scholarly journals Cryo-EM structure of the human PAC1 receptor coupled to an engineered heterotrimeric G protein

2019 ◽  
Author(s):  
Kazuhiro Kobayashi ◽  
Wataru Shihoya ◽  
Tomohiro Nishizawa ◽  
Francois Marie Ngako Kadji ◽  
Junken Aoki ◽  
...  
Keyword(s):  
G Protein ◽  
Structural Information ◽  
Receptor Activation ◽  
Peptide Ligand ◽  
Pac1 Receptor ◽  
Peripheral Tissues ◽  
Class B ◽  
Neuropeptide Hormone ◽  
The Central Nervous System ◽  
Glucagon Like Peptide 1

AbstractPituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide hormone functioning in the central nervous system and peripheral tissues. The PACAP receptor PAC1R, which belongs to the class B G-protein-coupled receptors (GPCRs), is a drug target for mental disorders and dry eye syndrome. Here we present a cryo-electron microscopy structure of human PAC1R bound to PACAP and an engineered Gs heterotrimer. The structure revealed that TM1 plays an essential role in PACAP recognition. The ECD (extracellular domain) of PAC1R tilts by ~40° as compared to that of the glucagon-like peptide-1 receptor (GLP1R), and thus does not cover the peptide ligand. A functional analysis demonstrated that the PAC1R-ECD functions as an affinity trap and is not required for receptor activation, whereas the GLP1R-ECD plays an indispensable role in receptor activation, illuminating the functional diversity of the ECDs in the class B GPCRs. Our structural information will facilitate the design and improvement of better PAC1R agonists for clinical applications.This article is a preprint version and has not been certified by peer review.

scholarly journals Molecular Basis of Class B GPCR Selectivity for the Neuropeptides PACAP and VIP

2021 ◽  
Vol 8 ◽  
Author(s):  
Chenyi Liao ◽  
Jacob M. Remington ◽  
Victor May ◽  
Jianing Li
Keyword(s):  
Rational Design ◽  
Structural Information ◽  
Receptor Activation ◽  
Binding Selectivity ◽  
Pac1 Receptor ◽  
Ligand Selectivity ◽  
Receptor Interactions ◽  
Class B ◽  
State Models ◽  
Class B Gpcr

The related neuropeptides PACAP and VIP, and their shared PAC1, VPAC1 and VPAC2 receptors, regulate a large array of physiological activities in the central and peripheral nervous systems. However, the lack of comparative and molecular mechanistic investigations hinder further understanding of their preferred binding selectivity and function. PACAP and VIP have comparable affinity at the VPAC1 and VPAC2 receptor, but PACAP is 400–1,000 fold more potent than VIP at the PAC1 receptor. A molecular understanding of the differing neuropeptide-receptor interactions and the details underlying the receptor transitions leading to receptor activation are much needed for the rational design of selective ligands. To these ends, we have combined structural information and advanced simulation techniques to study PACAP/VIP binding selectivity, full-length receptor conformation ensembles and transitions of the PACAP/VIP receptor variants and subtypes, and a few key interactions in the orthosteric-binding pocket. Our results reveal differential peptide-receptor interactions (at the atomistic detail) important for PAC1, VPAC1 and VPAC2 receptor ligand selectivity. Using microsecond-long molecular dynamics simulations and the Markov State Models, we have also identified diverse receptor conformational ensembles and microstate transition paths for each receptor, the potential mechanisms underlying receptor open and closed states, and the interactions and dynamics at the transmembrane orthosteric pocket for receptor activation. These analyses reveal important features in class B GPCR structure-dynamics-function relationships, which provide novel insights for structure-based drug discovery.

scholarly journals Structural basis for GLP-1 receptor activation by LY3502970, an orally active nonpeptide agonist

2020 ◽  
Vol 117 (47) ◽  
pp. 29959-29967 ◽  
Author(s):  
Takahiro Kawai ◽  
Bingfa Sun ◽  
Hitoshi Yoshino ◽  
Dan Feng ◽  
Yoshiyuki Suzuki ◽  
...  
Keyword(s):  
Oral Administration ◽  
G Protein ◽  
Partial Agonist ◽  
Pharmacokinetic Profile ◽  
Binding Pocket ◽  
Receptor Activation ◽  
Structural Basis ◽  
Protein Activation ◽  
Class B ◽  
Glucagon Like Peptide 1

