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

Rebecca E. Yarwood, Wendy L. Imlach, Tina Marie Lieu, Nicholas A. Veldhuis, Dane D. Jensen, Carmen Klein Herenbrink, Luigi Aurelio, Zhijian Cai, MacDonald J. Christie, Daniel P. Poole, Christopher J.H. Porter, Peter McLean, Gareth A. Hicks, Pierangelo Geppetti, Michelle L. Halls, Meritxell Canals, Nigel W. Bunnett

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)12309-12314
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume114
Issue number46
DOIs
StatePublished - Nov 14 2017

Keywords

  • Endocytosis
  • G protein-coupled receptors
  • Neuropeptides
  • Nociception
  • Pain

ASJC Scopus subject areas

  • General

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  • Cite this

    Yarwood, R. E., Imlach, W. L., Lieu, T. M., Veldhuis, N. A., Jensen, D. D., Herenbrink, C. K., Aurelio, L., Cai, Z., 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. (2017). Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission. Proceedings of the National Academy of Sciences of the United States of America, 114(46), 12309-12314. https://doi.org/10.1073/pnas.1706656114