Structures of full-length glycoprotein hormone receptor signaling complexes

Luteinizing hormone (LH) and chorionic gonadotropin (CG) are members of the glycoprotein hormone family essential to human reproduction and are important therapeutic drugs. They activate the same G protein-coupled receptor, LHCGR, by binding to the large extracellular domain (ECD). Here we report four cryo-EM structures of LHCGR, two wildtype receptor structures in the inactive and active states, and two constitutively active mutated receptor structures. The active structures are bound to CG and Gs heterotrimer, with one of the structure also containing the allosteric agonist, Org43553. The structures reveal a distinct ′push and pull′ mechanism of receptor activation, in which the ECD is pushed by the bound hormone and pulled by the extended hinge loop next to the transmembrane domain (TMD). A highly conserved 10-residue fragment (P10) from the hinge C-terminal loop at the ECD-TMD interface functions as a tethered agonist to induce conformational changes in TMD and G-protein coupling. Org43553 binds to a TMD pocket and interacts directly with P10 that further stabilizes the receptor in the active conformation. Together, these structures provide a common model for understanding glycoprotein hormone signal transduction and dysfunction, and inspire the search for clinically suitable small molecular compounds to treat endocrine diseases.

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Autoantibody and hormone activation of the thyrotropin G protein-coupled receptor

10.1101/2022.01.06.475289 ◽

2022 ◽

Author(s):

Bryan Faust ◽

Isha Singh ◽

Kaihua Zhang ◽

Nicholas Hoppe ◽

Antonio F.M. Pinto ◽

...

Keyword(s):

Thyroid Hormones ◽

G Protein ◽

Conformational Changes ◽

Hormone Receptors ◽

Transmembrane Domain ◽

Extracellular Domain ◽

Receptor Activation ◽

Glycoprotein Hormone ◽

Membrane Bilayer ◽

G Protein Coupled

Thyroid hormones are vital to growth and metabolism. Thyroid hormone synthesis is controlled by thyrotropin (TSH), which acts at the thyrotropin receptor (TSHR). Autoantibodies that activate the TSHR pathologically increase thyroid hormones in Graves' disease. How autoantibodies mimic TSH function remains unclear. We determined cryogenic-electron microscopy structures of active and inactive TSHR. In inactive TSHR, the extracellular domain lies close to the membrane bilayer. TSH selects an upright conformation of the extracellular domain due to steric clashes between a conserved hormone glycan and the membrane bilayer. An activating autoantibody selects a similar upright conformation of the extracellular domain. Conformational changes in the extracellular domain are transduced to the seven transmembrane domain via a conserved hinge domain, a tethered peptide agonist, and a phospholipid that binds within the seven transmembrane domain. Rotation of the TSHR ECD relative to the membrane bilayer is sufficient for receptor activation, revealing a shared mechanism for other glycoprotein hormone receptors that may also extend to G protein-coupled receptors with large extracellular domains.

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The Nematode Leucine-Rich Repeat-Containing, G Protein-Coupled Receptor (LGR) Protein Homologous to Vertebrate Gonadotropin and Thyrotropin Receptors is Constitutively Activated in Mammalian Cells

Molecular Endocrinology ◽

10.1210/mend.14.2.0422 ◽

2000 ◽

Vol 14 (2) ◽

pp. 272-284 ◽

Cited By ~ 35

Author(s):

Masataka Kudo ◽

Thomas Chen ◽

Koji Nakabayashi ◽

Sheau Yu Hsu ◽

Aaron J. W. Hsueh

Keyword(s):

