Stimulation of Gα q -coupled GPCRs releases PLCβ1 from stress granule–associated proteins, enabling their aggregation.
During adverse environmental conditions, mammalian cells regulate protein production by sequestering the translation machinery in membraneless organelles (i.e. stress granules) whose formation is carefully regulated. In this study, we show a direct connection between G protein signaling and stress granule formation through phospholipase Cβ (PLCβ). In cells, PLCβ localizes to both the cytoplasm and plasma membrane. We find that a major population of cytosolic PLCβ binds to stress granule proteins; specifically, eIF5A and Ago2, whose RNA-induced silencing activity is halted under stress. PLCβ1 is activated by Gαq in response to hormones and neurotransmitters and we find that activation of Gαq shifts the cytosolic population of PLCβ1 to the plasma membrane, releasing stress granule proteins. This release is accompanied by the formation of intracellular particles containing stress granule markers, an increase in the size and number of particles, and a shift of cytosolic RNAs to larger sizes consistent with cessation of transcription. Arrest of protein synthesis is seen when the cytosolic level of PLCβ1 is lowered by siRNA or by osmotic stress, but not cold, heat or oxidative stress causes similar behavior. Our results fit a simple thermodynamic model in which eIF5a and its associated proteins partition into particles after release from PLCβ1 due to Gαq stimulation. Taken together, our studies show a link between Gαq-coupled signals and transcription through stress granule formation.
Phospholipase Cβ (PLCβ) is the main effector of the Gαq signaling pathway relaying different extracellular sensory information to generate intracellular calcium signals. Besides this classic function, we have found that PLCβ plays an important but unknown role in regulating PC12 cell differentiation by interacting with components in the RNA-induced silencing machinery. In trying to understand the role of PLCβ in PC12 cell differentiation, we find that over-expressing PLCβ reduces PC12 cell proliferation while down-regulating PLCβ increases the rate of cell proliferation. However, this behavior is not seen in other cancerous cell lines. To determine the underlying mechanism, we carried out mass spectrometry analysis of PLCβ complexes in PC12 cells. We find that in unsynchronized cells, PLCβ primarily binds cyclin-dependent kinase (CDK)16 whose activity plays a key role in cell proliferation. In vitro studies show a direct association between the two proteins that result in loss in CDK16 activity. When cells are arrested in the G2/M phase, a large population of PLCβ is bound to Ago2 in a complex that contains C3PO and proteins commonly found in stress granules. Additionally, another population of PLCβ complexes with CDK18 and cyclin B1. Fluorescence lifetime imaging microscopy (FLIM) confirms cell cycle dependent associations between PLCβ and these other protein binding partners. Taken together, our studies suggest that PLCβ may play an active role in mediating interactions required to move through the cell cycle.
During adverse environmental conditions, mammalian cells regulate protein production by sequestering the translation machinery in membraneless organelles (i.e. stress granules) whose formation is carefully regulated. In this study, we show a direct connection between G protein signaling and stress granule formation through phospholipase Cβ (PLCβ). In cells, PLCβ localizes to both the cytoplasm and plasma membrane. We find that a major population of cytosolic PLCβ binds to stress granule proteins; specifically, eIF5A and Ago2, whose RNA‐induced silencing activity is halted under stress. PLCβ1 is activated by Gαq in response to hormones and neurotransmitters and we find that activation of Gαq shifts the cytosolic population of PLCβ1 to the plasma membrane, releasing stress granule proteins. This release is accompanied by the formation of intracellular particles containing stress granule markers, an increase in the size and number of particles, and a shift of cytosolic RNAs to larger sizes consistent with cessation of transcription. Arrest of protein synthesis is seen when the cytosolic level of PLCβ1 is lowered by siRNA or by osmotic stress, but not cold, heat or oxidative stress causes similar behavior. Our results fit a simple thermodynamic model in which eIF5a and its associated proteins partition into particles after release from PLCβ1 due to Gαq stimulation. Taken together, our studies show a link between Gαq‐coupled signals and transcription through stress granule formation.Support or Funding InformationNIH GM116187This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The epithelial single-pass transmembrane glycoprotein Mucin 1 (MUC1) is a main constituent of mucus and has roles in cell signaling and differentiation. In addition, overexpression and subsequent homodimerization of the C-terminal subunit of MUC1 has been implicated in the progression of many cancers, particularly breast cancer. Recent experiments have shown that strong dimerization is dependent on the formation of disulfide bonds between two cysteine residues in the juxtamembrane region, but weak dimers can also be formed without them. Certain mutations in the TMD can also partially disrupt the strength of the dimer. This suggests that protein-protein or protein-lipid interactions are also mediating dimer formation. However, the transmembrane domain of MUC1-C doesn't appear to contain common oligomerization motifs such as the Sm-X 3 -Sm sequence found in glycophorin A and many receptor tyrosine kinases. Understanding the physical mechanisms by which MUC1-C dimerizes within the surrounding plasma membrane environment will help in creating drug targets, as well as generating a more comprehensive model of protein-lipid interactions. To investigate this, atomistic replica exchange molecular dynamics simulations are being used to find stable conformations of wild-type and mutant MUC1 TMDs.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.