Publications by authors named "Denise Wootten"

72 Publications

Evolving cryo-EM structural approaches for GPCR drug discovery.

Structure 2021 May 2. Epub 2021 May 2.

Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, VIC, Australia; ARC Centre for Cryo-Electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, VIC, Australia. Electronic address:

G protein-coupled receptors (GPCRs) are the largest class of cell surface drug targets. Advances in stabilization of GPCR:transducer complexes, together with improvements in cryoelectron microscopy (cryo-EM) have recently been applied to structure-assisted drug design for GPCR agonists. Nonetheless, limitations in the commercial application of these approaches, including the use of nanobody 35 (Nb35) to aid complex stabilization and the high cost of 300 kV imaging, have restricted broad application of cryo-EM in drug discovery. Here, using the PF 06882961-bound GLP-1R as exemplar, we validated the formation of stable complexes with a modified Gs protein in the absence of Nb35. In parallel, we compare 200 versus 300 kV image acquisition using a Falcon 4 or K3 direct electron detector. Moreover, the 200 kV Glacios-Falcon 4 yielded a 3.2 Å map with clear density for bound drug and multiple structurally ordered waters. Our work paves the way for broader commercial application of cryo-EM for GPCR drug discovery.
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http://dx.doi.org/10.1016/j.str.2021.04.008DOI Listing
May 2021

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J Pharmacol Exp Ther 2021 Mar 16. Epub 2021 Mar 16.

Department of Pharmacology, Monash University, Australia

Obesity and associated co-morbidities are a major health burden and novel therapeutics to help treat obesity are urgently needed. There is increasing evidence that targeting the amylin receptors (AMYRs), heterodimers of the calcitonin G protein-coupled receptor (CTR) and receptor activity-modifying proteins (RAMPs), improves weight control, and has the potential to act additively with other treatments such as glucagon-like peptide-1 receptor agonists. Recent data indicates that AMYR agonists, which can also independently activate the CTR, may have improved efficacy for treating obesity, even though selective activation of CTRs is not efficacious. AM833 (cagrilintide) is a novel, lipidated, amylin analogue that is undergoing clinical trials as a non-selective AMYR and CTR agonist. In the current study, we have investigated the pharmacology of AM833 across 25 end points and compared this peptide with AMYR selective and non-selective lipidated analogues; AM1213 and AM1784, and the clinically used peptide agonists; pramlintide (AMYR selective), sCT (non-selective). We also profiled hCT and rat amylin as prototypical selective agonists of CTR and AMYRs, respectively. Our results demonstrate that AM833 has a unique pharmacological profile, across diverse measures of receptor binding, activation and regulation. AM833 is a novel non-selective agonist of calcitonin-family receptors that has demonstrated efficacy for the treatment of obesity in phase 2 clinical trials. In this study we demonstrate that AM833 has a unique pharmacological profile across diverse measures of receptor binding, activation and regulation, when compared to other selective and non-selective calcitonin receptor and amylin receptor agonists. Our data provide mechanistic insight into the actions of AM833.
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http://dx.doi.org/10.1124/jpet.121.000567DOI Listing
March 2021

Structure and dynamics of the CGRP receptor in apo and peptide-bound forms.

Science 2021 04 18;372(6538). Epub 2021 Feb 18.

Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia.

G protein-coupled receptors (GPCRs) are key regulators of information transmission between cells and organs. Despite this, we have only a limited understanding of the behavior of GPCRs in the apo state and the conformational changes upon agonist binding that lead to G protein recruitment and activation. We expressed and purified unmodified apo and peptide-bound calcitonin gene-related peptide (CGRP) receptors from insect cells to determine their cryo-electron microscopy (cryo-EM) structures, and we complemented these with analysis of protein conformational dynamics using hydrogen-deuterium exchange mass spectrometry and three-dimensional variance analysis of the cryo-EM data. Together with our previously published structure of the active, Gs-bound CGRP receptor complex, our work provides insight into the mechanisms of class B1 GPCR activation.
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http://dx.doi.org/10.1126/science.abf7258DOI Listing
April 2021

Differential GLP-1R Binding and Activation by Peptide and Non-peptide Agonists.

Mol Cell 2020 11 6;80(3):485-500.e7. Epub 2020 Oct 6.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia. Electronic address:

Peptide drugs targeting class B1 G-protein-coupled receptors (GPCRs) can treat multiple diseases; however, there remains substantial interest in the development of orally delivered non-peptide drugs. Here, we reveal unexpected overlap between signaling and regulation of the glucagon-like peptide-1 (GLP-1) receptor by the non-peptide agonist PF 06882961 and GLP-1 that was not observed for another compound, CHU-128. Compounds from these patent series, including PF 06882961, are currently in clinical trials for treatment of type 2 diabetes. High-resolution cryoelectron microscopy (cryo-EM) structures reveal that the binding sites for PF 06882961 and GLP-1 substantially overlap, whereas CHU-128 adopts a unique binding mode with a more open receptor conformation at the extracellular face. Structural differences involving extensive water-mediated hydrogen bond networks could be correlated to functional data to understand how PF 06882961, but not CHU-128, can closely mimic the pharmacological properties of GLP-1. These findings will facilitate rational structure-based discovery of non-peptide agonists targeting class B GPCRs.
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http://dx.doi.org/10.1016/j.molcel.2020.09.020DOI Listing
November 2020

The metabolic effects of mirabegron are mediated primarily by β -adrenoceptors.

Pharmacol Res Perspect 2020 10;8(5):e00643

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic., Australia.

