Regulation of the activity of the promoter of RNA-induced silencing, C3PO.

RNA-induced silencing is a process which allows cells to regulate the synthesis of specific proteins. RNA silencing is promoted by the protein C3PO (component 3 of RISC). We have previously found that phospholipase Cβ, which increases intracellular calcium levels in response to specific G protein signals, inhibits C3PO activity towards certain genes.

RNA-induced silencing attenuates G protein-mediated calcium signals.

Phospholipase Cβ (PLCβ) is activated by G protein subunits in response to environmental stimuli to increase intracellular calcium. In cells, a significant portion of PLCβ is cytosolic, where it binds a protein complex required for efficient RNA-induced silencing called C3PO (component 3 promoter of RISC). Binding between C3PO and PLCβ raises the possibility that RNA silencing activity can affect the ability of PLCβ to mediate calcium signals.

Phospholipase Cβ connects G protein signaling with RNA interference.

Phosphoinositide-specific-phospholipase Cβ (PLCβ) is the main effector of Gαq stimulation which is coupled to receptors that bind acetylcholine, bradykinin, dopamine, angiotensin II as well as other hormones and neurotransmitters. Using a yeast two-hybrid and other approaches, we have recently found that the same region of PLCβ that binds Gαq also interacts with Component 3 Promoter of RNA induced silencing complex (C3PO), which is required for efficient activity of the RNA-induced silencing complex. In purified form, C3PO competes with Gαq for PLCβ binding and at high concentrations can quench PLCβ activation.

Hydrolysis rates of different small interfering RNAs (siRNAs) by the RNA silencing promoter complex, C3PO, determines their regulation by phospholipase Cβ.

C3PO plays a key role in promoting RNA-induced gene silencing. C3PO consists of two subunits of the endonuclease translin-associated factor X (TRAX) and six subunits of the nucleotide-binding protein translin. We have found that TRAX binds strongly to phospholipase Cβ (PLCβ), which transmits G protein signals from many hormones and sensory inputs.

Role of phospholipase C-β in RNA interference.

Phospholipase C-β (PLCβ) enzymes are activated by G proteins in response to agents such as hormones and neurotransmitters, and have been implicated in leukemias and neurological disorders. PLCβ activity causes an increase in intracellular calcium which ultimately leads to profound changes in the cell. PLCβ localizes to three cellular compartments: the plasma membrane, the cytosol and the nucleus.

Phospholipase Cβ1 is linked to RNA interference of specific genes through translin-associated factor X.

Phospholipase Cβ1 (PLCβ1) is a G-protein-regulated enzyme whose activity results in proliferative and mitogenic changes in the cell. We have previously found that in solution PLCβ1 binds to the RNA processing protein translin-associated factor X (TRAX) with nanomolar affinity and that this binding competes with G proteins. Here, we show that endogenous PLCβ1 and TRAX interact in SK-N-SH cells and also in HEK293 cells induced to overexpress PLCβ1.

Synergistic Ca2+ responses by G{alpha}i- and G{alpha}q-coupled G-protein-coupled receptors require a single PLC{beta} isoform that is sensitive to both G{beta}{gamma} and G{alpha}q.

Cross-talk between Gα(i)- and Gα(q)-linked G-protein-coupled receptors yields synergistic Ca(2+) responses in a variety of cell types. Prior studies have shown that synergistic Ca(2+) responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase Cβ3 (PLCβ3), with a possible contribution of PLCβ2, whereas signaling through PLCβ4 interferes with synergy. We here show that synergy can be induced by the combination of Gβγ and Gα(q) activation of a single PLCβ isoform.

Synergistic activation of phospholipase C-beta3 by Galpha(q) and Gbetagamma describes a simple two-state coincidence detector.

Background: Receptors that couple to G(i) and G(q) often interact synergistically in cells to elicit cytosolic Ca(2+) transients that are several-fold higher than the sum of those driven by each receptor alone. Such synergism is commonly assumed to be complex, requiring regulatory interaction between components, multiple pathways, or multiple states of the target protein.

Results: We show that cellular G(i)-G(q) synergism derives from direct supra-additive stimulation of phospholipase C-beta3 (PLC-beta3) by G protein subunits Gbetagamma and Galpha(q), the relevant components of the G(i) and G(q) signaling pathways.

Caveolin-1 alters Ca(2+) signal duration through specific interaction with the G alpha q family of G proteins.

Caveolae are membrane domains having caveolin-1 (Cav1) as their main structural component. Here, we determined whether Cav1 affects Ca(2+) signaling through the Galpha(q)-phospholipase-Cbeta (PLCbeta) pathway using Fischer rat thyroid cells that lack Cav1 (FRTcav(-)) and a sister line that forms caveolae-like domains due to stable transfection with Cav1 (FRTcav(+)). In the resting state, we found that eCFP-Gbetagamma and Galpha(q)-eYFP are similarly associated in both cell lines by Forster resonance energy transfer (FRET).

Signaling through a G Protein-coupled receptor and its corresponding G protein follows a stoichiometrically limited model.

The bradykinin receptor is a G protein-coupled receptor (GPCR) that is coupled to the Galpha(q) family of heterotrimeric G proteins. In general, a GPCR can exert intracellular signals either by transiently associating with multiple diffusing G protein subunits or by activating a G protein that is stably bound to the receptor, thus generating a signal that is limited by the stoichiometry of the complex. Here we have distinguished between these models by monitoring the association of type 2 bradykinin receptor (B(2)R) and the Galpha(q)/Gbetagamma heterotrimer in living human embryonic kidney 293 cells expressing fluorescent-tagged proteins.

Real-time measurements of protein affinities on membrane surfaces by fluorescence spectroscopy.

Signal transduction in cells involves transitory interactions between proteins and membranes and between different proteins of the interacting species. These associations depend on the strength of the interactions and on the local concentration. Because the energy and intensity of the fluorescence of many probes are very sensitive to the local environment, fluorescence measurements can report on events, such as membrane binding and protein association, in real time.

Influence of membrane components in the binding of proteins to membrane surfaces.

We have quantified the enhancement of membrane binding of activated and deactivated Galpha(s) and Galpha(q) subunits, Gbetagamma subunits, and phospholipase Cbeta(2) by lipid rafts and by the presence of membrane-associated protein partners. Membrane binding studies show that lipid rafts do not affect the intrinsic membrane affinity of Galpha(q)(GDP) and Galpha(s)(GDP), supporting the idea that these proteins partition evenly between the domains. Visualization of lipid rafts on monolayers by use of a probe that does not enter raft domains shows that neither activated nor deactivated Galpha(q)(GDP) subunits distribute evenly between the raft and nonraft domains, contrary to previous suggestions.

The Pleckstrin homology domains of phospholipases C-beta and -delta confer activation through a common site.

Mammalian inositol-specific phospholipase C-beta2 (PLC beta 2) and PLC delta 1 differ in their cellular activators. PLC beta 2 can be activated by G beta gamma subunits, whereas PLC delta 1 can be activated by phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2). For both proteins, the N-terminal pleckstrin homology (PH) domain appears to mediate activation.

Multiple roles of pleckstrin homology domains in phospholipase Cbeta function.

Since their discovery almost 10 years ago pleckstrin homology (PH) domains have been identified in a wide variety of proteins. Here, we focus on two proteins whose PH domains play a defined functional role, phospholipase C (PLC)-beta(2) and PLCdelta(1). While the PH domains of both proteins are responsible for membrane targeting, their specificity of membrane binding drastically differs.