Glucagon-like peptide-1 receptor (GLP-1R) agonists are efficacious antidiabetic medications that work by enhancing glucose-dependent insulin secretion and improving energy balance. Currently approved GLP-1R agonists are peptide based, and it has proven difficult to obtain small-molecule activators possessing optimal pharmaceutical properties. We report the discovery and mechanism of action of LY3502970 (OWL833), a nonpeptide GLP-1R agonist. LY3502970 is a partial agonist, biased toward G protein activation over β-arrestin recruitment at the GLP-1R. The molecule is highly potent and selective against other class B G protein–coupled receptors (GPCRs) with a pharmacokinetic profile favorable for oral administration. A high-resolution structure of LY3502970 in complex with active-state GLP-1R revealed a unique binding pocket in the upper helical bundle where the compound is bound by the extracellular domain (ECD), extracellular loop 2, and transmembrane helices 1, 2, 3, and 7. This mechanism creates a distinct receptor conformation that may explain the partial agonism and biased signaling of the compound. Further, interaction between LY3502970 and the primate-specific Trp33 of the ECD informs species selective activity for the molecule. In efficacy studies, oral administration of LY3502970 resulted in glucose lowering in humanized GLP-1R transgenic mice and insulinotropic and hypophagic effects in nonhuman primates, demonstrating an effect size in both models comparable to injectable exenatide. Together, this work determined the molecular basis for the activity of an oral agent being developed for the treatment of type 2 diabetes mellitus, offering insights into the activation of class B GPCRs by nonpeptide ligands.

Mechanochemical Activation of Class-B G-Protein-Coupled Receptor upon Peptide–Ligand Binding

Nano Letters ◽  
2020 ◽  
Vol 20 (7) ◽  
pp. 5575-5582 ◽  
Author(s):  
Cristina Lo Giudice ◽  
Haonan Zhang ◽  
Beili Wu ◽  
David Alsteens
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Mechanochemical Activation ◽  
Peptide Ligand ◽  
G Protein Coupled Receptor ◽  
Class B ◽  
G Protein Coupled

scholarly journals Structural insights into ligand efficacy and activation of the glucagon receptor

2019 ◽  
Author(s):  
Daniel Hilger ◽  
Kaavya Krishna Kumar ◽  
Hongli Hu ◽  
Mie Fabricius Pedersen ◽  
Lise Giehm ◽  
...  
Keyword(s):  
G Protein ◽  
Partial Agonist ◽  
Transmembrane Helix ◽  
Peptide Hormone ◽  
Receptor Activation ◽  
Glucagon Receptor ◽  
Structural Framework ◽  
Class B ◽  
Treatment Of Diabetes ◽  
Sugar Levels

AbstractThe glucagon receptor family comprises Class B G protein-coupled receptors (GPCRs) that play a crucial role in regulating blood sugar levels. Receptors of this family represent important therapeutic targets for the treatment of diabetes and obesity. Despite intensive structural studies, we only have a poor understanding of the mechanism of peptide hormone-induced Class B receptor activation. This process involves the formation of a sharp kink in transmembrane helix 6 that moves out to allow formation of the nucleotide-free G protein complex. Here, we present the cryo-EM structure of the glucagon receptor (GCGR), a prototypical Class B GPCR, in complex with an engineered soluble glucagon derivative and the heterotrimeric G-protein, Gs. Comparison with the previously determined crystal structures of GCGR bound to a partial agonist reveals a structural framework to explain the molecular basis of ligand efficacy that is further supported by mutagenesis data.

scholarly journals Mechanisms of adhesion G protein–coupled receptor activation

2020 ◽  
Vol 295 (41) ◽  
pp. 14065-14083 ◽  
Author(s):  
Alexander Vizurraga ◽  
Rashmi Adhikari ◽  
Jennifer Yeung ◽  
Maiya Yu ◽  
Gregory G. Tall
Keyword(s):  
G Protein ◽  
Receptor Activation ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Physiological Processes ◽  
Class B ◽  
Peptide Agonist ◽  
G Protein Coupled ◽  
Existing Structure

