Activation biosensor for G protein-coupled receptors: a FRET-based m1 muscarinic activation sensor that regulates G(q).
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ABSTRACT:
SUBMITTER: Chang S
PROVIDER: S-EPMC3447775 | biostudies-literature | 2012
REPOSITORIES: biostudies-literature
Publications
Activation biosensor for G protein-coupled receptors: a FRET-based m1 muscarinic activation sensor that regulates G(q).
Chang Seungwoo S Ross Elliott M EM
PloS one 20120920 9
We describe the design, construction and validation of a fluorescence sensor to measure activation by agonist of the m1 muscarinic cholinergic receptor, a prototypical class I G(q)-coupled receptor. The sensor uses an established general design in which Förster resonance energy transfer (FRET) from a circularly permuted CFP mutant to FlAsH, a selectively reactive fluorescein, is decreased 15-20% upon binding of a full agonist. Notably, the sensor displays essentially wild-type capacity to cataly ...[more]
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Project description:Background and purposeAlthough clinically useful for their immunomodulatory, antiproliferative and antiviral properties, type I interferons (IFNs) are involved in the pathogenesis of several neurodegenerative/neuroinflammatory diseases. In the present study, we investigated the ability of cholinergic stimulation to protect from IFN-?-induced neuronal apoptosis.Experimental approachThe effects of the ACh receptor agonist carbachol (CCh) on IFN-?-induced apoptosis of human SH-SY5Y neuroblastoma cells were examined by using western blots, immunofluorescence and cytofluorimetry. The involvement of muscarinic acetylcholine receptors (mAChRs) was assessed by using selective antagonists and siRNA transfection. Pharmacological inhibitors and overexpression of ERK2 and an ERK2 constitutively active form (ERK2-CA) were employed to study ERK1/2 signalling. The effects of oxotremorine-M (Oxo-M) on IFN-?-induced apoptosis of mouse hippocampal neurons were examined by measuring cleaved caspase 3 expression.Key resultsIn SH-SY5Y cells, CCh inhibited IFN-?-induced mitochondrial cytochrome c release, activation of caspases 9, 7 and 3, PARP cleavage and DNA fragmentation. The anti-apoptotic effect of CCh was mediated by M3 receptors, blocked by Gq/11 antagonist YM254890 and PKC inhibitor Go 6983, impaired by inhibition of ERK1/2 pathway, potentiated by overexpression of ERK2 and mimicked by ERK2-CA. Blockade of JNK activation enhanced the CCh anti-apoptotic response. IFN-? inhibited JNK activation and up-regulated CCh-induced ERK1/2 signalling. In hippocampal neurons, Oxo-M reduced IFN-?-induced apoptosis; this effect was antagonized by blockade of M1 /M3 receptors and ERK1/2.Conclusions and implicationsStimulation of mAChRs counteracted IFN-?-induced neuronal apoptosis through the activation of ERK1/2 signalling. The data indicate that activation of ERK1/2-coupled mAChRs may be an effective strategy for preventing IFNs neurotoxicity.
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