The β -adrenoceptor agonist mirabegron is approved for use for overactive bladder and has been purported to be useful in the treatment of obesity-related metabolic diseases in humans, including those involving disturbances of glucose homeostasis. We investigated the effect of mirabegron on glucose homeostasis with in vitro and in vivo models, focusing on its selectivity at β-adrenoceptors, ability to cause browning of white adipocytes, and the role of UCP1 in glucose homeostasis. In mouse brown, white, and brite adipocytes, mirabegron-mediated effects were examined on cyclic AMP, UCP1 mRNA, [ H]-2-deoxyglucose uptake, cellular glycolysis, and O consumption. Mirabegron increased cyclic AMP levels, UCP1 mRNA content, glucose uptake, and cellular glycolysis in brown adipocytes, and these effects were either absent or reduced in white adipocytes. In brite adipocytes, mirabegron increased cyclic AMP levels and UCP1 mRNA content resulting in increased UCP1-mediated oxygen consumption, glucose uptake, and cellular glycolysis. The metabolic effects of mirabegron in both brown and brite adipocytes were primarily due to actions at β -adrenoceptors as they were largely absent in adipocytes derived from β -adrenoceptor knockout mice. In vivo, mirabegron increased whole body oxygen consumption, glucose uptake into brown and inguinal white adipose tissue, and improved glucose tolerance, all effects that required the presence of the β -adrenoceptor. Furthermore, in UCP1 knockout mice, the effects of mirabegron on glucose tolerance were attenuated. Thus, mirabegron had effects on cellular metabolism in adipocytes that improved glucose handling in vivo, and were primarily due to actions at the β -adrenoceptor.
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http://dx.doi.org/10.1002/prp2.643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437350PMC
October 2020

Structure and dynamics of the active Gs-coupled human secretin receptor.

Nat Commun 2020 08 18;11(1):4137. Epub 2020 Aug 18.

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, AZ, 85259, USA.

The class B secretin GPCR (SecR) has broad physiological effects, with target potential for treatment of metabolic and cardiovascular disease. Molecular understanding of SecR binding and activation is important for its therapeutic exploitation. We combined cryo-electron microscopy, molecular dynamics, and biochemical cross-linking to determine a 2.3 Å structure, and interrogate dynamics, of secretin bound to the SecR:Gs complex. SecR exhibited a unique organization of its extracellular domain (ECD) relative to its 7-transmembrane (TM) core, forming more extended interactions than other family members. Numerous polar interactions formed between secretin and the receptor extracellular loops (ECLs) and TM helices. Cysteine-cross-linking, cryo-electron microscopy multivariate analysis and molecular dynamics simulations revealed that interactions between peptide and receptor were dynamic, and suggested a model for initial peptide engagement where early interactions between the far N-terminus of the peptide and SecR ECL2 likely occur following initial binding of the peptide C-terminus to the ECD.
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http://dx.doi.org/10.1038/s41467-020-17791-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7435274PMC
August 2020

Evaluation of biased agonism mediated by dual agonists of the GLP-1 and glucagon receptors.

Biochem Pharmacol 2020 10 17;180:114150. Epub 2020 Jul 17.

Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia. Electronic address:

Metabolic diseases such as obesity, diabetes, and their comorbidities have converged as one of the most serious health concerns on a global scale. Selective glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists are one of the major therapeutics for type 2 diabetes and obesity. Polypharmacological approaches that enable modulation of multiple metabolic targets in a single drug have emerged as a potential avenue to improve therapeutic outcomes. Among numerous peptides under development are those targeting the GLP-1R and either the glucagon receptor (GCGR), glucose-dependent insulinotropic peptide receptor (GIPR) or all 3 receptors, as dual- or tri- peptide agonists. Despite many of them entering into clinical trials, current development has been based on only a limited understanding of the spectrum of potential pharmacological properties of these ligands beyond binding selectivity. In the present study, we examined the potential for agonists that target both GLP-1R and GCGR to exhibit biased agonism, comparing activity across proximal activation of Gs protein, cAMP accumulation, pERK1/2 and β-arrestin recruitment. Three distinct dual agonists that have different relative cAMP production potency for GLP-1R versus GCGR, "peptide 15", MEDI0382 and SAR425899, and one triagonist of the GLP-1R, GCGR and GIPR were examined. We demonstrated that all novel peptides have distinct biased agonism profiles relative to either of the cognate agonists of the receptors, and to each other. This is an important feature of the pharmacology of this drug class that needs to be considered alongside selectivity, bioavailability and pharmacokinetics for rational optimization of new therapeutics.
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http://dx.doi.org/10.1016/j.bcp.2020.114150DOI Listing
October 2020

Cryo-electron microscopy structure of the glucagon receptor with a dual-agonist peptide.

J Biol Chem 2020 07 5;295(28):9313-9325. Epub 2020 May 5.

Monash Institute of Pharmaceutical Sciences, Drug Discovery Biology, Monash University, Parkville, Victoria, Australia

Unimolecular dual agonists of the glucagon (GCG) receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) are a new class of drugs that are potentially superior to GLP-1R-specific agonists for the management of metabolic disease. The dual-agonist, peptide 15 (P15), is a glutamic acid 16 analog of GCG with GLP-1 peptide substitutions between amino acids 17 and 24 that has potency equivalent to those of the cognate peptide agonists at the GCGR and GLP-1R. Here, we have used cryo-EM to solve the structure of an active P15-GCGR-G complex and compared this structure to our recently published structure of the GCGR-G complex bound to GCG. This comparison revealed that P15 has a reduced interaction with the first extracellular loop (ECL1) and the top of transmembrane segment 1 (TM1) such that there is increased mobility of the GCGR extracellular domain and at the C terminus of the peptide compared with the GCG-bound receptor. We also observed a distinct conformation of ECL3 and could infer increased mobility of the far N-terminal His-1 residue in the P15-bound structure. These regions of conformational variance in the two peptide-bound GCGR structures were also regions that were distinct between GCGR structures and previously published peptide-bound structures of the GLP-1R, suggesting that greater conformational dynamics may contribute to the increased efficacy of P15 in activation of the GLP-1R compared with GCG. The variable domains in this receptor have previously been implicated in biased agonism at the GLP-1R and could result in altered signaling of P15 at the GCGR compared with GCG.
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http://dx.doi.org/10.1074/jbc.RA120.013793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363120PMC
July 2020

Pharmacological characterization of mono-, dual- and tri-peptidic agonists at GIP and GLP-1 receptors.

