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Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized, some GPCRs remain complexed with β-arrestins, while others interact only transiently; this difference affects GPCR signaling and recycling. Cell-based and in vitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in β-arrestin recruitment and GPCR-β-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one that requires PIP binding for β-arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of β-arrestin and stabilize GPCR-β-arrestin complexes by promoting a fully engaged state of the complex. As allosteric modulators of GPCR-β-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. For GPCRs that require membrane PIP binding for β-arrestin recruitment, this provides a mechanism for β-arrestin release upon translocation of the GPCR to endosomes, allowing for its rapid recycling. [Display omitted] • β-Arrestins coincidentally detect membrane PIPs for recruitment to GPCRs • GPCR phosphorylation affects dependence on membrane PIPs for β-arrestin recruitment • Membrane PIPs are allosteric modulators of GPCR-β-arrestin complexes • Plasma membrane PIPs alone promote β-arrestin activation By acting as allosteric regulators of arrestin conformation, membrane phosphoinositides potentiate an active conformation of arrestin and stabilize GPCR-arrestin complexes. Membrane PIPs can therefore drive GPCR conformational diversity beyond what is seen from GPCR phosphorylation alone and provide spatial control for GPCR-arrestin complex formation. [ABSTRACT FROM AUTHOR]

The M2 muscarinic acetylcholine receptor (M2R) is a prototypical GPCR that plays important roles in regulating heart rate and CNS functions. Crystal structures provide snapshots of the M2R in inactive and active states, but the allosteric link between the ligand binding pocket and cytoplasmic surface remains poorly understood. Here we used solution NMR to examine the structure and dynamics of the M2R labeled with 13CH 3 -ε-methionine upon binding to various orthosteric and allosteric ligands having a range of efficacy for both G protein activation and arrestin recruitment. We observed ligand-specific changes in the NMR spectra of 13CH 3 -ε-methionine probes in the M2R extracellular domain, transmembrane core, and cytoplasmic surface, allowing us to correlate ligand structure with changes in receptor structure and dynamics. We show that the M2R has a complex energy landscape in which ligands with different efficacy profiles stabilize distinct receptor conformations. • Analysis of conformational dynamics throughout the M2R using 13CH 3 -ε-methionine NMR • Ligands with various efficacies for G protein and arrestin signaling were examined • Each ligand stabilized a distinct conformation of the M2R • There is no clear linear correlation between ligand efficacy and chemical shifts Xu et al. reveal that the M2 muscarinic receptor is highly dynamic, with a complex energy landscape in which ligands with different signaling profiles stabilize distinct receptor conformations. [ABSTRACT FROM AUTHOR]

Summary Cannabis elicits its mood-enhancing and analgesic effects through the cannabinoid receptor 1 (CB1), a G protein-coupled receptor (GPCR) that signals primarily through the adenylyl cyclase-inhibiting heterotrimeric G protein G i. Activation of CB1-G i signaling pathways holds potential for treating a number of neurological disorders and is thus crucial to understand the mechanism of G i activation by CB1. Here, we present the structure of the CB1-G i signaling complex bound to the highly potent agonist MDMB-Fubinaca (FUB), a recently emerged illicit synthetic cannabinoid infused in street drugs that have been associated with numerous overdoses and fatalities. The structure illustrates how FUB stabilizes the receptor in an active state to facilitate nucleotide exchange in G i. The results compose the structural framework to explain CB1 activation by different classes of ligands and provide insights into the G protein coupling and selectivity mechanisms adopted by the receptor. Graphical Abstract Highlights • 3 Å cryo-EM structure of the CB1-G i complex bound to potent agonist MDMB-Fubinaca • MDMB-Fubinaca locks "toggle switch" residues F2003.36/W3566.48 in active conformation • Quantum mechanics calculations reveal the mechanism for the high affinity of Fubinaca • Molecular dynamic simulations reveal a path for ligand entry between TM1 and TM7 Looking at how a toxic, synthetic ligand locks cannabinoid receptor 1 into a signaling conformation points to ways to understand and modulate receptor activity. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) encodes a G protein-coupled receptor (GPCR) termed BILF1 that is essential for EBV-mediated immunosuppression and oncogenesis. BILF1 couples with inhibitory G protein (Gi), the major intracellular signaling effector for human chemokine receptors, and exhibits constitutive signaling activity; the ligand(s) for BILF1 are unknown. We studied the origins of BILF1's constitutive activity through structure determination of BILF1 bound to the inhibitory G protein (Gi) heterotrimer. The 3.2-Å resolution cryo-electron microscopy structure revealed an extracellular loop within BILF1 that blocked the typical chemokine binding site, suggesting ligand-autonomous receptor activation. Rather, amino acid substitutions within BILF1 transmembrane regions at hallmark ligand-activated class A GPCR "microswitches" stabilized a constitutively active BILF1 conformation for Gi coupling in a ligand-independent fashion. Thus, the constitutive activity of BILF1 promotes immunosuppression and virulence independent of ligand availability, with implications for the function of GPCRs encoded by related viruses and for therapeutic targeting of EBV., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)