Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief

Abstract

Typically considered to be cell surface sensors of extracellular signals, heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) control many pathophysiological processes and are the target of 30% of therapeutic drugs. Activated receptors redistribute to endosomes, but researchers have yet to explore whether endosomal receptors generate signals that control complex processes in vivo and are viable therapeutic targets. We report that the substance P (SP) neurokinin 1 receptor (NK₁R) signals from endosomes to induce sustained excitation of spinal neurons and pain transmission and that specific antagonism of the NK₁R in endosomes with membrane-anchored drug conjugates provides more effective and sustained pain relief than conventional plasma membrane-targeted antagonists. Pharmacological and genetic disruption of clathrin, dynamin, and β-arrestin blocked SP-induced NK₁R endocytosis and prevented SP-stimulated activation of cytosolic protein kinase C and nuclear extracellular signal-regulated kinase, as well as transcription. Endocytosis inhibitors prevented sustained SP-induced excitation of neurons in spinal cord slices in vitro and attenuated nociception in vivo. When conjugated to cholestanol to promote endosomal targeting, NK₁R antagonists selectively inhibited endosomal signaling and sustained neuronal excitation. Cholestanol conjugation amplified and prolonged the antinociceptive actions of NK₁R antagonists. These results reveal a critical role for endosomal signaling of the NK₁R in the complex pathophysiology of pain and demonstrate the use of endosomally targeted GPCR antagonists.

Fig. 1. N₁R endocytosis-dependent compartmentalized signaling

(A to I) Effect of inhibitors of dynamin (Dy4) and clathrin (PS2), and of inactive (inact) analogs, on SP-induced spatiotemporal signaling profiles for nuclear ERK (NucEKAR) (A to C), cytosolic PKC (CytoCKAR) (D to F), and cytosolic cAMP (CytoEpac2) (G to I) measured in HEK293 cells using FRET biosensors. (A, D, and G) Time course of responses. (B, E, and H). Representative ratiometric images and sensor localization. Max, response to positive controls. Yellow arrows denote localization of FRET sensor and white arrows show the SP-stimulated signals in control cells and cells treated with Dy4 inact. (C, F, and I) Area under the curve (AUC) of (A), (D), and (G). (J and K) Effect of dynamin WT (J) or dominant negative K44E (K) overexpression on the spatiotemporal profile of SP-induced nuclear ERK. (L) AUC of (J) and (K). (M) Effect of clathrin heavy chain and dynamin-1 siRNA on the spatiotemporal profile of SP-induced nuclear ERK. (N) AUC of (M). (O) Effect of dynamin WT or K44E overexpression on the SP-induced SRE-SEAP. *P < 0.05, **P < 0.01, ***P < 0.001, vehicle (Veh); ^^P < 0.01, ^^^P < 0.001, control to inhibitors. (A to N) Thirty to 354 cells, three to five experiments. (O) n = 3 experiments. ANOVA, Tukey’s test (C, F, I, and N); Sidak’s test (L); Dunnett’s test (O).

Fig. 2. G protein–dependent NK₁R signaling in endosomes

(A to D) SP-induced BRET between NK₁R-RLUC8 and KRAS-Venus (A) or RAB5A-Venus (B) and between Gα_q-RLUC8 and Gγ₂-Venus (C) or RAB5A-Venus (D) in HEK293 cells. *P < 0.05, **P < 0.01, ***P < 0.001 to baseline. Triplicate observations, n ≥ 3 experiments. (E) Localization of NK₁R-IR (green), Gα_q-IR (cyan), and EEA1-IR (red) in HEK293 cells by super-resolution microscopy. Blue boxes, plasma membrane; red boxes, endosomes. (F) Quantification of the proportion of endosomes containing NK₁R-IR and Gα_q-IR. Sixty to 66 cells per condition (20 to 22 cells from n = 3 experiments). ****P < 0.0001. (G to I) Effect of inhibitors of Gα_q (UBO-QIC) or PLC (U73122) and Ca²⁺ chelation (EGTA) or inhibitors of Gα_s (NF449) or PKC (GF109203X, GFX) on SP-induced nuclear ERK (G), cytosolic PKC (H), and cytosolic cAMP (I) measured using FRET biosensors. ***P < 0.001, SP to vehicle; ^^^P < 0.001, control to inhibitor. Thirty-five to 67 cells, three experiments. ANOVA, Dunnett’s test (A to D); Sidak’s test (F and G); Tukey’s test (H and I).