Biochem Pharmacol 2020 07 29;177:114001. Epub 2020 Apr 29.

Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, 3052 Victoria, Australia. Electronic address:

Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone with physiological roles in adipose tissue, the central nervous system and bone metabolism. While selective ligands for GIP receptor (GIPR) have not been advanced for disease treatment, dual and triple agonists of GIPR, in conjunction with that of glucagon-like peptide-1 (GLP-1) and glucagon receptors, are currently in clinical trials, with an expectation of enhanced efficacy beyond that of GLP-1 receptor (GLP-1R) agonist monotherapy for diabetic patients. Consequently, it is important to understand the pharmacological behavior of such drugs. In this study, we have explored signaling pathway specificity and the potential for biased agonism of mono-, dual- and tri-agonists of GIPR using human embryonic kidney 293 (HEK293) cells recombinantly expressing human GIPR or GLP-1R. Compared to GIP(1-42), the GIPR mono-agonists Pro3GIP and Lys3GIP are biased towards ERK1/2 phosphorylation (pERK1/2) relative to cAMP accumulation at GIPR, whereas the triple agonist at GLP-1R/GCGR/GIPR is biased towards pERK1/2 relative to β-arrestin2 recruitment. Moreover, the dual GIPR/GLP-1R agonist, LY3298176, is biased towards pERK1/2 relative to cAMP accumulation at both GIPR and GLP-1R compared to their respective endogenous ligands. These data reveal novel pharmacological properties of potential therapeutic agents that may impact on diversity in clinical responses.
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http://dx.doi.org/10.1016/j.bcp.2020.114001DOI Listing
July 2020

Structure and Dynamics of Adrenomedullin Receptors AM and AM Reveal Key Mechanisms in the Control of Receptor Phenotype by Receptor Activity-Modifying Proteins.

ACS Pharmacol Transl Sci 2020 Apr 20;3(2):263-284. Epub 2020 Mar 20.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia.

Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) receptors are critically important for metabolism, vascular tone, and inflammatory response. AM receptors are also required for normal lymphatic and blood vascular development and angiogenesis. They play a pivotal role in embryo implantation and fertility and can provide protection against hypoxic and oxidative stress. CGRP and AM receptors are heterodimers of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) (CGRPR), as well as RAMP2 or RAMP3 (AMR and AMR, respectively). However, the mechanistic basis for RAMP modulation of CLR phenotype is unclear. In this study, we report the cryo-EM structure of the AMR in complex with AM and Gs at a global resolution of 3.0 Å, and structures of the AMR in complex with either AM or intermedin/adrenomedullin 2 (AM2) and Gs at 2.4 and 2.3 Å, respectively. The structures reveal distinctions in the primary orientation of the extracellular domains (ECDs) relative to the receptor core and distinct positioning of extracellular loop 3 (ECL3) that are receptor-dependent. Analysis of dynamic data present in the cryo-EM micrographs revealed additional distinctions in the extent of mobility of the ECDs. Chimeric exchange of the linker region of the RAMPs connecting the TM helix and the ECD supports a role for this segment in controlling receptor phenotype. Moreover, a subset of the motions of the ECD appeared coordinated with motions of the G protein relative to the receptor core, suggesting that receptor ECD dynamics could influence G protein interactions. This work provides fundamental advances in our understanding of GPCR function and how this can be allosterically modulated by accessory proteins.
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http://dx.doi.org/10.1021/acsptsci.9b00080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7155201PMC
April 2020

Molecular Mechanisms of Class B GPCR Activation: Insights from Adrenomedullin Receptors.

ACS Pharmacol Transl Sci 2020 Apr 26;3(2):246-262. Epub 2020 Feb 26.

School of Biological Sciences, University of Auckland, Auckland, 1010, New Zealand.

Adrenomedullin (AM) is a 52 amino acid peptide that plays a regulatory role in the vasculature. Receptors for AM comprise the class B G protein-coupled receptor, the calcitonin-like receptor (CLR), in complex with one of three receptor activity-modifying proteins (RAMPs). The C-terminus of AM is involved in binding to the extracellular domain of the receptor, while the N-terminus is proposed to interact with the juxtamembranous portion of the receptor to activate signaling. There is currently limited information on the molecular determinants involved in AM signaling, thus we set out to define the importance of the AM N-terminus through five signaling pathways (cAMP production, ERK phosphorylation, CREB phosphorylation, Akt phosphorylation, and IP production). We characterized the three CLR:RAMP complexes through the five pathways, finding that each had a distinct repertoire of intracellular signaling pathways that it is able to regulate. We then performed an alanine scan of AM from residues 15-31 and found that most residues could be substituted with only small effects on signaling, and that most substitutions affected signaling through all receptors and pathways in a similar manner. We identify F18, T20, L26, and I30 as being critical for AM function, while also identifying an analogue (AM G19A) which has unique signaling properties relative to the unmodified AM. We interpret our findings in the context of new structural information, highlighting the complementary nature of structural biology and functional assays.
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http://dx.doi.org/10.1021/acsptsci.9b00083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7155197PMC
April 2020

Deconvoluting the Molecular Control of Binding and Signaling at the Amylin 3 Receptor: RAMP3 Alters Signal Propagation through Extracellular Loops of the Calcitonin Receptor.