Adhesion G protein–coupled receptors (AGPCRs) are a thirty-three-member subfamily of Class B GPCRs that control a wide array of physiological processes and are implicated in disease. AGPCRs uniquely contain large, self-proteolyzing extracellular regions that range from hundreds to thousands of residues in length. AGPCR autoproteolysis occurs within the extracellular GPCR autoproteolysis-inducing (GAIN) domain that is proximal to the N terminus of the G protein–coupling seven-transmembrane–spanning bundle. GAIN domain–mediated self-cleavage is constitutive and produces two-fragment holoreceptors that remain bound at the cell surface. It has been of recent interest to understand how AGPCRs are activated in relation to their two-fragment topologies. Dissociation of the AGPCR fragments stimulates G protein signaling through the action of the tethered-peptide agonist stalk that is occluded within the GAIN domain in the holoreceptor form. AGPCRs can also signal independently of fragment dissociation, and a few receptors possess GAIN domains incapable of self-proteolysis. This has resulted in complex theories as to how these receptors are activated in vivo, complicating pharmacological advances. Currently, there is no existing structure of an activated AGPCR to support any of the theories. Further confounding AGPCR research is that many of the receptors remain orphans and lack identified activating ligands. In this review, we provide a detailed layout of the current theorized modes of AGPCR activation with discussion of potential parallels to mechanisms used by other GPCR classes. We provide a classification means for the ligands that have been identified and discuss how these ligands may activate AGPCRs in physiological contexts.

scholarly journals Design and Synthesis of Brain Penetrant Glycopeptide Analogues of PACAP With Neuroprotective Potential for Traumatic Brain Injury and Parkinsonism

2022 ◽  
Vol 1 ◽  
Author(s):  
Christopher R. Apostol ◽  
Kelsey Bernard ◽  
Parthasaradhireddy Tanguturi ◽  
Gabriella Molnar ◽  
Mitchell J. Bartlett ◽  
...  
Keyword(s):  
Traumatic Brain Injury ◽  
Brain Injury ◽  
Animal Models ◽  
Pharmacokinetic Profile ◽  
Neuroprotective Effects ◽  
Drug Candidates ◽  
Design And Synthesis ◽  
Class B ◽  
Neuropeptide Hormone ◽  
The Central Nervous System

There is an unmet clinical need for curative therapies to treat neurodegenerative disorders. Most mainstay treatments currently on the market only alleviate specific symptoms and do not reverse disease progression. The Pituitary adenylate cyclase-activating polypeptide (PACAP), an endogenous neuropeptide hormone, has been extensively studied as a potential regenerative therapeutic. PACAP is widely distributed in the central nervous system (CNS) and exerts its neuroprotective and neurotrophic effects via the related Class B GPCRs PAC1, VPAC1, and VPAC2, at which the hormone shows roughly equal activity. Vasoactive intestinal peptide (VIP) also activates these receptors, and this close analogue of PACAP has also shown to promote neuronal survival in various animal models of acute and progressive neurodegenerative diseases. However, PACAP’s poor pharmacokinetic profile (non-linear PK/PD), and more importantly its limited blood-brain barrier (BBB) permeability has hampered development of this peptide as a therapeutic. We have demonstrated that glycosylation of PACAP and related peptides promotes penetration of the BBB and improves PK properties while retaining efficacy and potency in the low nanomolar range at its target receptors. Furthermore, judicious structure-activity relationship (SAR) studies revealed key motifs that can be modulated to afford compounds with diverse selectivity profiles. Most importantly, we have demonstrated that select PACAP glycopeptide analogues (2LS80Mel and 2LS98Lac) exert potent neuroprotective effects and anti-inflammatory activity in animal models of traumatic brain injury and in a mild-toxin lesion model of Parkinson’s disease, highlighting glycosylation as a viable strategy for converting endogenous peptides into robust and efficacious drug candidates.

scholarly journals Expression of mini-G proteins specifically halt cognate GPCR trafficking and intracellular signalling

2021 ◽  
Author(s):  
Yusman Manchanda ◽  
Zenouska Ramchunder ◽  
Maria M Shchepinova ◽  
Guy A Rutter ◽  
Asuka Inoue ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Pancreatic Beta Cell ◽  
Ease Of Use ◽  
Intracellular Signalling ◽  
Receptor Coupling ◽  
Cell Class ◽  
Class B ◽  
Cell Assays ◽  
Glucagon Like Peptide 1

