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 Cß, which increases intracellular calcium levels in response to specific G protein signals, inhibits C3PO activity towards certain genes. Understanding the parameters that control C3PO activity and which genes are impacted by G protein activation would help predict which genes are more vulnerable to downregulation. Here, using a library of 1018 oligonucleotides, we show that C3PO binds oligonucleotides with structural specificity but little sequence specificity. Alternately, C3PO hydrolyzes oligonucleotides with a rate that is sensitive to substrate stability. Importantly, we find that oligonucleotides with higher Tm values are inhibited by bound PLCß. This finding is supported by microarray analysis in cells over-expressing PLCß1. Taken together, this study allows predictions of the genes whose post-transcriptional regulation is responsive to the G protein/phospholipase Cß/calcium signaling pathway.

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

Phospholipase Cß (PLCß) is activated by G protein subunits in response to environmental stimuli to increase intracellular calcium. In cells, a significant portion of PLCß 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 PLCß raises the possibility that RNA silencing activity can affect the ability of PLCß to mediate calcium signals. By use of human and rat neuronal cell lines (SK-N-SH and PC12), we show that overexpression of one of the main components of C3PO diminishes Ca(2+) release in response to Gαq/PLCß stimulation by 30 to 40%. In untransfected SK-N-SH or PC12 cells, the introduction of siRNA(GAPDH) [small interfering RNA(glyceraldehyde 3-phosphate dehydrogenase)] reduces PLCß-mediated calcium signals by ∼30%, but addition of siRNA(Hsp90) (heat shock protein 90) had little effect. Fluorescence imaging studies suggest an increase in PLCß-C3PO association in cells treated with siRNA(GAPDH) but not siRNA(Hsp90). Taken together, our studies raise the possibility that Ca(2+) responses to extracellular stimuli can be modulated by components of the RNA silencing machinery.-Philip, F., Sahu, S., Golebiewska, U., Scarlata, S. RNA-induced silencing attenuates G protein-mediated calcium signals.

Phospholipase Cß connects G protein signaling with RNA interference.

Phosphoinositide-specific-phospholipase Cß (PLCß) 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 PLCß 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 PLCß binding and at high concentrations can quench PLCß activation. Additionally, we have found that the binding of PLCß to C3PO inhibits its nuclease activity leading to reversal of RNA-induced silencing of specific genes. In cells, we found that PLCß distributes between the plasma membrane where it localizes with Gαq, and in the cytosol where it localizes with C3PO. When cells are actively processing small interfering RNAs the interaction between PLCß and C3PO gets stronger and leads to changes in the cellular distribution of PLCß. The magnitude of attenuation is specific for different silencing RNAs. Our studies imply a direct link between calcium responses mediated through Gαq and post-transcriptional gene regulation through PLCß.

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

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 Cß (PLCß), which transmits G protein signals from many hormones and sensory inputs. The association between PLCß and TRAX is thought to underlie the ability of PLCß to reverse gene silencing by small interfering RNAs. However, this reversal only occurs for some genes (e.g. GAPDH and LDH) but not others (e.g. Hsp90 and cyclophilin A). To understand this specificity, we carried out studies using fluorescence-based methods. In cells, we find that PLCß, TRAX, and their complexes are identically distributed through the cytosol suggesting that selectivity is not due to large scale sequestration of either the free or complexed proteins. Using purified proteins, we find that PLCß binds ∼5-fold more weakly to translin than to TRAX but ∼2-fold more strongly to C3PO. PLCß does not alter TRAX-translin assembly to C3PO, and brightness studies suggest one PLCß binds to one C3PO octamer without a change in the number of TRAX/translin molecules suggesting that PLCß binds to an external site. Functionally, we find that C3PO hydrolyzes siRNA(GAPDH) at a faster rate than siRNA(Hsp90). However, when PLCß is bound to C3PO, the hydrolysis rate of siRNA(GAPDH) becomes comparable with siRNA(Hsp90). Our results show that the selectivity of PLCß toward certain genes lies in the rate at which the RNA is hydrolyzed by C3PO.

Role of phospholipase C-ß in RNA interference.