ACS Pharmacol Transl Sci 2019 Jun 18;2(3):183-197. Epub 2019 Mar 18.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia.

Amylin is coexpressed with insulin in pancreatic islet β-cells and has potent effects on gastric emptying and food intake. The effect of amylin on satiation has been postulated to involve AMY receptors (AMYR) that are heteromers of the calcitonin receptor (CTR) and receptor activity-modifying protein 3 (RAMP3). Understanding the molecular control of signaling through the AMYR is thus important for peptide drug targeting of this receptor. We have previously used alanine scanning mutagenesis to study the contribution of the extracellular surface of the CTR to binding and signaling initiated by calcitonin (CT) and related peptides (Dal Maso, E., . (2019) The molecular control of calcitonin receptor signaling. , 31-51). That work revealed ligand- and pathway-specific effects of mutation, with extracellular loops (ECLs) 2 and 3 particularly important in the distinct propagation of signaling mediated by individual peptides. In the current study, we have used equivalent alanine scanning of ECL2 and ECL3 of the CTR in the context of coexpression with RAMP3 to form AMYRs, to examine functional affinity and efficacy of peptides in cAMP accumulation and extracellular signal-regulated kinase (ERK) phosphorylation (pERK). The effect of mutation was determined on representatives of the three major distinct classes of CT peptide, salmon CT (sCT), human CT (hCT), and porcine CT (pCT), as well as rat amylin (rAmy) or human α-CGRP (calcitonin gene-related peptide, hCGRP) whose potency is enhanced by RAMP interaction. We demonstrate that the dynamic nature of CTR ECL2 and ECL3 in propagation of signaling is fundamentally altered when complexed with RAMP3 to form the AMYR, despite only having predicted direct interactions with ECL2. Moreover, the work shows that the role of these loops in receptor signaling is highly peptide dependent, illustrating that even subtle changes to peptide sequence may change signaling output downstream of the receptor.
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http://dx.doi.org/10.1021/acsptsci.9b00010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7088965PMC
June 2019

Rational development of a high-affinity secretin receptor antagonist.

Biochem Pharmacol 2020 07 23;177:113929. Epub 2020 Mar 23.

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, AZ 85259, United States. Electronic address:

The secretin receptor is a prototypic class B GPCR with substantial and broad pharmacologic importance. The aim of this project was to develop a high affinity selective antagonist as a new and important pharmacologic tool and to aid stabilization of this receptor in an inactive conformation for ultimate structural characterization. Amino-terminal truncation of the natural 27-residue ligand reduced biological activity, but also markedly reduced binding affinity. This was rationally and experimentally overcome with lactam stabilization of helical structure and with replacement of residues with natural and unnatural amino acids. A key new step in this effort was the replacement of peptide residue Leu with L-cyclohexylalanine (Cha) to enhance potential hydrophobic interactions with receptor residues Leu, Val, and Phe that were predicted from molecular modeling. Alanine-replacement mutagenesis of these residues markedly affected ligand binding and biological activity. The optimal antagonist ligand, (Y,c[E,K],I,Cha,R)sec(6-27), exhibited high binding affinity (4 nM), similar to natural secretin, and exhibited no demonstrable biological activity to stimulate cAMP accumulation, intracellular calcium mobilization, or β-arrestin-2 translocation. It acts as an orthosteric competitive antagonist, predicted to bind within the peptide-binding groove in the receptor extracellular domain. The analogous peptide that was one residue longer, retaining Thr, exhibited partial agonist activity, while further truncation of even a single residue (Phe) reduced binding affinity. This sec(6-27)-based peptide will be an important new tool for pharmacological and structural studies.
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http://dx.doi.org/10.1016/j.bcp.2020.113929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299832PMC
July 2020

Structural basis of G and G recognition by the human glucagon receptor.

Science 2020 03;367(6484):1346-1352

CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

Class B G protein-coupled receptors, an important class of therapeutic targets, signal mainly through the G class of heterotrimeric G proteins, although they do display some promiscuity in G protein binding. Using cryo-electron microscopy, we determined the structures of the human glucagon receptor (GCGR) bound to glucagon and distinct classes of heterotrimeric G proteins, G or G These two structures adopt a similar open binding cavity to accommodate G and G The G binding selectivity of GCGR is explained by a larger interaction interface, but there are specific interactions that affect G more than G binding. Conformational differences in the receptor intracellular loops were found to be key selectivity determinants. These distinctions in transducer engagement were supported by mutagenesis and functional studies.
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http://dx.doi.org/10.1126/science.aaz5346DOI Listing
March 2020

Molecular Basis for Hormone Recognition and Activation of Corticotropin-Releasing Factor Receptors.

Mol Cell 2020 02 30;77(3):669-680.e4. Epub 2020 Jan 30.

The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address:

Corticotropin-releasing factor (CRF) and the three related peptides urocortins 1-3 (UCN1-UCN3) are endocrine hormones that control the stress responses by activating CRF1R and CRF2R, two members of class B G-protein-coupled receptors (GPCRs). Here, we present two cryoelectron microscopy (cryo-EM) structures of UCN1-bound CRF1R and CRF2R with the stimulatory G protein. In both structures, UCN1 adopts a single straight helix with its N terminus dipped into the receptor transmembrane bundle. Although the peptide-binding residues in CRF1R and CRF2R are different from other members of class B GPCRs, the residues involved in receptor activation and G protein coupling are conserved. In addition, both structures reveal bound cholesterol molecules to the receptor transmembrane helices. Our structures define the basis of ligand-binding specificity in the CRF receptor-hormone system, establish a common mechanism of class B GPCR activation and G protein coupling, and provide a paradigm for studying membrane protein-lipid interactions for class B GPCRs.
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http://dx.doi.org/10.1016/j.molcel.2020.01.013DOI Listing
February 2020

Toward a Structural Understanding of Class B GPCR Peptide Binding and Activation.

