PubMed Health⌕ Search

Biomedical subjects

Elliott M Ross

Publications and source records attributed to Elliott M Ross.

10 recordsLinked to original sources
PubMed2006

Structural determinants for phosphatidic acid regulation of phospholipase C-beta1.

Signaling from G protein-coupled receptors to phospholipase C-beta (PLC-beta) is regulated by coordinate interactions among multiple intracellular signaling molecules. Phosphatidic acid (PA), a signaling phospholipid, binds to and stimulates PLC-beta(1) through a mechanism that requires the PLC-beta(1) C-terminal domain. PA also modulates Galpha(q) stimulation of PLC-beta(1). These data suggest that PA may have a key role in the regulation of PLC-beta(1) signaling in cells. The present studies addressed the structural requirements and the mechanism for PA regulation of PLC-beta(1). We used a combination of enzymatic assays, PA-binding assays, and circular dichroism spectroscopy to evaluate the interaction of PA with wild-type and mutant PLC-beta(1) proteins and with fragments of the Galpha(q) binding domain. The results identify a region that includes the alphaA helix and flexible loop of the Galpha(q)-binding domain as necessary for PA regulation. A mutant PLC-beta(1) with multiple alanine/glycine replacements for residues (944)LIKEHTTKYNEIQN(957) was markedly impaired in PA regulation. The high affinity and low affinity component of PA stimulation was reduced 70% and PA binding was reduced 45% in this mutant. Relative PLC stimulation by PA increased with PLC-beta(1) concentration in a manner suggesting cooperative binding to PA. Similar concentration dependence was observed in the PLC-beta(1) mutant. These data are consistent with a model for PA regulation of PLC-beta(1) that involves cooperative interactions, probably PLC homodimerization, that require the flexible loop region, as is consistent with the dimeric structure of the Galpha(q)-binding domain. PA regulation of PLC-beta(1) requires unique residues that are not required for Galpha(q) stimulation or GTPase-activating protein activity.

Binding Sites↗
PubMed2005

Gbetagamma inhibits Galpha GTPase-activating proteins by inhibition of Galpha-GTP binding during stimulation by receptor.

Gbetagamma subunits modulate several distinct molecular events involved with G protein signaling. In addition to regulating several effector proteins, Gbetagamma subunits help anchor Galpha subunits to the plasma membrane, promote interaction of Galpha with receptors, stabilize the binding of GDP to Galpha to suppress spurious activation, and provide membrane contact points for G protein-coupled receptor kinases. Gbetagamma subunits have also been shown to inhibit the activities of GTPase-activating proteins (GAPs), both phospholipase C (PLC)-betas and RGS proteins, when assayed in solution under single turnover conditions. We show here that Gbetagamma subunits inhibit G protein GAP activity during receptor-stimulated, steady-state GTPase turnover. GDP/GTP exchange catalyzed by receptor requires Gbetagamma in amounts approximately equimolar to Galpha, but GAP inhibition was observed with superstoichiometric Gbetagamma. The potency of inhibition varied with the GAP and the Galpha subunit, but half-maximal inhibition of the GAP activity of PLC-beta1 was observed with 5-10 nM Gbetagamma, which is at or below the concentrations of Gbetagamma needed for regulation of physiologically relevant effector proteins. The kinetics of GAP inhibition of both receptor-stimulated GTPase activity and single turnover, solution-based GAP assays suggested a competitive mechanism in which Gbetagamma competes with GAPs for binding to the activated, GTP-bound Galpha subunit. An N-terminal truncation mutant of PLC-beta1 that cannot be directly regulated by Gbetagamma remained sensitive to inhibition of its GAP activity, suggesting that the Gbetagamma binding site relevant for GAP inhibition is on the Galpha subunit rather than on the GAP. Using fluorescence resonance energy transfer between cyan or yellow fluorescent protein-labeled G protein subunits and Alexa532-labeled RGS4, we found that Gbetagamma directly competes with RGS4 for high-affinity binding to Galpha(i)-GDP-AlF4.

