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Importance of the second extracellular loop for melatonin MT1 receptor function and absence of melatonin binding in GPR50.

ABSTRACT: BACKGROUND AND PURPOSE:Recent crystal structures of GPCRs have emphasized the previously unappreciated role of the second extracellular (E2) loop in ligand binding and gating and receptor activation. Here, we have assessed the role of the E2 loop in the activation of the melatonin MT1 receptor and in the inactivation of the closely related orphan receptor GPR50. EXPERIMENTAL APPROACH:Chimeric MT1 -GPR50 receptors were generated and functionally analysed in terms of 2-[125 I]iodomelatonin binding, Gi /cAMP signalling and ?-arrestin2 recruitment. We also used computational molecular dynamics (MD) simulations. KEY RESULTS:MD simulations of 300 ns revealed (i) the tight hairpin structure of the E2 loop of the MT1 receptor (ii) the most suitable features for melatonin binding in MT1 receptors and (iii) major predicted rearrangements upon MT1 receptor activation, stabilizing interaction networks between Phe179 or Gln181 in the E2 loop and transmembrane helixes 5 and 6. Functional assays confirmed these predictions, because reciprocal replacement of MT1 and GPR50 residues/domains led to the predicted loss- and gain-of-melatonin action of MT1 receptors and GPR50 respectively. CONCLUSIONS AND IMPLICATIONS:Our work demonstrated the crucial role of the E2 loop for MT1 receptor and GPR50 function by proposing a model in which the E2 loop is important in stabilizing active MT1 receptor conformations and by showing how evolutionary processes appear to have selected for modifications in the E2 loop in order to make GPR50 unresponsive to melatonin. LINKED ARTICLES:This article is part of a themed section on Recent Developments in Research of Melatonin and its Potential Therapeutic Applications. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.16/issuetoc.

SUBMITTER: Clement N

PROVIDER: S-EPMC6057912 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Importance of the second extracellular loop for melatonin MT<sub>1</sub> receptor function and absence of melatonin binding in GPR50.

Clement Nathalie N Renault Nicolas N Guillaume Jean-Luc JL Cecon Erika E Journé Anne-Sophie AS Laurent Xavier X Tadagaki Kenjiro K Cogé Francis F Gohier Arnaud A Delagrange Philippe P Chavatte Philippe P Jockers Ralf R

British journal of pharmacology 20171027 16

<h4>Background and purpose</h4>Recent crystal structures of GPCRs have emphasized the previously unappreciated role of the second extracellular (E2) loop in ligand binding and gating and receptor activation. Here, we have assessed the role of the E2 loop in the activation of the melatonin MT<sub>1</sub> receptor and in the inactivation of the closely related orphan receptor GPR50.<h4>Experimental approach</h4>Chimeric MT<sub>1</sub> -GPR50 receptors were generated and functionally analysed in te ...[more]

PMID: 28898928

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Project description:Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.

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Importance of the second extracellular loop for melatonin MT1 receptor function and absence of melatonin binding in GPR50.

Publications

Importance of the second extracellular loop for melatonin MT<sub>1</sub> receptor function and absence of melatonin binding in GPR50.

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Importance of the second extracellular loop for melatonin MT1 receptor function and absence of melatonin binding in GPR50.

Publications

Importance of the second extracellular loop for melatonin MT&lt;sub&gt;1&lt;/sub&gt; receptor function and absence of melatonin binding in GPR50.

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OmicsDI is part of the ELIXIR infrastructure

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Importance of the second extracellular loop for melatonin MT<sub>1</sub> receptor function and absence of melatonin binding in GPR50.