Mol Cell 2020 02 30;77(3):656-668.e5. Epub 2020 Jan 30.

Drug Discovery Biology and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, VIC, Australia; School of Pharmacy, Fudan University, Shanghai 201203, China. Electronic address:

Class B G protein-coupled receptors (GPCRs) are important therapeutic targets for major diseases. Here, we present structures of peptide and Gs-bound pituitary adenylate cyclase-activating peptide, PAC1 receptor, and corticotropin-releasing factor (CRF), (CRF1) receptor. Together with recently solved structures, these provide coverage of the major class B GPCR subfamilies. Diverse orientations of the extracellular domain to the receptor core in different receptors are at least partially dependent on evolutionary conservation in the structure and nature of peptide interactions. Differences in peptide interactions to the receptor core also influence the interlinked TM2-TM1-TM6/ECL3/TM7 domain, and this is likely important in their diverse signaling. However, common conformational reorganization of ECL2, linked to reorganization of ICL2, modulates G protein contacts. Comparison between receptors reveals ICL2 as a key domain forming dynamic G protein interactions in a receptor- and ligand-specific manner. This work advances our understanding of class B GPCR activation and Gs coupling.
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http://dx.doi.org/10.1016/j.molcel.2020.01.012DOI Listing
February 2020

Activation of the GLP-1 receptor by a non-peptidic agonist.

Nature 2020 01 8;577(7790):432-436. Epub 2020 Jan 8.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, including diabetes and obesity. Structures of active receptors reveal peptide agonists engage deep within the receptor core, leading to an outward movement of extracellular loop 3 and the tops of transmembrane helices 6 and 7, an inward movement of transmembrane helix 1, reorganization of extracellular loop 2 and outward movement of the intracellular side of transmembrane helix 6, resulting in G-protein interaction and activation. Here we solved the structure of a non-peptide agonist, TT-OAD2, bound to the glucagon-like peptide-1 (GLP-1) receptor. Our structure identified an unpredicted non-peptide agonist-binding pocket in which reorganization of extracellular loop 3 and transmembrane helices 6 and 7 manifests independently of direct ligand interaction within the deep transmembrane domain pocket. TT-OAD2 exhibits biased agonism, and kinetics of G-protein activation and signalling that are distinct from peptide agonists. Within the structure, TT-OAD2 protrudes beyond the receptor core to interact with the lipid or detergent, providing an explanation for the distinct activation kinetics that may contribute to the clinical efficacy of this compound series. This work alters our understanding of the events that drive the activation of class B receptors.
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http://dx.doi.org/10.1038/s41586-019-1902-zDOI Listing
January 2020

Use of Backbone Modification To Enlarge the Spatiotemporal Diversity of Parathyroid Hormone Receptor-1 Signaling via Biased Agonism.

J Am Chem Soc 2019 09 9;141(37):14486-14490. Epub 2019 Sep 9.

Department of Chemistry , University of Wisconsin-Madison , Madison , Wisconsin 53706 , United States.

The type-1 parathyroid hormone receptor (PTHR1), which regulates calcium homeostasis and tissue development, has two native agonists, parathyroid hormone (PTH) and PTH-related protein (PTHrP). PTH forms a complex with the PTHR1 that is rapidly internalized and induces prolonged cAMP production from endosomes. In contrast, PTHrP induces only transient cAMP production, which primarily arises from receptors on the cell surface. We show that backbone modification of PTH(1-34)-NH and abaloparatide (a PTHrP derivative) with a single homologous β-amino acid residue can generate biased agonists that induce prolonged cAMP production from receptors at the cell surface. This unique spatiotemporal profile could be useful for distinguishing effects associated with the duration of cAMP production from effects associated with the site of cAMP production.
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http://dx.doi.org/10.1021/jacs.9b04179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6930011PMC
September 2019

Structural Basis for Allosteric Modulation of Class B G Protein-Coupled Receptors.

Annu Rev Pharmacol Toxicol 2020 01 27;60:89-107. Epub 2019 Aug 27.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, and Department of Pharmacology, Monash University, Parkville 3052, Australia; email:

Recent advances in our understanding of the structure and function of class B G protein-coupled receptors (GPCRs) provide multiple opportunities for targeted development of allosteric modulators. Given the pleiotropic signaling patterns emanating from these receptors in response to a variety of natural agonist ligands, modulators have the potential to sculpt the responses to meet distinct needs of different groups of patients. In this review, we provide insights into how this family of GPCRs differs from the rest of the superfamily, how orthosteric agonists bind and activate these receptors, the potential for allosteric modulators to interact with various regions of these targets, and the allosteric influence of endogenous proteins on the pharmacology of these receptors, all of which are important considerations when developing new therapies.
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http://dx.doi.org/10.1146/annurev-pharmtox-010919-023301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7092389PMC
January 2020

O-GlcNAc Engineering of GPCR Peptide-Agonists Improves Their Stability and in Vivo Activity.

J Am Chem Soc 2019 09 28;141(36):14210-14219. Epub 2019 Aug 28.

Bridge Institute , University of Southern California , Los Angeles , California 90089 , United States.

