Project description:Biased G protein-coupled receptor agonists engender a restricted repertoire of downstream events from their cognate receptors, permitting them to produce mixed agonist-antagonist effects in vivo. While this opens the possibility of novel therapeutics, it complicates rational drug design, since the in vivo response to a biased agonist cannot be reliably predicted from its in vitro efficacy. We have employed novel informatic approaches to characterize the in vivo transcriptomic signature of the arrestin pathway-selective parathyroid hormone analog [D-Trp12, Tyr34]-bPTH(7-34) in six different murine tissues after chronic drug exposure. We find that [D-Trp12, Tyr34]-bPTH(7-34) elicits a distinctive arrestin-signaling focused transcriptomic response that is more coherently regulated across tissues than that of the pluripotent agonist, hPTH(1-34). This arrestin-focused network is closely associated with transcriptional control of cell growth and development. Our demonstration of a conserved arrestin-dependent transcriptomic signature suggests a framework within which the in vivo outcomes of arrestin-biased signaling may be generalized.
Project description:Antimuscarinic drugs such as pirenzepine (PZ) and muscarinic toxin 7 (MT7), long considered M1 muscarinic acetylcholine receptor (M1R) antagonists, exhibit beta-arrestin-biased agonism. Using HEK293 cells and adult dorsal root ganglion (DRG) neurons, we show that PZ and MT7 selectively recruit beta-arrestin to M1R, activate ERK1/2 signaling, and promote neurite outgrowth without engaging G-alpha-q pathways or receptor internalization. Mass spectrometry identified six key M1R phosphorylation sites (T230, S251, T254, S321, T354, S356) required for beta-arrestin recruitment, with S251/T254 essential for PZ signaling. Casein kinase 2 (CK2), but not GRKs, mediates this phosphorylation; CK2 inhibition blocks beta-arrestin binding, ERK activation, and neurite outgrowth. These findings redefine antimuscarinic drugs as partial, beta-arrestin-biased M1R agonists and identify CK2 as a critical modulator of non-canonical M1R signaling relevant to sensory neuron regeneration and neuropathy therapy.
Project description:Biased agonists targeting the α2A-adrenergic receptor (α2AAR) have therapeutic potential by preferentially engaging G protein signaling over β-arrestin pathways. Here, we used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to compare α2AAR conformational dynamics across apo, agonist-bound, and GoA-coupled states. We analyzed HDX-MS changes induced by norepinephrine (NorEpi), the endogenous full agonist; dexmedetomidine (Dex), a clinically used full agonist that activates both Gi/o and β-arrestin signaling; and PS75, a Gi/o-biased partial agonist. By quantifying ligand-dependent differences in deuterium uptake and local dynamics, we aimed to define ligand-specific conformational ensembles. These findings provide mechanistic insight into partial agonism and suggest potential implications for biased signaling.
Project description:Biased GPCR agonists are orthosteric ligands that possess pathway-selective efficacy, activating or inhibiting only a subset of the signaling repertoire of their cognate receptors. In vitro, D-Trp12,Tyr34-bPTH(7-34) (PTH-{beta}arr), a biased agonist for the type 1 parathyroid hormone receptor, antagonizes receptor-G protein coupling but activates arrestin-dependent signaling. In vivo, both PTH-{beta}arr and the conventional agonist PTH(1-34) stimulate anabolic bone formation. To understand how two PTH1R ligands with markedly different in vitro efficacy could elicit similar in vivo responses, we analyzed transcriptional profiles from calvarial bone of mice treated for 8 weeks with vehicle, PTH-{beta}arr or PTH(1-34). Treatment of wild type mice with PTH-{beta}arr primarily affected pathways that promote expansion of the osteoblast pool, notably cell cycle regulation, cell survival and migration. These responses were absent in beta-arrestin2 null mice, identifying them as downstream targets of beta-arrestin2-mediated signaling. In contrast, PTH(1-34) primarily affected pathways classically associated with enhanced bone formation, including collagen synthesis and matrix mineralization. PTH(1-34) actions were less dependent on beta-arrestin2, as might be expected of a ligand capable of G protein activation. These results illustrate the uniqueness of biased agonism in vivo and demonstrate that functional selectivity can be exploited to change the quality of GPCR efficacy.
