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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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

α-Synuclein is found in plaques associated with Parkinson's and other neurodegenerative diseases. Changes in α-synuclein oligomerization are thought to give rise to nucleation of neurodegenerative plaques. Here, we investigated the effect of hydrostatic pressure on the aggregation of α-synuclein in cultured neuronal cells. We found that hydrostatic pressure is associated with a transition from monomeric to higher order α-synuclein aggregates. We then tested whether this aggregation is associated with the loss of binding partners, such as phospholipase Cβ. We found that increased pressure reduces the level of PLCβ1 and the amount of α-synuclein/PLCβ1 complexes. These studies suggest that pressure promotes release of α-synuclein from protein partners promoting its oligomerization., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Phospholipase Cβ2 (PLC β2) is activated by G proteins and generates calcium signals in cells. PLCβ2 is absent in normal breast tissue, but is highly expressed in breast tumors where its expression is correlated with the progression and migration of the tumor. This pattern of expression parallels the expression of the breast cancer specific gene protein 1 which is also known as γ-synuclein. The cellular function of γ-synuclein and the role it plays in proliferation are unknown. Here, we determined whether γ-synuclein can interact with PLCβ2 and affect its activity. Using co-immunprecitation and co-immunofluorescence, we find that in both benign and aggressive breast cancer cell lines γ-synuclein and PLCβ2 are associated. In solution, purified γ-synuclein binds to PLCβ2 with high affinity as measured by fluorescence methods. Protease digestion and mass spectrometry studies show that γ-synuclein binds to a site on the C-terminus of PLCβ2 that overlaps with the Gαq binding site. Additionally, γ-synuclein competes for Gαq association, but not for activators that bind to the N-terminus (i.e. Rac1 and Gβγ). Binding of γ-synuclein reduces the catalytic activity of PLCβ2 by mechanism that involves inhibition of product release without affecting membrane interactions. Since activated Gαq binds more strongly to PLCβ2 than γ-synuclein, addition of Gαq(GTPγS) to the γ-synuclein -PLCβ2 complex allows for relief of enzyme inhibition along with concomitant activation. We also find that Gβγ can reverse γ-synuclein inhibition without dissociating the γ-synuclein- PLCβ2- complex. These studies point to a role of γ-synuclein in promoting a more robust G protein activation of PLCβ2.

Phospholipase C (PLC) β2, a well studied member of the family of enzymes that catalyze the hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) into secondary messengers, can be activated by the Gβγ subunits of heterotrimeric G-proteins in a manner that depends on the presence and composition of the associated phospholipid membrane surface. The N-terminal pleckstrin homology (PH) domain of PLCβ2 mediates both the response to Gβγ and membrane binding, but how these interactions are coupled to yield an activated catalytic core remains unknown. Here we propose a mechanism based on molecular models of truncated PLCβ2 in its activated form complexed with Gβγ and in the catalytically inactive/membrane-bound form, obtained with the application of protein-protein docking algorithms and coarse-grained molecular dynamics simulations. These models were probed experimentally, and the inferences were confirmed by results from a combination of molecular biology and fluorescence assays. Results from the dynamic simulations of the molecular models and their interactions with various lipid bilayers identify the determinants of PLCβ2-PH domain specificity for Gβγ and lipid membranes and suggest a mechanism for the previously reported dependence of Gβγ activation on the associated membrane composition. Together, these findings explain the roles of the different activators in terms of their effect on the orientations of the PH and catalytic core domains relative to the lipid membranes.

Rac1, which is associated with cytoskeletal pathways, can activate phospholipase Cbeta2 (PLCbeta2) to increase intracellular Ca(2+) levels. This increased Ca(2+) can in turn activate the very robust PLCdelta1 to synergize Ca(2+) signals. We have previously found that PLCbeta2 will bind to and inhibit PLCdelta1 in solution by an unknown mechanism and that PLCbeta2.PLCdelta1 complexes can be disrupted by Gbetagamma subunits. However, because the major populations of PLCbeta2 and PLCdelta1 are cytosolic, their regulation by Gbetagamma subunits is not clear. Here, we have found that the pleckstrin homology (PH) domains of PLCbeta2 and PLCbeta3 are the regions that result in PLCdelta1 binding and inhibition. In cells, PLCbeta2.PLCdelta1 form complexes as seen by Förster resonance energy transfer and co-immunoprecipitation, and microinjection of PHbeta2 dissociates the complex. Using PHbeta2 as a tool to assess the contribution of PLCbeta inhibition of PLCdelta1 to Ca(2+) release, we found that, although PHbeta2 only results in a 25% inhibition of PLCdelta1 in solution, in cells the presence of PHbeta2 appears to eliminates Ca(2+) release suggesting a large threshold effect. We found that the small plasma membrane population of PLCbeta2.PLCdelta1 is disrupted by activation of heterotrimeric G proteins, and that the major cytosolic population of the complexes are disrupted by Rac1 activation. Thus, the activity of PLCdelta1 is controlled by the amount of bound PLCbeta2 that changes with displacement of the enzyme by heterotrimeric or small G proteins. Through PLCbeta2, PLCdelta1 activation is linked to surface receptors as well as signals that mediate cytoskeletal pathways.

The plasma membrane serves as a barrier to limit the exit and entry of components into and out of the cell, offering protection from the external environment. Communication between the cell and the external environment is mediated by multiple signaling pathways. While the plasma membrane was historically viewed as a lipid bilayer with freely diffusing proteins, the last decade has shown that the lipids and proteins in the plasma membrane are organized in a non-random manner, and that this organization can direct and modify various signaling pathways in the cell. In this review, we qualitatively discuss the ways that membrane domains can affect cell signaling. We then focus on how membrane domains can affect a specific signaling pathway--the G protein-phospholipase Cbeta pathway and show how membrane domains can play an active role in directing or redirecting G protein signals.

G-proteins transduce signals along diverse pathways, but the factors involved in pathway selection are largely unknown. Here, we have studied the ability of Galpha(q) to select between two effectors-mammalian inositide-specific phospholipase Cbeta (PLCbeta) and phosphoinositide-3-kinase (PI3K)-in human embryonic kidney 293 cells. These studies were carried out by measuring interactions between eCFP- and eYFP-tagged proteins using Forster resonance energy transfer in the basal state and during stimulation. Instead of association of Galpha(q) with effectors through diffusion and exchange, we found separate and stable pools of Galpha(q)-PLCbeta and Galpha(q)-PI3K complexes existing throughout the stimulation cycle. These separate complexes existed despite the ability of Galpha(q) to simultaneously bind both effectors as determined by in vitro measurements using purified proteins. Preformed G-protein/effector complexes will limit the number of pathways that a given signal will take, which may simplify predictive models.