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, CGRP, 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. CGRP-cholestanol, but not unconjugated CGRP, prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP-cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund's adjuvant more effectively than unconjugated CGRP 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.

When trafficking and signaling mix: How subcellular location shapes G protein‐coupled receptor activation of heterotrimeric G proteins

“…Another is to deliberately exploit the potential of conformationally selective nanobodies to block GPCR signaling reactions, specifically localizing them to a defined target membrane using chemical recruitment tools and assessing effect on the cellular response . A third approach, which may also have therapeutic potential with further development, is to generate antagonist ligands that accumulate in endosomes to inhibit activation in this compartment selectively …”

“…13,37 Over the last decade, however, the hypothesis that GPCRs can initiate G protein-coupled signaling from endosomes as well as the plasma membrane has gained considerable experimental support. [38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55] Evidence for GPCR-G protein signaling from endosomes. Broadly considered, four experimental approaches have produced evidence supporting endosomal GPCR-G protein signaling.…”

“…48,54 In a third approach, GPCR endocytosis was inhibited using genetic or chemical manipulations; endocytic blockade was found to reduce the strength and/or duration of downstream cellular responses. 39,40,42,45,46,49,50 In a fourth approach, biosensors derived from single-domain antibodies (nanobodies) were used to localize active-conformation GPCRs as well as detect conformational activation of G protein;…”

Signalling in response to sub‐picomolar concentrations of active compounds: Pushing the boundaries of GPCR sensitivity

“…Recent studies have revealed the importance of the texture of the initiated intracellular signal in controlling unique physiological outcomes, including both spatial (i.e., subcellular compartmentalisation of the signal) and temporal (i.e., changes in the signal over time) properties (Calebiro et al, ; DeFea, ; Godbole, Lyga, Lohse, & Calebiro, ; Irannejad et al, ; Irannejad et al, ; Kotowski, Hopf, Seif, Bonci, & von Zastrow, ; Mullershausen et al, ; Nikolaev, Bünemann, Schmitteckert, Lohse, & Engelhardt, ; Tsvetanova & von Zastrow, ; Wehbi et al, ). These shifts in the way we understand canonical GPCR activation and downstream signalling are already opening exciting new avenues for drug discovery (Jensen et al, ; Jimenez‐Vargas et al, ; Yarwood et al, ).…”

“…Recent advances in the understanding of compartmentalised GPCR signalling have illustrated the importance of receptor location (within different plasma membrane domains or within different subcellular compartments) for the control of distinct signalling outcomes (Halls et al, 2016;Jensen et al, 2017;Jimenez-Vargas et al, 2018;Stoeber et al, 2018;Tsvetanova & von Zastrow, 2014;Yarwood et al, 2017). Cellular context also appears to be very important in order for GPCRs to respond to ultra-low concentrations of ligand ( Figure 1).…”