G Protein ◽

Hormone Receptors ◽

Receptor Activation ◽

Leucine Rich Repeat ◽

Camp Production ◽

Glycoprotein Hormone ◽

Lh Receptor ◽

Glycoprotein Hormone Receptors ◽

Leucine Rich Repeats ◽

G Protein Coupled

Abstract The receptors for LH, FSH, and TSH belong to the large G protein-coupled, seven-transmembrane (TM) protein family and are unique in having a large N-terminal extracellular (ecto-) domain containing leucine-rich repeats important for interactions with the large glycoprotein hormone ligands. Recent studies indicated the evolution of an expanding family of homologous leucine-rich repeat-containing, G protein-coupled receptors (LGRs), including the three known glycoprotein hormone receptors; mammalian LGR4 and LGR5; and LGRs in sea anemone, fly, and snail. We isolated nematode LGR cDNA and characterized its gene from the Caenorhabditis elegans genome. This receptor cDNA encodes 929 amino acids consisting of a signal peptide for membrane insertion, an ectodomain with nine leucine-rich repeats, a seven-TM region, and a long C-terminal tail. The nematode LGR has five potential N-linked glycosylation sites in its ectodomain and multiple consensus phosphorylation sites for protein kinase A and C in the cytoplasmic loop and C tail. The nematode receptor gene has 13 exons; its TM region and C tail, unlike mammalian glycoprotein hormone receptors, are encoded by multiple exons. Sequence alignments showed that the TM region of the nematode receptor has 30% identity and 50% similarity to the same region in mammalian glycoprotein hormone receptors. Although human 293T cells expressing the nematode LGR protein do not respond to human glycoprotein hormones, these cells exhibited major increases in basal cAMP production in the absence of ligand stimulation, reaching levels comparable to those in cells expressing a constitutively activated mutant human LH receptor found in patients with familial male-limited precocious puberty. Analysis of cAMP production mediated by chimeric receptors further indicated that the ectodomain and TM region of the nematode LGR and human LH receptor are interchangeable and the TM region of the nematode LGR is responsible for constitutive receptor activation. Thus, the identification and characterization of the nematode receptor provides the basis for understanding the evolutionary relationship of diverse LGRs and for future analysis of mechanisms underlying the activation of glycoprotein hormone receptors and related LGRs.

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Dissecting the allosteric networks governing agonist efficacy and potency in G protein-coupled receptors

10.1101/2021.09.14.460253 ◽

2021 ◽

Author(s):

Franziska Marie Heydenreich ◽

Maria Marti-Solano ◽

Manbir Sandhu ◽

Brian K Kobilka ◽

Michel Bouvier ◽

...

Keyword(s):

G Protein ◽

Conformational Changes ◽

Structural Changes ◽

Receptor Activation ◽

Residue Level ◽

G Protein Coupled Receptors ◽

Maximal Response ◽

Pharmacological Properties ◽

Receptor Ligand ◽

G Protein Coupled

G protein-coupled receptors (GPCRs) translate binding of extracellular ligands into intracellular responses through conformational changes. Ligand properties are described by the maximum response (efficacy) and the agonist concentration at half-maximal response (potency). Integrating structural changes with pharmacological properties remains challenging and has not yet been performed at the resolution of individual amino acids. We use epinephrine and β2-adrenergic receptor as a model to integrate residue-level pharmacology data with intramolecular residue contact data describing receptor activation. This unveils the allosteric networks driving ligand efficacy and potency. We provide detailed insights into how structural rearrangements are linked to fundamental pharmacological properties at single-residue level in a receptor-ligand system. Our approach can be used to determine such pharmacological networks for any receptor-ligand complex.

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The Role of ICL1 and H8 in Class B1 GPCRs; Implications for Receptor Activlation

Frontiers in Endocrinology ◽

10.3389/fendo.2021.792912 ◽

2022 ◽

Vol 12 ◽

Author(s):

Ian Winfield ◽

Kerry Barkan ◽

Sarah Routledge ◽

Nathan J. Robertson ◽

Matthew Harris ◽

...

Keyword(s):

Molecular Dynamics ◽

G Protein ◽

Conformational Changes ◽

Transmembrane Helix ◽

Receptor Activation ◽

Cgrp Receptor ◽

Intracellular Loop ◽

Glucagon Receptor ◽

G Protein Coupled

The first intracellular loop (ICL1) of G protein-coupled receptors (GPCRs) has received little attention, although there is evidence that, with the 8th helix (H8), it is involved in early conformational changes following receptor activation as well as contacting the G protein β subunit. In class B1 GPCRs, the distal part of ICL1 contains a conserved R12.48KLRCxR2.46b motif that extends into the base of the second transmembrane helix; this is weakly conserved as a [R/H]12.48KL[R/H] motif in class A GPCRs. In the current study, the role of ICL1 and H8 in signaling through cAMP, iCa2+ and ERK1/2 has been examined in two class B1 GPCRs, using mutagenesis and molecular dynamics. Mutations throughout ICL1 can either enhance or disrupt cAMP production by CGRP at the CGRP receptor. Alanine mutagenesis identified subtle differences with regard elevation of iCa2+, with the distal end of the loop being particularly sensitive. ERK1/2 activation displayed little sensitivity to ICL1 mutation. A broadly similar pattern was observed with the glucagon receptor, although there were differences in significance of individual residues. Extending the study revealed that at the CRF1 receptor, an insertion in ICL1 switched signaling bias between iCa2+ and cAMP. Molecular dynamics suggested that changes in ICL1 altered the conformation of ICL2 and the H8/TM7 junction (ICL4). For H8, alanine mutagenesis showed the importance of E3908.49b for all three signal transduction pathways, for the CGRP receptor, but mutations of other residues largely just altered ERK1/2 activation. Thus, ICL1 may modulate GPCR bias via interactions with ICL2, ICL4 and the Gβ subunit.