Aluminum Compounds↗
PubMed2005

Spinophilin regulates Ca2+ signalling by binding the N-terminal domain of RGS2 and the third intracellular loop of G-protein-coupled receptors.

Signalling by G proteins is controlled by the regulator of G-protein signalling (RGS) proteins that accelerate the GTPase activity of Galpha subunits and act in a G-protein-coupled receptor (GPCR)-specific manner. The conserved RGS domain accelerates the G subunit GTPase activity, whereas the variable amino-terminal domain participates in GPCR recognition. How receptor recognition is achieved is not known. Here, we show that the scaffold protein spinophilin (SPL), which binds the third intracellular loop (3iL) of several GPCRs, binds the N-terminal domain of RGS2. SPL also binds RGS1, RGS4, RGS16 and GAIP. When expressed in Xenopus laevis oocytes, SPL markedly increased inhibition of alpha-adrenergic receptor (alphaAR) Ca2+ signalling by RGS2. Notably, the constitutively active mutant alphaAR(A293E) (the mutation being in the 3iL) did not bind SPL and was relatively resistant to inhibition by RGS2. Use of betaAR-alphaAR chimaeras identified the 288REKKAA293 sequence as essential for the binding of SPL and inhibition of Ca2+ signalling by RGS2. Furthermore, alphaAR-evoked Ca2+ signalling is less sensitive to inhibition by SPL in rgs2-/- cells and less sensitive to inhibition by RGS2 in spl-/- cells. These findings provide a general mechanism by which RGS proteins recognize GPCRs to confer signalling specificity.

Animals↗
PubMed2004

Uncoupling conformational change from GTP hydrolysis in a heterotrimeric G protein alpha-subunit.

Heterotrimeric G protein alpha (G alpha) subunits possess intrinsic GTPase activity that leads to functional deactivation with a rate constant of approximately 2 min(-1) at 30 degrees C. GTP hydrolysis causes conformational changes in three regions of G alpha, including Switch I and Switch II. Mutation of G202-->A in Switch II of G alpha(i1) accelerates the rates of both GTP hydrolysis and conformational change, which is measured by the loss of fluorescence from Trp-211 in Switch II. Mutation of K180-->P in Switch I increases the rate of conformational change but decreases the GTPase rate, which causes transient but substantial accumulation of a low-fluorescence G alpha(i1).GTP species. Isothermal titration calorimetric analysis of the binding of (G202A)G alpha(i1) and (K180P)G alpha(i1) to the GTPase-activating protein RGS4 indicates that the G202A mutation stabilizes the pretransition state-like conformation of G alpha(i1) that is mimicked by the complex of G alpha(i1) with GDP and magnesium fluoroaluminate, whereas the K180P mutation destabilizes this state. The crystal structures of (K180P)G alpha(i1) bound to a slowly hydrolyzable GTP analog, and the GDP.magnesium fluoroaluminate complex provide evidence that the Mg(2+) binding site is destabilized and that Switch I is torsionally restrained by the K180P mutation. The data are consistent with a catalytic mechanism for G alpha in which major conformational transitions in Switch I and Switch II are obligate events that precede the bond-breaking step in GTP hydrolysis. In (K180P)G alpha(i1), the two events are decoupled kinetically, whereas in the native protein they are concerted.

Amino Acid Substitution↗
PubMed2003

Phosphorylation-regulated inhibition of the Gz GTPase-activating protein activity of RGS proteins by synapsin I.