Mini-G proteins are engineered thermostable variants of Gα subunits designed to specifically stabilise G protein-coupled receptors (GPCRs) in their active conformation for structural analyses. Due to their smaller size and ease of use, they have become popular tools in recent years to assess specific GPCR behaviours in cells, both as reporters of receptor coupling to each G protein subtype and for in-cell assays designed to quantify compartmentalised receptor signalling from a range of subcellular locations. Here, we describe a previously unappreciated consequence of the co-expression of mini-G proteins with their cognate GPCRs, namely a profound disruption in GPCR trafficking and intracellular signalling caused by the co-expression of the specific mini-G subtype coupled to the affected receptor. We studied the Gαs-coupled pancreatic beta cell class B GPCR glucagon-like peptide-1 receptor (GLP-1R) as a model to describe in detail the molecular consequences derived from this effect, including a complete halt in β-arrestin-2 recruitment and receptor internalisation, despite near-normal levels of receptor GRK2 recruitment and lipid nanodomain segregation, as well as the disruption of endosomal GLP-1R signalling by mini-Gs co-expression. We also extend our analysis to a range of other prototypical GPCRs covering the spectrum of Gα subtype coupling preferences, to unveil a widely conserved phenomenon of GPCR internalisation blockage by specific mini-G proteins coupled to a particular receptor. Our results have important implications for the design of methods to assess intracellular GPCR signalling. We also present an alternative adapted bystander intracellular signalling assay for the GLP-1R in which we substitute the mini-Gs by a nanobody, Nb37, with specificity for active Gαs:GPCR complexes and no deleterious effect on the capacity for GLP-1R internalisation.

scholarly journals Targeting the PAC1 Receptor for Neurological and Metabolic Disorders

2019 ◽  
Vol 19 (16) ◽  
pp. 1399-1417 ◽  
Author(s):  
Chenyi Liao ◽  
Mathilde P. de Molliens ◽  
Severin T. Schneebeli ◽  
Matthias Brewer ◽  
Gaojie Song ◽  
...  
Keyword(s):  
Rational Design ◽  
Metabolic Diseases ◽  
Structural Information ◽  
Tissue Injury ◽  
Current Knowledge ◽  
Biological Activities ◽  
Receptor Activation ◽  
Pac1 Receptor ◽  
Activation Mechanisms ◽  
And Function

The pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor (PAC1R, ADCYAP1R1) is a member of the vasoactive intestinal peptide (VIP)/secretin/glucagon family of G protein-coupled receptors (GPCRs). PAC1R has been shown to play crucial roles in the central and peripheral nervous systems. The activation of PAC1R initiates diverse downstream signal transduction pathways, including adenylyl cyclase, phospholipase C, MEK/ERK, and Akt pathways that regulate a number of physiological systems to maintain functional homeostasis. Accordingly, at times of tissue injury or insult, PACAP/PAC1R activation of these pathways can be trophic to blunt or delay apoptotic events and enhance cell survival. Enhancing PAC1R signaling under these conditions has the potential to mitigate cellular damages associated with cerebrovascular trauma (including stroke), neurodegeneration (such as Parkinson’s and Alzheimer's disease), or peripheral organ insults. Conversely, maladaptive PACAP/PAC1R signaling has been implicated in a number of disorders, including stressrelated psychopathologies (i.e., depression, posttraumatic stress disorder, and related abnormalities), chronic pain and migraine, and metabolic diseases; abrogating PAC1R signaling under these pathological conditions represent opportunities for therapeutic intervention. Given the diverse PAC1R-mediated biological activities, the receptor has emerged as a relevant pharmaceutical target. In this review, we first describe the current knowledge regarding the molecular structure, dynamics, and function of PAC1R. Then, we discuss the roles of PACAP and PAC1R in the activation of a variety of signaling cascades related to the physiology and diseases of the nervous system. Lastly, we examine current drug design and development of peptides and small molecules targeting PAC1R based on a number of structure- activity relationship studies and key pharmacophore elements. At present, the rational design of PAC1R-selective peptide or small-molecule therapeutics is largely hindered by the lack of structural information regarding PAC1R activation mechanisms, the PACAP-PAC1R interface, and the core segments involved in receptor activation. Understanding the molecular basis governing the PACAP interactions with its different cognate receptors will undoubtedly provide a basis for the development and/or refinement of receptor-selective therapeutics.

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