Fig. 3. NK₁R endocytosis and neuronal excitation in spinal cord slices

(A) Effect of Dy4 and Dy4 inact on SP-induced endocytosis of NK₁R-IR in rat spinal neurons. Arrows, internalized; arrowheads, cell surface NK₁R. (B) Quantification of endocytosis. ****P < 0.0001. Eighteen neurons per group (six neurons in slices from n = 3 rats). (C to H) Effects of Dy4, Dy4 inact, U0126 (MEK inhibitor), and GF109203X (PKC inhibitor) on SP-induced firing of rat spinal neurons. (C and F) Representative traces. (D and G) Firing rate normalized to 2 min. (E and H) Firing duration to last action potential. Six to 7 neurons per group from n = 8 to 17 rats. *P < 0.05, **P < 0.01. (I and J) Effect of Dy4 and Dy4 inact on EPSC in lamina I/II_o induced by primary afferent stimulation, n = 11 neurons. (K and L) Effects of Dy4, PS2, and inactive analogs on capsaicin-stimulated SP-IR (K) and CGRP-IR (L) release from segments of mouse spinal cord. n = 6 experiments. ANOVA, Tukey’s test (B); Sidak’s test (D and G); Dunn’s test (E and H).

Fig. 4. NK₁R endocytosis, ERK signaling, and nociception in vivo

Effects of intrathecal (i.t.) injections of inhibitors or siRNA. (A and B) Localization of NK₁R-IR (A) and pERK-IR (B) in rat spinal neurons 10 min after intraplantar (i.pl.) vehicle or capsaicin (Cap). L, lamina. (A) Arrowheads show cell surface and arrows show endosomal NK₁R. (B) Arrows show pERK-IR (green) and red shows NeuN (neuronal marker). (C and D) Quantification of NK₁R endocytosis (C) and pERK-expressing neurons (D). **P < 0.01, ***P < 0.001. Neuronal numbers: Veh, 54; capsaicin, 52; Dy4, 28; Dy4 inact, 18; PS2, 22; PS2 inact, 19 (≥6 neurons in sections from n = 3 rats). (E, F, H, to K) Nociception in mice after intrathecal injection of endocytic inhibitors (Dy4, PS2), NK₁R antagonist (SR140,333; SR), dynamin-1 siRNA, βARR1/2 siRNA, endothelin-converting enzyme-1 inhibitor (SM-19712; SM), or MEK inhibitor (U0126). von Frey withdrawal responses of capsaicin-injected (E and H to K) or contralateral (F) paw. (G) Rotarod latency. (L) Formalin (form) nocifensive behavior. (M) von Frey withdrawal responses of the CFA-injected paw. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, to control. Student’s t test (C and D); ANOVA, Dunnett’s test (E to M).

Fig. 5. Disruption of NK₁R/βARR interactions

(A) Mouse NK₁R C terminus, indicating NK₁Rδ311 truncation and sequences of Tat-conjugated NK₁R and control peptides. (B and C) SP-induced BRET between WT NK₁R-RLUC8 or NK₁Rδ311-RLUC8 and βARR2-YFP (B) or RAB5A-Venus (C). Triplicate observations, n ≥ 3 experiments. (D) SP-induced cytosolic ERK (CytoEKAR) and nuclear ERK (NucEKAR) measured using FRET biosensors. *P < 0.05. Forty-nine to 99 cells, three experiments. (E) Effect of SP on SRE-SEAP release from HEK-NK₁Rδ311 cells. (F and G) Effect of control and three NK₁R peptides on SP-induced NK₁R-RLUC8/βARR2-YFP BRET (F) and NK₁R endocytosis (G). (H to J) Effects of intrathecally administered control and NK₁R peptides on capsaicin-induced mechanical allodynia (H), formalin-evoked nocifensive behavior (I), and CFA-induced mechanical hyperalgesia (J) in mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 to control. ANOVA, Sidak’s test (D and G); Dunnett’s test (H to J).