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A Microdomain Formed by the Extracellular Ends of the Transmembrane Domains Promotes Activation of the G Protein-Coupled α-Factor Receptor

Molecular and Cellular Biology ◽

10.1128/mcb.24.5.2041-2051.2004 ◽

2004 ◽

Vol 24 (5) ◽

pp. 2041-2051 ◽

Cited By ~ 18

Author(s):

Jennifer C. Lin ◽

Ken Duell ◽

James B. Konopka

Keyword(s):

Ligand Binding ◽

G Protein ◽

Transmembrane Domain ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Transmembrane Domains ◽

Consecutive Series ◽

Subsequent Step ◽

G Protein Coupled ◽

Specific Reagent

ABSTRACT The α-factor receptor (Ste2p) that promotes mating in Saccharomyces cerevisiae is similar to other G protein-coupled receptors (GPCRs) in that it contains seven transmembrane domains. Previous studies suggested that the extracellular ends of the transmembrane domains are important for Ste2p function, so a systematic scanning mutagenesis was carried out in which 46 residues near the ends of transmembrane domains 1, 2, 3, 4, and 7 were replaced with cysteine. These mutants complement mutations constructed previously near the ends of transmembrane domains 5 and 6 to analyze all the extracellular ends. Eight new mutants created in this study were partially defective in signaling (V45C, N46C, T50C, A52C, L102C, N105C, L277C, and A281C). Treatment with 2-([biotinoyl] amino) ethyl methanethiosulfonate, a thiol-specific reagent that reacts with accessible cysteine residues but not membrane-embedded cysteines, identified a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. An unusual prolonged zone of intermediate reactivity near the extracellular end of transmembrane domain 2 suggests that this region may adopt a special structure. Interestingly, residues implicated in ligand binding were mainly accessible, whereas residues involved in the subsequent step of promoting receptor activation were mainly inaccessible. These results define a receptor microdomain that provides an important framework for interpreting the mechanisms by which functionally important residues contribute to ligand binding and activation of Ste2p and other GPCRs.

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Conformational changes involved in G-protein-coupled-receptor activation

Trends in Pharmacological Sciences ◽

10.1016/j.tips.2008.08.006 ◽

2008 ◽

Vol 29 (12) ◽

pp. 616-625 ◽

Cited By ~ 52

Author(s):

Jürgen Wess ◽

Sung-Jun Han ◽

Soo-Kyung Kim ◽

Kenneth A. Jacobson ◽

Jian Hua Li

Keyword(s):

G Protein ◽

Conformational Changes ◽

Receptor Activation ◽

G Protein Coupled Receptor ◽

G Protein Coupled

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CB1 Cannabinoid Receptor Signaling and Biased Signaling

Molecules ◽

10.3390/molecules26175413 ◽

2021 ◽

Vol 26 (17) ◽

pp. 5413

Author(s):

Luciana M. Leo ◽

Mary E. Abood

Keyword(s):

Adverse Effects ◽

G Protein ◽

Conformational Changes ◽

Cannabinoid Receptor ◽

Therapeutic Potential ◽

Receptor Activation ◽

Cb1 Receptor ◽

Cb1 Cannabinoid Receptor ◽

Biased Signaling ◽

G Protein Coupled

The CB1 cannabinoid receptor is a G-protein coupled receptor highly expressed throughout the central nervous system that is a promising target for the treatment of various disorders, including anxiety, pain, and neurodegeneration. Despite the wide therapeutic potential of CB1, the development of drug candidates is hindered by adverse effects, rapid tolerance development, and abuse potential. Ligands that produce biased signaling—the preferential activation of a signaling transducer in detriment of another—have been proposed as a strategy to dissociate therapeutic and adverse effects for a variety of G-protein coupled receptors. However, biased signaling at the CB1 receptor is poorly understood due to a lack of strongly biased agonists. Here, we review studies that have investigated the biased signaling profile of classical cannabinoid agonists and allosteric ligands, searching for a potential therapeutic advantage of CB1 biased signaling in different pathological states. Agonist and antagonist bound structures of CB1 and proposed mechanisms of action of biased allosteric modulators are used to discuss a putative molecular mechanism for CB1 receptor activation and biased signaling. Current studies suggest that allosteric binding sites on CB1 can be explored to yield biased ligands that favor or hinder conformational changes important for biased signaling.