Phospholipase C-ß (PLCß) enzymes are activated by G proteins in response to agents such as hormones and neurotransmitters, and have been implicated in leukemias and neurological disorders. PLCß activity causes an increase in intracellular calcium which ultimately leads to profound changes in the cell. PLCß localizes to three cellular compartments: the plasma membrane, the cytosol and the nucleus. Under most cell conditions, the majority of PLCß localizes to the plasma membrane where it interacts with G proteins. In trying to determine the factors that localize PLCß to the cytosol and nucleus, we have recently identified the binding partner, TRAX. TRAX is a nuclease and part of the machinery involved in RNA interference. This review discusses the interaction between PLCß and TRAX, and its repercussions in G protein signaling and RNA silencing.

Phospholipase Cß1 is linked to RNA interference of specific genes through translin-associated factor X.

Phospholipase Cß1 (PLCß1) is a G-protein-regulated enzyme whose activity results in proliferative and mitogenic changes in the cell. We have previously found that in solution PLCß1 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 PLCß1 and TRAX interact in SK-N-SH cells and also in HEK293 cells induced to overexpress PLCß1. In HEK293 cells, TRAX overexpression ablates Ca(2+) signals generated by G protein-PLCß1 activation. TRAX plays a key role in down-regulation of proteins by small, interfering RNA, and PLCß1 overexpression completely reverses the 2- to 4-fold down-regulation of GAPDH by siRNA in HEK293 and HeLa cells as seen by an ∼4-fold recovery in both the transcript and protein levels. Also, down-regulation of endogenous PLCß1 in HEK293 and HeLa cells allows for an ∼20% increase in siRNA(GAPDH) silencing. While PLCß1 overexpression results in a 50% reversal of cell death caused by siRNA(LDH), it does not affect cell survival or silencing of other genes (e.g., cyclophilin, Hsp90, translin). PLCß1 overexpression in HEK293 and HeLa cells causes a 30% reduction in the total amount of small RNAs. LDH and GAPDH are part of a complex that promotes H2B synthesis that allows cells to progress through the S phase. We find that PLCß1 reverses the cell death and completely rescues H2B levels caused by siRNA knockdown of LDH or GAPDH. Taken together, our study shows a novel role of PLCß1 in gene regulation through TRAX association.

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 Cß3 (PLCß3), with a possible contribution of PLCß2, whereas signaling through PLCß4 interferes with synergy. We here show that synergy can be induced by the combination of Gßγ and Gα(q) activation of a single PLCß isoform. Synergy was absent in macrophages lacking both PLCß2 and PLCß3, but it was fully reconstituted following transduction with PLCß3 alone. Mechanisms of PLCß-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCß2. RNAi-mediated knockdown of endogenous PLCßs demonstrated that synergy in these cells was dependent on PLCß3, but PLCß1 and PLCß4 did not contribute, and overexpression of either isoform inhibited Ca(2+) synergy. When synergy was blocked by RNAi of endogenous PLCß3, it could be reconstituted by expression of either human PLCß3 or mouse PLCß2. In contrast, it could not be reconstituted by human PLCß3 with a mutation of the Y box, which disrupted activation by Gßγ, and it was only partially restored by human PLCß3 with a mutation of the C terminus, which partly disrupted activation by Gα(q). Thus, both Gßγ and Gα(q) contribute to activation of PLCß3 in cells for Ca(2+) synergy. We conclude that Ca(2+) synergy between Gα(i)-coupled and Gα(q)-coupled receptors requires the direct action of both Gßγ and Gα(q) on PLCß and is mediated primarily by PLCß3, although PLCß2 is also competent.