Synapsins are neuronal proteins that bind and cluster synaptic vesicles in the presynaptic space, presumably by anchoring to actin filaments, but specific regulatory functions of the synapsins are unknown. We found that a sub-population of brain synapsin Ia, a splice variant of one of three synapsin isoforms, inhibits the GTPase-activating protein (GAP) activity of several RGS proteins. Inhibition is highly selective for Galphaz, a member of the Gi family that is found in neurons, platelets, adrenal chromaffin cells, and a few other neurosecretory cells. Gz has been indirectly implicated in the regulation of secretion. Synapsin Ia constitutes a major fraction of the total GAP-inhibitory activity in brain, and its inhibitory activity is absent from the brains of synapsin I(-/-)/II(-/-) mice. Inhibition depends on the cationic D/E domain of synapsin. Phosphorylation of synapsin Ia at serine 9 by either cyclic AMP-dependent protein kinase or p21-activated protein kinase (PAK1) attenuates its potency as a GAP inhibitor more than 7-fold. Synapsin can thus act as a phosphorylation-modulated mediator of feedback regulation of Gz signaling by the synaptic machinery.

Alternative Splicing↗
PubMed2003

Homer 2 tunes G protein-coupled receptors stimulus intensity by regulating RGS proteins and PLCbeta GAP activities.

Homers are scaffolding proteins that bind G protein-coupled receptors (GPCRs), inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs), ryanodine receptors, and TRP channels. However, their role in Ca2+ signaling in vivo is not known. Characterization of Ca2+ signaling in pancreatic acinar cells from Homer2-/- and Homer3-/- mice showed that Homer 3 has no discernible role in Ca2+ signaling in these cells. In contrast, we found that Homer 2 tunes intensity of Ca2+ signaling by GPCRs to regulate the frequency of [Ca2+]i oscillations. Thus, deletion of Homer 2 increased stimulus intensity by increasing the potency for agonists acting on various GPCRs to activate PLCbeta and evoke Ca2+ release and oscillations. This was not due to aberrant localization of IP3Rs in cellular microdomains or IP3R channel activity. Rather, deletion of Homer 2 reduced the effectiveness of exogenous regulators of G proteins signaling proteins (RGS) to inhibit Ca2+ signaling in vivo. Moreover, Homer 2 preferentially bound to PLCbeta in pancreatic acini and brain extracts and stimulated GAP activity of RGS4 and of PLCbeta in an in vitro reconstitution system, with minimal effect on PLCbeta-mediated PIP2 hydrolysis. These findings describe a novel, unexpected function of Homer proteins, demonstrate that RGS proteins and PLCbeta GAP activities are regulated functions, and provide a molecular mechanism for tuning signal intensity generated by GPCRs and, thus, the characteristics of [Ca2+]i oscillations.

Animals↗
PubMed2002

Overview of the Alliance for Cellular Signaling.

The Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells--B lymphocytes (the cells of the immune system) and cardiac myocytes--to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community.

B-Lymphocytes↗
PubMed2001

Mutations in the carboxyl-terminal domain of phospholipase C-beta 1 delineate the dimer interface and a potential Galphaq interaction site.

The carboxyl-terminal domain of phospholipase C-beta is required for its stimulation by Galpha(q) and for its Galpha(q)-specific GTPase-activating protein (GAP) activity. We subjected this domain to a combination of deletion and alanine/glycine scanning mutagenesis to detect mutations that would inhibit either responsiveness to G(q) or G(q) GAP activity. Most mutations that altered either response or GAP activity diminished both in parallel. Many of these mutations map at the interface at which the carboxyl-terminal domain was recently shown to form a dimer (Singer, A. U., et al. (2001) Nat. Struct. Biol., 9, 32-36). Most others clustered in an area that is a plausible Galpha(q) binding site. In addition, one mutation that differentially inhibited GAP activity relative to responsiveness to Galpha(q) mapped in this region at a location modeled to be in close contact with the switch II region of Galpha(q). This is the site at which RGS proteins are thought to exert their GAP activity. Last, a deletion mutation differentially inhibited the response of phospholipase C-beta1 to Galpha(q) without blocking GAP activity. Its location in the molecule suggests that moving the attachment point of the catalytic domain can disrupt its ability to be activated by Galpha(q).

Animals↗