Journal Article

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

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

2017

Overview

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.

• Publisher: National Academy of Sciences
• Subject: Adrenergic Antagonists - pharmacology / Amino acids / Animals / Biological Sciences / Brain slice preparation / Calcitonin / Calcitonin gene-related peptide / Calcitonin Gene-Related Peptide - pharmacology / Calcitonin Receptor-Like Protein - antagonists & inhibitors / Calcitonin Receptor-Like Protein - genetics / Calcitonin Receptor-Like Protein - metabolism / Capsaicin / Capsaicin - antagonists & inhibitors / Capsaicin - pharmacology / Cholestanols - pharmacology / Clathrin / Clathrin - antagonists & inhibitors / Clathrin - genetics / Clathrin - metabolism / Cytosol / Drug development / Dynamin / Dynamins - genetics / Dynamins - metabolism / Endocytosis / Endocytosis - drug effects / Endosomes / Endosomes - drug effects / Endosomes - metabolism / Excitation / Extracellular signal-regulated kinase / Formaldehyde - antagonists & inhibitors / Formaldehyde - pharmacology / Freund's adjuvant / Freund's Adjuvant - antagonists & inhibitors / Freund's Adjuvant - pharmacology / G protein-coupled receptors / Gene Expression Regulation / Genes / In vivo methods and tests / Inhibitors / Injections, Spinal / Male / Mice / Microtomy / Mitogen-Activated Protein Kinase 1 - genetics / Mitogen-Activated Protein Kinase 1 - metabolism / Mitogen-Activated Protein Kinase 3 - genetics / Mitogen-Activated Protein Kinase 3 - metabolism / Nociception - drug effects / Nociception - physiology / Pain / Pain - chemically induced / Pain - genetics / Pain - physiopathology / Pain - prevention & control / Pain perception / Peptide Fragments - pharmacology / Peptides / Pharmacology / Protein kinase C / Protein Kinase C - genetics / Protein Kinase C - metabolism / Proteins / Rats / Receptor mechanisms / Receptors / Signal transduction / Signaling / Spinal cord / Spinal Cord - cytology / Spinal Cord - drug effects / Spinal Cord - metabolism / Synaptic Transmission - drug effects / Tissue Culture Techniques