Cellular functions of arrestins are determined in part by the pattern of phosphorylation on the G protein-coupled receptors (GPCRs) to which arrestins bind. Despite high-resolution structural data of arrestins bound to phosphorylated receptor C-termini, the functional role of each phosphorylation site remains obscure. Here, we employ a library of synthetic phosphopeptide analogues of the GPCR rhodopsin C-terminus and determine the ability of these peptides to bind and activate arrestins using a variety of biochemical and biophysical methods. We further characterize how these peptides modulate the conformation of arrestin-1 by nuclear magnetic resonance (NMR). Our results indicate different functional classes of phosphorylation sites: ‘key sites’ required for arrestin binding and activation, an ‘inhibitory site’ that abrogates arrestin binding, and ‘modulator sites’ that influence the global conformation of arrestin. These functional motifs allow a better understanding of how different GPCR phosphorylation patterns might control how arrestin functions in the cell.

No abstract

The human M5muscarinic acetylcholine receptor (mAChR) has recently emerged as an exciting therapeutic target for treating a range of disorders, including drug addiction. However, a lack of structural information for this receptor subtype has limited further drug development and validation. Here we report a high-resolution crystal structure of the human M5mAChR bound to the clinically used inverse agonist, tiotropium. This structure allowed for a comparison across all 5 mAChR family members that revealed important differences in both orthosteric and allosteric sites that could inform the rational design of selective ligands. These structural studies, together with chimeric swaps between the extracellular regions of the M2and M5mAChRs, provided structural insight into kinetic selectivity, where ligands show differential residency times between related family members. Collectively, our study provides important insights into the nature of orthosteric and allosteric ligand interaction across the mAChR family that could be exploited for the design of selective drugs.

G protein-coupled receptors (GPCRs) are flexible integral membrane proteins involved in transmembrane signaling. Their involvement in many physiological processes makes them interesting targets for drug development. Determination of the structure of these receptors will help to design more specific drugs, however, their structural characterization has so far been hampered by the low expression and their inherent instability in detergents which made protein engineering indispensable for structural and biophysical characterization. Several approaches to stabilize the receptors in a particular conformation have led to breakthroughs in GPCR structure determination. These include truncations of the flexible regions, stabilization by antibodies and nanobodies, fusion partners, high affinity and covalently bound ligands as well as conformational stabilization by mutagenesis. In this review we focus on stabilization of GPCRs by insertion of point mutations, which lead to increased conformational and thermal stability as well as improved expression levels. We summarize existing mutagenesis strategies with different coverage of GPCR sequence space and depth of information, design and transferability of mutations and the molecular basis for stabilization. We also discuss whether mutations alter the structure and pharmacological properties of GPCRs.

Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular-level understanding of the general principles that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M4 muscarinic acetylcholine receptor (M4 mAChR) is a validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. In this study, we rigorously quantified the affinity, efficacy, and magnitude of modulation of two different positive allosteric modulators, LY2033298 (LY298) and VU0467154 (VU154), combined with the endogenous agonist acetylcholine (ACh) or the high-affinity agonist iperoxo (Ipx), at the human M4 mAChR. By determining the cryo-electron microscopy structures of the M4 mAChR, bound to a cognate Gi1 protein and in complex with ACh, Ipx, LY298-Ipx, and VU154-Ipx, and applying molecular dynamics simulations, we determine key molecular mechanisms underlying allosteric pharmacology. In addition to delineating the contribution of spatially distinct binding sites on observed pharmacology, our findings also revealed a vital role for orthosteric and allosteric ligand–receptor–transducer complex stability, mediated by conformational dynamics between these sites, in the ultimate determination of affinity, efficacy, cooperativity, probe dependence, and species variability. There results provide a holistic framework for further GPCR mechanistic studies and can aid in the discovery and design of future allosteric drugs.

32The human M5 muscarinic acetylcholine receptor (mAChR) has recently emerged as an 33 exciting therapeutic target for treating a range of disorders, including drug addiction. However, 34 a lack of structural information for this receptor subtype has limited further drug development 35 and validation. Here we report a high-resolution crystal structure of the human M5 mAChR 36 bound to the clinically used inverse agonist, tiotropium. This structure allowed for a 37 comparison across all five mAChR family members that revealed important differences in both 38 orthosteric and allosteric sites that could inform the rational design of selective ligands. These 39 structural studies together with chimeric swaps between the extracellular regions of the M2 and 40 M5 mAChR further revealed the structural insight into "kinetic-selectivity", where ligands 41show differential residency times between related family members. Collectively, our study 42 provides important insights into the nature of orthosteric and allosteric ligand interaction across 43 the mAChR family that could be exploited for the design of selective ligands. 44 45 Significance Statement 46The five subtypes of the muscarinic acetylcholine receptors (mAChRs) are expressed 47 throughout the central and peripheral nervous system where they play a vital role in physiology 48 and pathologies. Recently, the M5 mAChR subtype has emerged as an exciting drug target for 49 the treatment of drug addiction. We have determined the atomic structure of the M5 mAChR 50 bound to the clinically used inverse agonist tiotropium. The M5 mAChR structure now allows 51 for a full comparison of all five mAChR subtypes and reveals subtle differences in the 52 extracellular loop (ECL) regions of the receptor that mediate orthosteric and allosteric ligand 53 selectivity. Together these findings open the door for future structure-based design of selective

No abstract

The M 5 muscarinic acetylcholine receptor (mAChR) has emerged as an exciting therapeutic target for the treatment of addiction and behavioral disorders. This has been in part due to promising preclinical studies with the M 5 mAChR selective negative allosteric modulator (NAM), ML375. The binding site of ML375 remains unknown, however, making it difficult to develop improved M 5 mAChR selective modulators. To determine the possible location of the ML375 binding site, we used radioligand binding and functional assays to show that ML375 does not interact with the well-characterized "common" mAChR allosteric site located in the receptor's extracellular vestibule, nor a previously proposed second allosteric site recognized by the modulator, amiodarone. Molecular docking was used to predict potential allosteric sites within the transmembrane (TM) domain of the M 5 mAChR. These predicted sites were assessed using M 5 −M 2 mAChR receptor chimeras and further targeted with site-directed mutagenesis, which enabled the identification of a putative binding site for ML375 at the interface of TMs 2−4. Collectively, these results identify a third allosteric site at the M 5 mAChR and highlight the ability of allosteric modulators to selectively target highly conserved proteins.