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. 2017 Nov 14;114(46):12309-12314.
doi: 10.1073/pnas.1706656114. Epub 2017 Oct 30.

Endosomal Signaling of the Receptor for Calcitonin Gene-Related Peptide Mediates Pain Transmission

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Free PMC article

Endosomal Signaling of the Receptor for Calcitonin Gene-Related Peptide Mediates Pain Transmission

Rebecca E Yarwood et al. Proc Natl Acad Sci U S A. .
Free PMC article

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: G protein-coupled receptors; endocytosis; neuropeptides; nociception; pain.

Conflict of interest statement

Conflict of interest statement: Research in the N.W.B. laboratory is funded in part by Takeda Pharmaceuticals.

Figures

Fig. 1.
Fig. 1.
CLR endocytosis. (AD) BRET assays of CLR-RLuc8 and βARR2-YFP (A), Rab5a-Venus and KRas-Venus (B and D), and Rab11-Venus (C) proximity in HEK cells. (D) CLR-RLuc8 and Rab5a-Venus or KRas-Venus BRET (100 nM CGRP, 15 min). (E) Cell-surface HA-CLR ELISA in HEK cells (100 nM CGRP, 15 min). n = 3 experiments. (F) Confocal images of HA-CLR-IR and EEA1-IR in HEK cells. Dy4a, dynamin inhibitor; DynK44E, dominant-negative dynamin; inact, inactive analog; PS2, clathrin inhibitor. n = 3–6 experiments. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001 to basal or control. ANOVA, Sidak’s test.
Fig. 2.
Fig. 2.
CLR compartmentalized signaling. FRET assays of CGRP-induced activation of cytosolic PKC (cytoCKAR, A, B, and G), cytosolic ERK (cytoEKAR, C and D), and nuclear ERK (nucEKAR, E, F, and H) in individual HEK-HA-CLR/myc-RAMP1 cells. (A, C, and E) Kinetics; (B, D, and FH) area under curve (AUC). Cells were treated with inhibitors of dynamin (DynK44E, Dy4a), Gαs (NF449), Gαi (NF023), Gαq (UBO-QIC), phospholipase Cβ (U73122), or with a chelator of intracellular Ca2+ (EGTA-AM). n = 29–401 cells, n = 3 experiments. **P < 0.01, ***P < 0.001 to vehicle; ∧∧∧P < 0.001 to CGRP control. ANOVA, Tukey’s test.
Fig. 3.
Fig. 3.
Tripartite probes. (A) Probe structure. (BD) Confocal images of live HEK cells. (B and C) HEK-HA-CLR/myc-RAMP1 cells were incubated with Cy5-Chol (B) or Cy5-Ethyl Ester (C). CLR was labeled with HA-Alexa488 antibody. Cells were stimulated with CGRP (50 nM, 30 or 60 min) to induce endocytosis. Insets (white boxes) show magnified regions and colocalization (arrows). Traces (Left) show relative overlap of pixel intensities for HA-CLR and Cy5-Chol along dashed lines. (D) Untransfected HEK cells incubated with Cy5-Chol. (E) Overlap coefficient for HA-CLR and Cy5-Chol or Cy5-Ethyl Ester. n = 6–14 cells, n = 4 experiments. **P < 0.01 to 0 min. ANOVA, Dunnett’s test.
Fig. 4.
Fig. 4.
Tripartite antagonism of CLR. ERK activity was assessed in individual HEK-HA-CLR/myc-RAMP1 cells expressing FRET biosensors for cytosolic ERK (cytoEKAR, A, C, and E) or nuclear (nucEKAR, B, D, and F) ERK. Cells were preincubated with vehicle, CGRP8–17, or CGRP8–37–Chol for 30 min and washed. CGRP-stimulated ERK activity was assessed immediately after washing (A and B, 30 min preincubation) or 4 h after washing (C and D, 4 h prepulse). (AD) Kinetics. (E and F) Area under curve (AUC). (G and H) Effects of graded concentrations of CGRP8–37–Chol on cytosolic (G) and nuclear (H) ERK signaling in populations of HEK-HA-CLR/myc-RAMP1 cells. (AF) n = 159–417 cells, n = 3 experiments; (G and H) n = 4–9 experiments. ***P < 0.001 to vehicle; ∧∧∧P < 0.001 to antagonist vehicle control. ANOVA, Tukey’s test.
Fig. 5.
Fig. 5.
CLR endocytosis and excitation of spinal neurons. (AF) CGRP-induced activation of lamina I neurons in rat spinal cord slices. Dy4a, dynamin inhibitor; GF109203X, PKC inhibitor; U0126, MEK inhibitor. (A and D) Representative traces. (B and E) Firing rate normalized to 2 min. (C and F) Firing duration to last action potential. n = 5–8 neurons per group; n = 19 rats. *P < 0.05, ***P < 0.001. ANOVA, Sidak’s multiple comparisons test (firing rate), or Dunn’s multiple comparisons test (firing time). (G) Confocal images of CLR-IR. Arrow, endosomes. Arrowhead, plasma membrane. (H) Quantification of CLR endocytosis. n = 6–8 neurons per group, n = 3 rats. *P < 0.05, ***P < 0.001. ANOVA, Tukey’s test.
Fig. 6.
Fig. 6.
Tripartite antagonism of CGRP-induced excitation of spinal neurons. Spinal cord slices were incubated with vehicle (Veh), CGRP8–37, or CGRP8–37–Chol for 60 min; washed; and challenged with CGRP 60 min later. (A) Representative traces. (B) Firing rate normalized to 2 min. (C) Firing duration to last action potential. n = 5–7 neurons per group, n = 21 rats. *P < 0.05. ANOVA, Sidak’s test (firing rate), or Dunn’s test (firing time). ns, not significant.
Fig. 7.
Fig. 7.
Tripartite antagonism of nociception. Antagonists were injected intrathecally 3 h before intraplantar injection of capsaicin (Cap, A and B) or formalin (Form, C and D) or 36 h after CFA (E and F). von Frey withdrawal responses to stimulation of the planar surface of the injected paws (A, E, and F) or noninjected paws (B) and nocifensive behavior (C and D) were assessed. Number of mice used in each group is indicated in parentheses. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 to basal or vehicle control. ANOVA, Dunnett’s test.

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