Peptide agonists of GPCRs and other receptors are powerful signaling molecules with high potential as biological tools and therapeutics, but they are typically plagued by instability and short half-lives in vivo. Nature uses protein glycosylation to increase the serum stability of secreted proteins. However, these extracellular modifications are complex and heterogeneous in structure, making them an impractical solution. In contrast, intracellular proteins are subjected to a simple version of glycosylation termed O-GlcNAc modification. In our studies of this modification, we found that O-GlcNAcylation inhibits proteolysis, and strikingly, this stabilization occurs despite large distances in primary sequence (10-15 amino acids) between the O-GlcNAc and the site of cleavage. We therefore hypothesized that this "remote stabilization" concept could be useful to engineer the stability and potentially additional properties of peptide or protein therapeutics. Here, we describe the application of O-GlcNAcylation to two clinically important peptides: glucagon-like peptide-1 (GLP-1) and the parathyroid hormone (PTH), which respectively help control glucose and calcium levels in the blood. For both peptides, we found O-GlcNAcylated analogs that are equipotent to unmodified peptide in cell-based activation assays, while several GLP-1 analogs were biased agonists relative to GLP-1. As we predicted, O-GlcNAcylation can improve the stability of both GLP-1 and PTH in serum despite the fact that the O-GlcNAc can be quite remote from characterized sites of peptide cleavage. The O-GlcNAcylated GLP-1 and PTH also displayed significantly improved in vivo activity. Finally, we employed structure-based molecular modeling and receptor mutagenesis to predict how O-GlcNAcylation can be accommodated by the receptors and the potential interactions that contribute to peptide activity. This approach demonstrates the potential of O-GlcNAcylation for generating analogs of therapeutic peptides with enhanced proteolytic stability.
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http://dx.doi.org/10.1021/jacs.9b05365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6860926PMC
September 2019

Expression and activity of the calcitonin receptor family in a sample of primary human high-grade gliomas.

BMC Cancer 2019 Feb 18;19(1):157. Epub 2019 Feb 18.

Drug Discovery Biology and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, 3052, Australia.

Background: Glioblastoma (GBM) is the most common and aggressive type of primary brain cancer. With median survival of less than 15 months, identification and validation of new GBM therapeutic targets is of critical importance.

Results: In this study we tested expression and performed pharmacological characterization of the calcitonin receptor (CTR) as well as other members of the calcitonin family of receptors in high-grade glioma (HGG) cell lines derived from individual patient tumours, cultured in defined conditions. Previous immunohistochemical data demonstrated CTR expression in GBM biopsies and we were able to confirm CALCR (gene encoding CTR) expression. However, as assessed by cAMP accumulation assay, only one of the studied cell lines expressed functional CTR, while the other cell lines have functional CGRP (CLR/RAMP1) receptors. The only CTR-expressing cell line (SB2b) showed modest coupling to the cAMP pathway and no activation of other known CTR signaling pathways, including ERK and p38 MAP kinases, and Ca mobilization, supportive of low cell surface receptor expression. Exome sequencing data failed to account for the discrepancy between functional data and expression on the cell lines that do not respond to calcitonin(s) with no deleterious non-synonymous polymorphisms detected, suggesting that other factors may be at play, such as alternative splicing or rapid constitutive receptor internalisation.

Conclusions: This study shows that GPCR signaling can display significant variation depending on cellular system used, and effects seen in model recombinant cell lines or tumour cell lines are not always reproduced in a more physiologically relevant system and vice versa.
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http://dx.doi.org/10.1186/s12885-019-5369-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379965PMC
February 2019

The Molecular Control of Calcitonin Receptor Signaling.

ACS Pharmacol Transl Sci 2019 Feb 11;2(1):31-51. Epub 2019 Jan 11.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria Australia.

The calcitonin receptor (CTR) is a class B G protein-coupled receptor (GPCR) that responds to the peptide hormone calcitonin (CT). CTs are clinically approved for the treatment of bone diseases. We previously reported a 4.1 Å structure of the activated CTR bound to salmon CT (sCT) and heterotrimeric Gs protein by cryo-electron microscopy (Liang, Y.-L., . Phase-plate cryo- EM structure of a class B GPCR-G protein complex. , , 118-123). In the current study, we have reprocessed the electron micrographs to yield a 3.3 Å map of the complex. This has allowed us to model extracellular loops (ECLs) 2 and 3, and the peptide N-terminus that previously could not be resolved. We have also performed alanine scanning mutagenesis of ECL1 and the upper segment of transmembrane helix 1 (TM1) and its extension into the receptor extracellular domain (TM1 stalk), with effects on peptide binding and function assessed by cAMP accumulation and ERK1/2 phosphorylation. These data were combined with previously published alanine scanning mutagenesis of ECL2 and ECL3 and the new structural information to provide a comprehensive 3D map of the molecular surface of the CTR that controls binding and signaling of distinct CT and related peptides. The work highlights distinctions in how different, related, class B receptors may be activated. The new mutational data on the TM1 stalk and ECL1 have also provided critical insights into the divergent control of cAMP versus pERK signaling and, collectively with previous mutagenesis data, offer evidence that the conformations linked to these different signaling pathways are, in many ways, mutually exclusive. This study furthers our understanding of the complex nature of signaling elicited by GPCRs and, in particular, that of the therapeutically important class B subfamily.
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http://dx.doi.org/10.1021/acsptsci.8b00056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7088896PMC
February 2019

Molecular Basis of Action of a Small-Molecule Positive Allosteric Modulator Agonist at the Type 1 Cholecystokinin Holoreceptor.

Mol Pharmacol 2019 03 27;95(3):245-259. Epub 2018 Dec 27.