Fig. 6. Antagonism of endosomal NK₁R

(A) Structure of tripartite probes. (B) Cy5–ethyl ester or Cy5-Chol uptake in HEK293 cells. (C) Cy5-Chol or Cy5-Span-Chol (red) trafficking to RAB5A–red fluorescent protein (RFP)–positive (blue) and NK₁R-GFP–positive (green) endosomes. Asterisk, extracellular; arrowheads, plasma membrane; arrows, endosomes. (D and E) Cy5-Chol:SNAP549-NK₁R FRET, indicating localization of SNAP549-NK₁R, Cy5-Chol, and FRET signals (D) and time course of FRET in the cytosol (E). Six to nine cells, n = 3 experiments. (F to H) FRET assays of nuclear ERK activity (NucEKAR) immediately after (0 min) (F) or 4 hours after (4 hours) (G) 30 min of preincubation with Span, Span-Chol, or SR140,333 (SR). (H) AUC of (G). **P < 0.01, ***P < 0.001, to vehicle; ^^^P < 0.001, to antagonists. Thirty-one to 417 cells, n = 3 to 5 experiments. (I) Effects of Span or Span-Chol on SP-induced SRE-SEAP. HEK-NK₁R cells were pulse-incubated with Span or Span-Chol for 30 min, washed, recovered for 4 hours, and then stimulated with SP for 20 hours (pulse incubation) or were coincubated with antagonists throughout the experiment (coincubation). n = 3 experiments. ANOVA, Sidak’s test (H).

Fig. 7. Antagonism of endosomal NK₁R in spinal cord slices and in vivo

(A to C) Effects of tripartite antagonists on SP-induced firing of rat spinal neurons. (A) Representative traces. (B) Firing rate normalized to 2 min. (C) Firing duration to last action potential. Six to seven neurons per group from n = 5 to 18 rats. (D) Localization of Cy5-Chol (arrows, red) and DAP (blue) in superficial laminae (L) 6 hours after intrathecal injection in mouse. Top panel shows phase contrast superimposed on a fluorescence image; bottom panels show fluorescence images. White box denotes magnified region. (E to J) Effects of intrathecally administered Cy5-Chol, SR140,333 (SR), Span, Span-Chol, L-733,066 (L733), or L-733,0660–Chol on nociception in mice. (E to G) von Frey withdrawal responses of capsaicin-injected paw. (H) Nocifensive behavior after intraplantar formalin. (I and J) von Frey withdrawal responses of CFA-injected paw. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, to control. (K) Kinetics of degradation of SP, Span, and Span-Chol by membranes prepared from mouse spinal cord (n = 3). (L) Kinetics of degradation of Span and Span-Chol in human cerebrospinal fluid. n = 2, mean ± SD. ANOVA, Sidak’s test (B); Dunn’s test (C); Dunnett’s test (E to J).

Fig. 8. Endosomal platforms for signaling pain

(A) Nociceptive signaling. NK₁R couples to Gα_q (1), PLC-dependent Ca²⁺ mobilization (2), and a disintegrin and metalloproteinase (ADAM)–dependent epidermal growth factor receptor (EGFR) transactivation (3), which stimulates cytosolic ERK (4). Ca²⁺ activates PKC, which stimulates adenylyl cyclase (AC) to produce plasma membrane cAMP (5). GRK-phosphorylated NK₁R interacts with βARRs (6), which scaffold clathrin and adaptor protein 2 (AP2), leading to SP/NK₁R endocytosis (7). Endosomal SP/NK₁R (8) stimulates cytosolic PKC activity, cytosolic cAMP, and nuclear ERK activity (9), which drive neuronal excitation and nociceptive transmission. (B) Antinociception, endocytic inhibitors. Inhibition of dynamin (1), clathrin (2), or βARRs (3) prevents SP/NK₁R endocytosis, endosomal signaling, and nociceptive transmission. (C) Antinociception, tripartite antagonists. Tripartite antagonists incorporate into the plasma membrane (1) and traffic to endosomes (2), where they suppress SP/NK₁R nociceptive signaling.