Finly Philip

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 Gαq 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 (B2R) and the Gαq/Gβγ heterotrimer in living human embryonic kidney 293 cells expressing fluorescent-tagged proteins. Stable B2R-Gαq·Gβγ complexes are observed in resting cells by fluorescence resonance energy transfer from either Gαq-eCFP or eCFP-Gβγ to B2R-eYFP. Stimulating the cells with bradykinin causes detachment of B2R from the G protein subunits as the receptor internalizes into early endosomes, with a corresponding elimination of B2R-G protein fluorescence resonance energy transfer because Gαq and its associated Gβγ remain on the plasma membrane. Single point and scanning fluorescence correlation spectroscopy measurements show that a portion of B2R 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.

Caveolin-1 alters Ca2+ signal duration through specific interaction with the Gαq family of G proteins

Caveolae are membrane domains having caveolin-1 (Cav1) as their main structural component. Here, we determined whether Cav1 affects Ca2+ signaling through the Gαq–phospholipase-Cβ (PLCβ) 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-Gβγ and Gαq-eYFP are similarly associated in both cell lines by Forster resonance energy transfer (FRET). Upon stimulation, the amount of FRET between Gαq-eYFP and eCFP-Gβγ remains high in FRTcav– cells, but decreases almost completely in FRTcav+ cells, suggesting that Cav1 is increasing the separation between Gαq-Gβγ subunits. In FRTcav– cells overexpressing PLCβ, a rapid recovery of Ca2+ is observed after stimulation. However, FRTcav+ cells show a sustained level of elevated Ca2+. FRET and colocalization show specific interactions between Gαq and Cav1 that increase upon stimulation. Fluorescence correlation spectroscopy studies show that the mobility of Gαq-eGFP is unaffected by activation in either cell type. The mobility of eGFP-Gβγ remains slow in FRTcav– cells but increases in FRTcav+ cells. Together, our data suggest that, upon stimulation, Gαq(GTP) switches from having strong interactions with Gβγ to Cav1, thereby releasing Gβγ. This prolongs the recombination time for the heterotrimer, thus causing a sustained Ca2+ signal.

Multiple roles of pleckstrin homology domains in phospholipase Cβ 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)‐β2 and PLCδ1. 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.

Synergistic Ca2+ responses by Gαi- and Gαq-coupled GPCRs require a single PLCβ isoform that is sensitive to both Gβγ and Gαq

Cross-talk between Gαi- and Gαq-linked G-protein-coupled receptors yields synergistic Ca2+ responses in a variety of cell types. Prior studies have shown that synergistic Ca2+ 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 Gαq activation of a single PLCβ isoform. Synergy was absent in macrophages lacking both PLCβ2 and PLCβ3, but it was fully reconstituted following transduction with PLCβ3 alone. Mechanisms of PLCβ-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCβ2. RNAi-mediated knockdown of endogenous PLCβs demonstrated that synergy in these cells was dependent on PLCβ3, but PLCβ1 and PLCβ4 did not contribute, and overexpression of either isoform inhibited Ca2+ synergy. When synergy was blocked by RNAi of endogenous PLCβ3, it could be reconstituted by expression of either human PLCβ3 or mouse PLCβ2. In contrast, it could not be reconstituted by human PLCβ3 with a mutation of the Y box, which disrupted activation by Gβγ, and it was only partially restored by human PLCβ3 with a mutation of the C terminus, which partly disrupted activation by Gαq. Thus, both Gβγ and Gαq contribute to activation of PLCβ3 in cells for Ca2+ synergy. We conclude that Ca2+ synergy between Gαi-coupled and Gαq-coupled receptors requires the direct action of both Gβγ and Gαq on PLCβ and is mediated primarily by PLCβ3, although PLCβ2 is also competent.

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. In HEK293 cells, TRAX overexpression ablates Ca2+ signals generated by G protein‐PLCβ1 activation. TRAX plays a key role in down‐regulation of proteins by small, interfering RNA, and PLCβ1 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 PLCβ1 in HEK293 and HeLa cells allows for an ~20% increase in siRNA(GAPDH) silencing. While PLCβ1 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). PLCβ1 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 PLCβ1 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 PLCβ1 in gene regulation through TRAX association.—Philip, F., Guo, Y., Aisiku, O., Scarlata, S. Phospholipase Cβ1 is linked to RNA interference of specific genes through translin‐associated factor X. FASEB J. 26, 4903–4913 (2012). www.fasebj.org

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. Additionally, we have found that the binding of PLCβ to C3PO inhibits its nuclease activity leading to reversal of RNA-induced silencing of specific genes. In cells, we found that PLCβ 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 PLCβ and C3PO gets stronger and leads to changes in the cellular distribution of PLCβ. 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 PLCβ.

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.

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. Under most cell conditions, the majority of PLCβ localizes to the plasma membrane where it interacts with G proteins. In trying to determine the factors that localize PLCβ 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 PLCβ and TRAX, and its repercussions in G protein signaling and RNA silencing.

Hydrolysis Rates of Different Small Interfering RNAs (siRNAs) by the RNA Silencing Promoter Complex, C3PO, Determines Their Regulation by Phospholipase Cβ

Background: PLCβ reverses siRNA down-regulation possibly by binding to the RNA silencing promoter C3PO. Results: PLCβ binds externally to C3PO to reduce rapid hydrolysis of specific siRNAs. Conclusion: A model for PLCβ effects on C3PO-mediated siRNA hydrolysis is presented. Significance: The studies provide a clue of the genes that may be regulated by cytosolic PLCβ. 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. The association between PLCβ and TRAX is thought to underlie the ability of PLCβ 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 PLCβ, 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 PLCβ binds ∼5-fold more weakly to translin than to TRAX but ∼2-fold more strongly to C3PO. PLCβ does not alter TRAX-translin assembly to C3PO, and brightness studies suggest one PLCβ binds to one C3PO octamer without a change in the number of TRAX/translin molecules suggesting that PLCβ binds to an external site. Functionally, we find that C3PO hydrolyzes siRNA(GAPDH) at a faster rate than siRNA(Hsp90). However, when PLCβ is bound to C3PO, the hydrolysis rate of siRNA(GAPDH) becomes comparable with siRNA(Hsp90). Our results show that the selectivity of PLCβ toward certain genes lies in the rate at which the RNA is hydrolyzed by C3PO.

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.