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The Molecular Basis of G Protein–Coupled Receptor Activation

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-060614-033910 ◽

2018 ◽

Vol 87 (1) ◽

pp. 897-919 ◽

Cited By ~ 207

Author(s):

William I. Weis ◽

Brian K. Kobilka

Keyword(s):

Ligand Binding ◽

G Protein ◽

Conformational Changes ◽

Cellular Responses ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Active Conformation ◽

Allosteric Coupling ◽

External Stimuli ◽

G Protein Coupled

G protein–coupled receptors (GPCRs) mediate the majority of cellular responses to external stimuli. Upon activation by a ligand, the receptor binds to a partner heterotrimeric G protein and promotes exchange of GTP for GDP, leading to dissociation of the G protein into α and βγ subunits that mediate downstream signals. GPCRs can also activate distinct signaling pathways through arrestins. Active states of GPCRs form by small rearrangements of the ligand-binding, or orthosteric, site that are amplified into larger conformational changes. Molecular understanding of the allosteric coupling between ligand binding and G protein or arrestin interaction is emerging from structures of several GPCRs crystallized in inactive and active states, spectroscopic data, and computer simulations. The coupling is loose, rather than concerted, and agonist binding does not fully stabilize the receptor in an active conformation. Distinct intermediates whose populations are shifted by ligands of different efficacies underlie the complex pharmacology of GPCRs.

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Emerging roles of adhesion G protein-coupled receptors

Biochemical Society Transactions ◽

10.1042/bst20201144 ◽

2021 ◽

Author(s):

Matthew Rosa ◽

Timothy Noel ◽

Matthew Harris ◽

Graham Ladds

Keyword(s):

G Protein ◽

Conformational Changes ◽

Current Knowledge ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Extracellular Region ◽

Extracellular Matrix Components ◽

Ligand Interactions ◽

Tethered Ligand ◽

G Protein Coupled

Adhesion G protein-coupled receptors (aGPCRs) form a sub-group within the GPCR superfamily. Their distinctive structure contains an abnormally large N-terminal, extracellular region with a GPCR autoproteolysis-inducing (GAIN) domain. In most aGPCRs, the GAIN domain constitutively cleaves the receptor into two fragments. This process is often required for aGPCR signalling. Over the last two decades, much research has focussed on aGPCR-ligand interactions, in an attempt to deorphanize the family. Most ligands have been found to bind to regions N-terminal to the GAIN domain. These receptors may bind a variety of ligands, ranging across membrane-bound proteins and extracellular matrix components. Recent advancements have revealed a conserved method of aGPCR activation involving a tethered ligand within the GAIN domain. Evidence for this comes from increased activity in receptor mutants exposing the tethered ligand. As a result, G protein-coupling partners of aGPCRs have been more extensively characterised, making use of their tethered ligand to create constitutively active mutants. This has led to demonstrations of aGPCR function in, for example, neurodevelopment and tumour growth. However, questions remain around the ligands that may bind many aGPCRs, how this binding is translated into changes in the GAIN domain, and the exact mechanism of aGPCR activation following GAIN domain conformational changes. This review aims to examine the current knowledge around aGPCR activation, including ligand binding sites, the mechanism of GAIN domain-mediated receptor activation and how aGPCR transmembrane domains may relate to activation. Other aspects of aGPCR signalling will be touched upon, such as downstream effectors and physiological roles.

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Structural insight into the activation of a class B G-protein-coupled receptor by peptide hormones in live human cells

eLife ◽

10.7554/elife.27711 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 17

Author(s):

Lisa Seidel ◽

Barbara Zarzycka ◽

Saheem A Zaidi ◽

Vsevolod Katritch ◽

Irene Coin

Keyword(s):

G Protein ◽

Conformational Changes ◽

Transmembrane Domain ◽

Peptide Hormones ◽

Human Cells ◽

Activation Mechanism ◽

Surface Mapping ◽

General Role ◽

Class B ◽

G Protein Coupled

The activation mechanism of class B G-protein-coupled receptors (GPCRs) remains largely unknown. To characterize conformational changes induced by peptide hormones, we investigated interactions of the class B corticotropin-releasing factor receptor type 1 (CRF1R) with two peptide agonists and three peptide antagonists obtained by N-truncation of the agonists. Surface mapping with genetically encoded photo-crosslinkers and pair-wise crosslinking revealed distinct footprints of agonists and antagonists on the transmembrane domain (TMD) of CRF1R and identified numerous ligand-receptor contact sites, directly from the intact receptor in live human cells. The data enabled generating atomistic models of CRF- and CRF(12-41)-bound CRF1R, further explored by molecular dynamics simulations. We show that bound agonist and antagonist adopt different folds and stabilize distinct TMD conformations, which involves bending of helices VI and VII around flexible glycine hinges. Conservation of these glycine hinges among all class B GPCRs suggests their general role in activation of these receptors.

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