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. No additional pathway or proteins are required. Synergism is quantitatively explained by the classical and simple two-state (inactive<-->active) allosteric mechanism. We show generally that synergistic activation of a two-state enzyme reflects enhanced conversion to the active state when both ligands are bound, not merely the enhancement of ligand affinity predicted by positive cooperativity. The two-state mechanism also explains why synergism is unique to PLC-beta3 among the four PLC-beta isoforms and, in general, why one enzyme may respond synergistically to two activators while another does not. Expression of synergism demands that an enzyme display low basal activity in the absence of ligand and becomes significant only when basal activity is

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). Upon stimulation, the amount of FRET between Galpha(q)-eYFP and eCFP-Gbetagamma remains high in FRTcav(-) cells, but decreases almost completely in FRTcav(+) cells, suggesting that Cav1 is increasing the separation between Galpha(q)-Gbetagamma subunits. In FRTcav(-) cells overexpressing PLCbeta, a rapid recovery of Ca(2+) is observed after stimulation. However, FRTcav(+) cells show a sustained level of elevated Ca(2+). FRET and colocalization show specific interactions between Galpha(q) and Cav1 that increase upon stimulation. Fluorescence correlation spectroscopy studies show that the mobility of Galpha(q)-eGFP is unaffected by activation in either cell type. The mobility of eGFP-Gbetagamma remains slow in FRTcav(-) cells but increases in FRTcav(+) cells. Together, our data suggest that, upon stimulation, Galpha(q)(GTP) switches from having strong interactions with Gbetagamma to Cav1, thereby releasing Gbetagamma. This prolongs the recombination time for the heterotrimer, thus causing a sustained Ca(2+) signal.

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. Stable B(2)R-Galpha(q) x Gbetagamma complexes are observed in resting cells by fluorescence resonance energy transfer from either Galpha(q)-eCFP or eCFP-Gbetagamma to B(2)R-eYFP. Stimulating the cells with bradykinin causes detachment of B(2)R from the G protein subunits as the receptor internalizes into early endosomes, with a corresponding elimination of B(2)R-G protein fluorescence resonance energy transfer because Galpha(q) and its associated Gbetagamma remain on the plasma membrane. Single point and scanning fluorescence correlation spectroscopy measurements show that a portion of B(2)R molecules diffuses with a mobility corresponding to dimers or small oligomers, whereas a second fraction diffuses in higher order molecular assemblies. Our studies support a model in which receptors are pre-coupled with their corresponding G proteins in the basal state of cells thereby limiting the response to an external signal to a defined stoichiometry that allows for a rapid and directed cellular response.

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. We describe methods to monitor associations both in vitro and in vivo by fluorescence.

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. Membrane binding of deactivated Galpha(q) and Galpha(s)(GDP) became weaker when Gbetagamma subunits were present, in contrast with the behavior predicted by thermodynamics. However, activated Galpha subunits and phospholipase Cbeta(2) were recruited to membrane surfaces by protein partners by predicted amounts. Our studies suggest that the anomalous behavior seen for deactivated Galpha subunits in the presence of Gbetagamma subunits may be due to conformational changes in the N-terminus and/or occlusion of a portion of its membrane interaction region by Gbetagamma. Even though membrane recruitment was clearly observed for one protein partner, the presence of a second partner of lower affinity did not further promote membrane binding. For these proteins, the formation of larger protein complexes with very high membrane affinities is unlikely.

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. Here, we have constructed a chimera in which we placed the N-terminal PH domain of PLC delta 1 into remaining C-terminal regions of PLC beta 2. The PH delta PLC beta chimera showed PI(4,5)P2-dependent membrane binding similar to PLC delta 1 and a G beta gamma interaction energy close to that of PLC delta 1. Like PLC delta 1, the chimera was activated by PI(4,5)P2 through the PH domain but not by G beta gamma. Because these and previous results indicate a common site of contact between the PH and catalytic domains in these two enzymes, we computationally docked the known structures of the PH and catalytic domains of PLC delta 1. A synthetic peptide whose sequence matches a potential interaction site between the two domains inhibited the basal activity of PLC beta 2, PLC delta 1, and a G beta gamma-activable PH beta 2-PLC delta 1 chimera. Also, the peptide was able to inhibit PI(4,5)P2 and G beta gamma activation of the PH-PLC delta 1 PH-PLC beta 2 enzymes in a concentration-dependent manner, suggesting that this is the region responsible for PH domain-mediated activation of the catalytic core.

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. However, in both these proteins the PH domains work to modulate the activity of their catalytic core upon interaction with either phosphoinositol lipids or G protein activators. These observations show that these PH domains are not simply binding sites tethered onto their host enzyme but are intimately associated with their catalytic core. This property may be true for other PH domains.