Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission

Yarwood, R.E.; Imlach, W.L.; Lieu, T.; Veldhuis, N.A.; Jensen, D.D.; Herenbrink, C.K.; Aurelio, L.; Cai, Zhijian; Christie, M.J.; Poole, D.P.; Porter, C.J.H.; McLean, P.; Hicks, G.A.; Geppetti, P.; Halls, M.L.; Canals, M.; Bunnett, N.W.

Abstract

G protein-coupled receptors (GPCRs) are considered to function primarily at the plasma membrane, where they interact with extracellular ligands and couple to G proteins that transmit intracellular signals. Consequently, therapeutic drugs are designed to target GPCRs at the plasma membrane. Activated GPCRs undergo clathrin-dependent endocytosis. Whether GPCRs in endosomes control pathophysiological processes in vivo and are therapeutic targets remains uncertain. We investigated the contribution of endosomal signaling of the calcitonin receptor-like receptor (CLR) to pain transmission. Calcitonin gene-related peptide (CGRP) stimulated CLR endocytosis and activated protein kinase C (PKC) in the cytosol and extracellular signal regulated kinase (ERK) in the cytosol and nucleus. Inhibitors of clathrin and dynamin prevented CLR endocytosis and activation of cytosolic PKC and nuclear ERK, which derive from endosomal CLR. A cholestanol-conjugated antagonist, CGRP8–37, accumulated in CLR-containing endosomes and selectively inhibited CLR signaling in endosomes. CGRP caused sustained excitation of neurons in slices of rat spinal cord. Inhibitors of dynamin, ERK, and PKC suppressed persistent neuronal excitation. CGRP8–37–cholestanol, but not unconjugated CGRP8–37, prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP8–37–cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund’s adjuvant more effectively than unconjugated CGRP8–37. Our results show that CLR signals from endosomes to control pain transmission and identify CLR in endosomes as a therapeutic target for pain. Thus, GPCRs function not only at the plasma membrane but also in endosomes to control complex processes in vivo. Endosomal GPCRs are a drug target that deserve further attention. © 2017, National Academy of Sciences. All rights reserved.

Citation

Yarwood, R., Imlach, W., Lieu, T., Veldhuis, N., Jensen, D., Herenbrink, C., Aurelio, L., Cai, Z., Christie, M., Poole, D., Porter, C., McLean, P., Hicks, G., Geppetti, P., Halls, M., Canals, M., & Bunnett, N. (2017). Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission. Proceedings of the National Academy of Sciences, 114(46), 12309-12314. https://doi.org/10.1073/pnas.1706656114

• Journal Article Type: Article
• Acceptance Date: Oct 6, 2017
• Online Publication Date: Oct 30, 2017
• Publication Date: Nov 14, 2017
• Deposit Date: Mar 5, 2019
• Publicly Available Date: Mar 5, 2019
• Journal: Proceedings of the National Academy of Sciences
• Print ISSN: 0027-8424
• Electronic ISSN: 1091-6490
• Publisher: National Academy of Sciences
• Peer Reviewed: Peer Reviewed
• Volume: 114
• Issue: 46
• Pages: 12309-12314
• DOI: https://doi.org/10.1073/pnas.1706656114
• Keywords: Endocytosis; G protein-coupled receptors; Neuropeptides; Nociception; Pain; calcitonin gene related peptide; calcitonin gene related peptide receptor; cholestanol; clathrin; dynamin; early endosome antigen 1; mitogen activated protein kinase; protein kinase C; adrenergic receptor blocking agent; calcitonin gene related peptide; calcitonin gene-related peptide (8-37); calcitonin receptor like receptor; Calcrl protein, rat; capsaicin; cholestanol; clathrin; dynamin; formaldehyde; Freund adjuvant; mitogen activated protein kinase 1; mitogen activated protein kinase 3; peptide fragment; protein kinase C; animal cell; Article; bioluminescence resonance energy transfer; cell membrane; confocal microscopy; controlled study; cytosol; endocytosis; endosome; enzyme linked immunosorbent assay; fluorescence resonance energy transfer; gene expression; immunoreactivity; nociception; nonhuman; pain; priority journal; rat; signal transduction; spinal cord; spinal cord nerve cell; animal; antagonists and inhibitors; chemically induced; cytology; drug effect; endosome; gene expression regulation; genetics; intraspinal drug administration; male; metabolism; microtomy; mouse; pain; pathophysiology; physiology; synaptic transmission; tissue culture technique; Adrenergic Antagonists; Animals; Calcitonin Gene-Related Peptide; Calcitonin Receptor-Like Protein; Capsaicin; Cholestanols; Clathrin; Dynamins; Endocytosis; Endosomes; Formaldehyde; Freund's Adjuvant; Gene Expression Regulation; Injections, Spinal; Male; Mice; Microtomy; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nociception; Pain; Peptide Fragments; Protein Kinase C; Rats; Spinal Cord; Synaptic Transmission; Tissue Culture Techniques
• Public URL: https://nottingham-repository.worktribe.com/output/1311753
• Publisher URL: https://www.pnas.org/content/114/46/12309
• Related Public URLs: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85033680288&doi=10.1073%2fpnas.1706656114&partnerID=40&md5=3e2db7f21c0a28c3ac334f0fe56198a3
• Contract Date: Mar 5, 2019