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

Rebecca E. Yarwood, Wendy L. Imlach, TinaMarie 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 journalArticleResearchpeer-review

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
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
Publication statusPublished - 14 Nov 2017

Keywords

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

Cite this

Yarwood, Rebecca E. ; Imlach, Wendy L. ; Lieu, TinaMarie ; Veldhuis, Nicholas A. ; Jensen, Dane D. ; Herenbrink, Carmen Klein ; Aurelio, Luigi ; Cai, Zhijian ; Christie, MacDonald J. ; Poole, Daniel P. ; Porter, Christopher J H ; McLean, Peter ; Hicks, Gareth A. ; Geppetti, Pierangelo ; Halls, Michelle L. ; Canals, Meritxell ; Bunnett, Nigel W. / Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission. In: Proceedings of the National Academy of Sciences of the United States of America. 2017 ; Vol. 114, No. 46. pp. 12309-12314.
@article{b994ac5829c74a3da612e3d01f6a9bc4,
title = "Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission",
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.",
keywords = "Endocytosis, G protein-coupled receptors, Neuropeptides, Nociception, Pain",
author = "Yarwood, {Rebecca E.} and Imlach, {Wendy L.} and TinaMarie Lieu and Veldhuis, {Nicholas A.} and Jensen, {Dane D.} and Herenbrink, {Carmen Klein} and Luigi Aurelio and Zhijian Cai and Christie, {MacDonald J.} and Poole, {Daniel P.} and Porter, {Christopher J H} and Peter McLean and Hicks, {Gareth A.} and Pierangelo Geppetti and Halls, {Michelle L.} and Meritxell Canals and Bunnett, {Nigel W.}",
year = "2017",
month = "11",
day = "14",
doi = "10.1073/pnas.1706656114",
language = "English",
volume = "114",
pages = "12309--12314",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
publisher = "National Academy of Sciences",
number = "46",

}

Yarwood, RE, Imlach, WL, Lieu, T, Veldhuis, NA, Jensen, DD, Herenbrink, CK, Aurelio, L, Cai, Z, Christie, MJ, Poole, DP, Porter, CJH, McLean, P, Hicks, GA, Geppetti, P, Halls, ML, Canals, M & Bunnett, NW 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, vol. 114, no. 46, pp. 12309-12314. https://doi.org/10.1073/pnas.1706656114

Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission. / Yarwood, Rebecca E.; Imlach, Wendy L.; Lieu, TinaMarie; Veldhuis, Nicholas A.; Jensen, Dane D.; Herenbrink, Carmen Klein; Aurelio, Luigi; Cai, Zhijian; Christie, MacDonald J.; Poole, Daniel P.; Porter, Christopher J H; McLean, Peter; Hicks, Gareth A.; Geppetti, Pierangelo; Halls, Michelle L.; Canals, Meritxell; Bunnett, Nigel W.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 114, No. 46, 14.11.2017, p. 12309-12314.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Endosomal signaling of the receptor for calcitonin gene-related peptide mediates pain transmission

AU - Yarwood, Rebecca E.

AU - Imlach, Wendy L.

AU - Lieu, TinaMarie

AU - Veldhuis, Nicholas A.

AU - Jensen, Dane D.

AU - Herenbrink, Carmen Klein

AU - Aurelio, Luigi

AU - Cai, Zhijian

AU - Christie, MacDonald J.

AU - Poole, Daniel P.

AU - Porter, Christopher J H

AU - McLean, Peter

AU - Hicks, Gareth A.

AU - Geppetti, Pierangelo

AU - Halls, Michelle L.

AU - Canals, Meritxell

AU - Bunnett, Nigel W.

PY - 2017/11/14

Y1 - 2017/11/14

N2 - 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.

AB - 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.

KW - Endocytosis

KW - G protein-coupled receptors

KW - Neuropeptides

KW - Nociception

KW - Pain

UR - http://www.scopus.com/inward/record.url?scp=85033680288&partnerID=8YFLogxK

U2 - 10.1073/pnas.1706656114

DO - 10.1073/pnas.1706656114

M3 - Article

VL - 114

SP - 12309

EP - 12314

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 46

ER -