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.

Some proteins can serve multiple functions depending on different cellularconditions. An example of a bifunctional protein is inositide-specific mammalian phospholipase Cβ (PLCβ). PLCβ is activated by G proteins in response to hormones and neurotransmitters to increase intracellular calcium. Recently, alternate cellular function(s) of PLCβ have become uncovered. However, the conditions that allow these different functions to be operative are unclear. Like many mammalian proteins, PLCβ has a conserved catalytic core along with several regulatory domains. These domains modulate the intensity and duration of calcium signals in response to external sensory information, and allow this enzyme to inhibit protein translation in a noncatalytic manner. In this review, we first describe PLCβ's cellular functions and regulation of the switching between these functions, and then discuss the thermodynamic considerations that offer insight into how cells manage multiple and competitive associations allowing them to rapidly shift between functional states.

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This study investigated the effect of sodium nitroprusside (SNP) preexposure on vasodilation via the β-adrenergic receptor (BAR) system. SNP was used as a nitrosative/oxidative proinflammatory insult. Small arterioles were visualized by intravital microscopy in the hamster cheek pouch tissue (isoflurane, n = 45). Control dilation to isoproterenol (EC: 10 mol/l) became biphasic as a function of concentration after 2 min of exposure to SNP (10 M), with increased potency at picomolar dilation uncovered and decreased efficacy at the micromolar dilation. Control dilation to curcumin was likewise altered after SNP, but only the increased potency at a low dose was uncovered, whereas micromolar dilation was eliminated. The picomolar dilations were blocked by the potent BAR-2 inverse agonist carazolol (10 mol/l). Dynamin inhibition with dynasore mimicked this effect, suggesting that SNP preexposure prevented BAR agonist internalization. Using HeLa cells transfected with BAR-2 tagged with monomeric red fluorescent protein, exposure to 10-10 mol/l curcumin resulted in internalization and colocalization of BAR-2 and curcumin (FRET) that was prevented by oxidative stress (10 mol/l CoCl), supporting that stress prevented internalization of the BAR agonist with the micromolar agonist. This study presents novel data supporting that distinct pools of BARs are differentially available after inflammatory insult. NEW & NOTEWORTHY Preexposure to an oxidative/nitrosative proinflammatory insult provides a "protective preconditioning" against future oxidative damage. We examined immediate vasoactive and molecular consequences of a brief preexposure via β-adrenergic receptor signaling in small arterioles. Blocked receptor internalization with elevated reactive oxygen levels coincides with a significant and unexpected vasodilation to β-adrenergic agonists at picomolar doses.