to PI ligation, these domains often insert hydrophobic elements into the membrane bilayer, as has been demonstrated for the C2 (10), ENTH (11), FERM (12), FYVE (13), and PX (14) domains and the vinculin tail (15). Insertion into the membrane can be accompanied by interactions with multiple lipid head groups. For example, the PX domain of the p47 subunit of the NADPH oxidase binds cooperatively to PtdIns(3)P and phosphatidic acid (16), and the vinculin tail co-ligates phosphatidylinositol 4,5-bisphosphate and PtdSer (15). Although it is becoming evident that insertion of proteins into membranes is widespread, the three-dimensional orientations and quantitative binding properties remain challenging to characterize. The most common electron paramagnetic resonance and fluorescence approaches have provided important insights (17-20) but require covalent attachment of paramagnetic groups to various positions of the protein or mutations of residues. The inevitable effects of these modifications on lipi...

Specific recognition of phosphatidylinositol 3-phosphate [PtdIns(3)P] by the FYVE domain targets cytosolic proteins to endosomal membranes during key signaling and trafficking events within eukaryotic cells. Here, we show that this membrane targeting is regulated by the acidic cellular environment. Lowering the cytosolic pH enhances PtdIns(3)P affinity of the FYVE domain, reinforcing the anchoring of early endosome antigen 1 (EEA1) to endosomal membranes. Reversibly, increasing the pH disrupts phosphoinositide binding and leads to cytoplasmic redistribution of EEA1. pH dependency is due to a pair of conserved His residues, the successive protonation of which is required for PtdIns(3)P head group recognition as revealed by NMR. Substitution of the His residues abolishes PtdIns(3)P binding by the FYVE domain in vitro and in vivo. Another PtdIns(3)P-binding module, the PX domain of Vam7 and p40 phox is shown to be pH-independent. This provides the fundamental functional distinction between the two phosphoinositide-recognizing domains. The presented mode of FYVE regulation establishes the unique function of FYVE proteins as low pH sensors of PtdIns(3)P and reveals the critical role of the histidine switch in targeting of these proteins to endosomal membranes.phosphoinositide ͉ early endosome antigen 1 T he specific recruitment of FYVE proteins to endosomes, multivesicular bodies, and phagosomes is primarily mediated by FYVE domain binding to membrane-embedded phosphatidylinositol 3-phosphate [PtdIns(3)P] (1-3). Additional membrane anchoring is provided by hydrophobic insertion into the bilayer, electrostatic interactions with acidic lipids (4, 5), and dimerization (6, 7). These synergistic factors are thought to be largely responsible for directing FYVE proteins to PtdIns(3)P-enriched membranes. Yet, the current model of FYVE domain function has several limitations and is not predictive. Some FYVE proteins localize inexplicably to sites that contain little PtdIns(3)P, such as the Golgi and endoplasmic reticulum (8, 9). On the other hand, places with significant PtdIns(3)P concentrations, such as the nucleus and mitochondria (10), do not appear to attract FYVE proteins. All canonical FYVE domains have been found to target PtdIns(3)P (refs. 6, 10-16); however, their binding properties were investigated at pH values of 6.5-8.0, leading to a discrepancy in estimated affinities despite the highly conserved sequences and structures of the FYVE proteins.The FYVE domain is defined by the three conserved elements: the N-terminal WxxD, the central RR͞KHHCR, and the Cterminal RVC motifs that form the PtdIns(3)P binding site seen in the solution and crystal structures of early endosome antigen 1 (EEA1), Hrs, and Vps27p FYVE (6,13,17,18). The PtdIns(3)P headgroup is coordinated by a cluster of four Arg͞Lys and two His residues and the N-terminal Asp. Among them, the two adjacent His residues are the most conserved in the FYVE domain sequences. In this work, we demonstrate that anchoring of the FYVE domain to PtdIns(3)P-enriched ...