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona (A.J.D., L.J.M.); In Silico Drug Discovery Department, Icagen Tucson Innovation Center, Oro Valley, Arizona (I.M., K.N., A.N.); Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Australia (C.V., D.W., P.M.S., A.C., L.J.M.); Molsoft, La Jolla, California (P.C.H.L., A.O., R.A.); Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California (R.A.); and School of Pharmacy, Fudan University, Shanghai, China (D.W., P.M.S.)

Allosteric modulation of receptors provides mechanistic safety while effectively achieving biologic endpoints otherwise difficult or impossible to obtain by other means. The theoretical case has been made for the development of a positive allosteric modulator (PAM) of the type 1 cholecystokinin receptor (CCK1R) having minimal intrinsic agonist activity to enhance meal-induced satiety for the treatment of obesity, while reducing the risk of side effects and/or toxicity. Unfortunately, such a drug does not currently exist. In this work, we have identified a PAM agonist of the CCK1R, SR146131, and determined its putative binding mode and receptor activation mechanism by combining molecular modeling, chimeric CCK1R/CCK2R constructs, and site-directed mutagenesis. We probed the structure-activity relationship of analogs of SR146131 for impact on agonism versus cooperativity of the analogs. This identified structural features that might be responsible for binding affinity and potency while retaining PAM activity. SR146131 and several of its analogs were docked into the receptor structure, which had the natural endogenous peptide agonist, cholecystokinin, already in the bound state (by docking), providing a refined structural model of the intact CCK1R holoreceptor. Both SR146131 and its analogs exhibited unique probe-dependent cooperativity with orthosteric peptide agonists and were simultaneously accommodated in this model, consistent with the derived structure-activity relationships. This provides improved understanding of the molecular basis for CCK1R-directed drug development.
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http://dx.doi.org/10.1124/mol.118.114082DOI Listing
March 2019

Cryo-EM structure of the active, G-protein complexed, human CGRP receptor.

Nature 2018 09 12;561(7724):492-497. Epub 2018 Sep 12.

Drug Discovery Biology and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide that has a major role in sensory neurotransmission. The CGRP receptor is a heterodimer of the calcitonin receptor-like receptor (CLR) class B G-protein-coupled receptor and a type 1 transmembrane domain protein, receptor activity-modifying protein 1 (RAMP1). Here we report the structure of the human CGRP receptor in complex with CGRP and the G-protein heterotrimer at 3.3 Å global resolution, determined by Volta phase-plate cryo-electron microscopy. The receptor activity-modifying protein transmembrane domain sits at the interface between transmembrane domains 3, 4 and 5 of CLR, and stabilizes CLR extracellular loop 2. RAMP1 makes only limited direct contact with CGRP, consistent with its function in allosteric modulation of CLR. Molecular dynamics simulations indicate that RAMP1 provides stability to the receptor complex, particularly in the positioning of the extracellular domain of CLR. This work provides insights into the control of G-protein-coupled receptor function.
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http://dx.doi.org/10.1038/s41586-018-0535-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166790PMC
September 2018

Glucagon-like peptide-1 receptor internalisation controls spatiotemporal signalling mediated by biased agonists.

Biochem Pharmacol 2018 10 7;156:406-419. Epub 2018 Sep 7.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, Parkville, Melbourne, Victoria 3052, Australia; School of Pharmacy, Fudan University, Shanghai 201203, China. Electronic address:

The glucagon-like peptide-1 receptor (GLP-1R) is a major therapeutic target in the treatment of type 2 diabetes due to its roles in regulating blood glucose and in promoting weight loss. Like many GPCRs, it is pleiotropically coupled, can be activated by multiple ligands and is subject to biased agonism. The GLP-1R undergoes agonist mediated receptor internalisation that may be associated with spatiotemporal control of signalling and biased agonism, although to date, this has not been extensively explored. Here, we investigate GLP-1R trafficking and its importance with regard to signalling, including the localisation of key signalling molecules, mediated by biased peptide agonists that are either endogenous GLP-1R ligands or are used clinically. Each of the agonists promoted receptor internalisation through a dynamin and caveolae dependent mechanism and traffic the receptor to both degradative and recycling pathways. This internalisation is important for signalling, with cAMP and ERK1/2 phoshorylation (pERK1/2) generated by both plasma membrane localised and internalised receptors. Further assessment of pERK1/2 revealed that all peptides induced nuclear ERK activity, but ligands, liraglutide and oxyntomodulin that are biased towards pERK1/2 relative to cAMP (when compared to GLP-1 and exendin-4), also stimulated pERK1/2 activity in the cytosol. This compartmentalisation of ERK1/2 signalling was reliant on receptor internalisation, with restriction of receptor localisation to the plasma membrane limiting ERK1/2 signalling to the cytosol. Thus, this study implicates a role of receptor internalisation in spatiotemporal control of ERK1/2 signalling that may contribute to GLP-1R biased agonism.
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http://dx.doi.org/10.1016/j.bcp.2018.09.003DOI Listing
October 2018

Rules of Engagement: GPCRs and G Proteins.

ACS Pharmacol Transl Sci 2018 Nov 7;1(2):73-83. Epub 2018 Sep 7.

Drug Discovery Biology and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.

G protein-coupled receptors (GPCRs) are a key drug target class. They account for over one-third of current pharmaceuticals, and both drugs that inhibit and promote receptor function are important therapeutically; in some cases, the same GPCR can be targeted with agonists and inhibitors, depending upon disease context. There have been major breakthroughs in understanding GPCR structure and drug binding through advances in X-ray crystallography, and membrane protein stabilization. Nonetheless, these structures have predominately been of inactive receptors bound to inhibitors. Efforts to capture structures of fully active GPCRs, in particular those in complex with the canonical, physiological transducer G protein, have been limited via this approach. Very recently, advances in cryo-electron microscopy have provided access to agonist:GPCR:G protein complex structures. These promise to revolutionize our understanding of GPCR:G protein engagement and provide insight into mechanisms of efficacy and coupling selectivity and how these might be controlled by biased agonists. Here we review what we have currently learned from the new GPCR:Gs and GPCR:Gi/o complex structures.
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http://dx.doi.org/10.1021/acsptsci.8b00026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7089011PMC
November 2018

Mechanisms of signalling and biased agonism in G protein-coupled receptors.

