Stimulation of phospholipase Cβ1 by Gαq promotes the assembly of stress granule proteins

Abstract

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.

Simulation of the Gαq /Phospholipase Cβ1 Signaling Pathway Returns Differentiated PC12 Cells to a Stem-like State

“…Our studies indicate that the levels of miRs begin to return to undifferentiated ones although the timeline appears to be much longer. Our studies also show that carbachol reduces the hydrolysis of the fluorescent-tagged miR103 and this observation correlates well with studies showing that reduction of carbachol stimulation increases the number and size of stress granule particles [24]. The link between Gαq and stress granules is through the ability of cytosolic PLCβ1 to bind stress granule proteins inhibiting their aggregation.…”

Regulation of bifunctional proteins in cells: Lessons from the phospholipase Cβ/G protein pathway

“…Stress granules are mainly composed of untranslated mRNA, but also include proteins such as Argonaute 2 (Ago2), polyadenylate binding protein 1 (PABPC1), fragile X mental retardation proteins 1 and 2 (FXR1/2), Ras, GTPase activating protein‐binding Protein 1 (G3BP1), and multiple eukaryotic translation initiation factors . We find that all of these proteins are bound to cytosolic PLCβ1 . Furthermore, stress granules are independent of P bodies (processing bodies), which are similar membraneless aggregates that traditionally do not contain translation initiation factors and only store degraded mRNA …”

“…To this end, we isolated the cytosolic fractions of both undifferentiated PC12 cells and A10 cells (smooth muscle cell line derived from thoracic aorta of rats) and pulled‐down PLCβ with a monoclonal antibody to identify the bound proteins by mass spectrometry. Surprisingly, we find that approximately one third of the bound proteins are classified as stress granule proteins …”

“…Our studies show that reducing cytosolic levels of PLCβ1 by downregulation or by activating Gαq to drive PLCβ to the membrane, increases the formation of stress granules containing Ago2, PABPC1 and G3BP1 in several types of cultured cells (Figure ). These observations suggest that the binding of PLCβ to stress granule proteins helps to prevent their aggregation .…”

“…We have found that the flattening of these domains by mechanical stretch or hypo‐osmotic conditions eliminates stabilization of activated Gαq by caveolin molecules reducing Ca 2+ signals . Additionally, osmotic stress reduces the cytosolic level of PLCβ eliminating its cytosolic function . Thus, caveolae have the potential to regulate the cytosolic versus plasma membrane functions of PLCβ by modulating the localization of Gαq and the duration of its activated state (Figure ).…”