Nat Rev Mol Cell Biol 2018 10;19(10):638-653

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

G protein-coupled receptors (GPCRs) are the largest group of cell surface receptors in humans that signal in response to diverse inputs and regulate a plethora of cellular processes. Hence, they constitute one of the primary drug target classes. Progress in our understanding of GPCR dynamics, activation and signalling has opened new possibilities for selective drug development. A key advancement has been provided by the concept of biased agonism, which describes the ability of ligands acting at the same GPCR to elicit distinct cellular signalling profiles by preferentially stabilizing different active conformational states of the receptor. Application of this concept raises the prospect of 'designer' biased agonists as optimized therapeutics with improved efficacy and/or reduced side-effect profiles. However, this application will require a detailed understanding of the spectrum of drug actions and a structural understanding of the drug-receptor interactions that drive distinct pharmacologies. The recent revolution in GPCR structural biology provides unprecedented insights into ligand binding, conformational dynamics and the control of signalling outcomes. These insights, together with new approaches to multi-dimensional analysis of drug action, are allowing refined classification of drugs according to their pharmacodynamic profiles, which can be linked to receptor structure and predictions of preclinical drug efficacy.
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http://dx.doi.org/10.1038/s41580-018-0049-3DOI Listing
October 2018

Dominant Negative G Proteins Enhance Formation and Purification of Agonist-GPCR-G Protein Complexes for Structure Determination.

ACS Pharmacol Transl Sci 2018 Sep 26;1(1):12-20. Epub 2018 Jul 26.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, 3052, Australia.

Advances in structural biology have yielded exponential growth in G protein-coupled receptor (GPCR) structure solution. Nonetheless, the instability of fully active GPCR complexes with cognate heterotrimeric G proteins has made them elusive. Existing structures have been limited to nanobody-stabilized GPCR:Gs complexes. Here we present methods for enhanced GPCR:G protein complex stabilization via engineering G proteins with reduced nucleotide affinity, limiting Gα:Gβγ dissociation. We illustrate the application of dominant negative G proteins of Gαs and Gαi2 to the purification of stable complexes where this was not possible with wild-type G protein. Active state complexes of adenosine:A1 receptor:Gαi2βγ and calcitonin gene-related peptide (CGRP):CLR:RAMP1:Gαsβγ:Nb35 were purified to homogeneity and were stable in negative stain electron microscopy. These were suitable for structure determination by cryo-electron microscopy at 3.6 and 3.3 Å resolution, respectively. The dominant negative Gα-proteins are thus high value tools for structure determination of agonist:GPCR:G protein complexes that are critical for informed translational drug discovery.
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http://dx.doi.org/10.1021/acsptsci.8b00017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7089020PMC
September 2018

Structure of the adenosine-bound human adenosine A receptor-G complex.

Nature 2018 06 20;558(7711):559-563. Epub 2018 Jun 20.

Drug Discovery Biology and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

The class A adenosine A receptor (AR) is a G-protein-coupled receptor that preferentially couples to inhibitory G heterotrimeric G proteins, has been implicated in numerous diseases, yet remains poorly targeted. Here we report the 3.6 Å structure of the human AR in complex with adenosine and heterotrimeric G protein determined by Volta phase plate cryo-electron microscopy. Compared to inactive AR, there is contraction at the extracellular surface in the orthosteric binding site mediated via movement of transmembrane domains 1 and 2. At the intracellular surface, the G protein engages the AR primarily via amino acids in the C terminus of the Gα α5-helix, concomitant with a 10.5 Å outward movement of the AR transmembrane domain 6. Comparison with the agonist-bound β adrenergic receptor-G-protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active AR structure provides molecular insights into receptor and G-protein selectivity.
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http://dx.doi.org/10.1038/s41586-018-0236-6DOI Listing
June 2018

Two distinct domains of the glucagon-like peptide-1 receptor control peptide-mediated biased agonism.

J Biol Chem 2018 06 1;293(24):9370-9387. Epub 2018 May 1.

the Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia,

G protein-coupled receptors (GPCRs) can be differentially activated by ligands to generate multiple and distinct downstream signaling profiles, a phenomenon termed biased agonism. The glucagon-like peptide-1 receptor (GLP-1R) is a class B GPCR and a key drug target for managing metabolic disorders; however, its peptide agonists display biased signaling that affects their relative efficacies. In this study, we combined mutagenesis experiments and mapping of surface mutations onto recently described GLP-1R structures, which revealed two major domains in the GLP-1/GLP-1R/G protein active structure that are differentially important for both receptor quiescence and ligand-specific initiation and propagation of biased agonism. Changes to the conformation of transmembrane helix (TM) 5 and TM 6 and reordering of extracellular loop 2 were essential for the propagation of signaling linked to cAMP formation and intracellular calcium mobilization, whereas ordering and packing of residues in TMs 1 and 7 were critical for extracellular signal-regulated kinase 1/2 (pERK) activity. On the basis of these findings, we propose a model of distinct peptide-receptor interactions that selectively control how these different signaling pathways are engaged. This work provides important structural insight into class B GPCR activation and biased agonism.
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http://dx.doi.org/10.1074/jbc.RA118.003278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6005446PMC
June 2018