Opioid-Induced Signaling and Antinociception Are Modulated by the Recently Deorphanized Receptor, GPR171

Abstract

ProSAAS is one of the most widely expressed proteins throughout the brain and was recently found to be upregulated in chronic fibromyalgia patients. BigLEN is a neuropeptide that is derived from ProSAAS and was recently discovered to be the endogenous ligand for the orphan G protein-coupled receptor GPR171. Although BigLEN-GPR171 has been found to play a role in feeding and anxiety behaviors, it has not yet been explored in pain and opioid modulation. The purpose of this study was to evaluate this novel neuropeptide-receptor system in opioid-induced antinociception. We found that GPR171 is expressed in GABAergic neurons within the periaqueductal gray, which is a key brain area involved in pain modulation and opioid functions. We also found that, although the GPR171 agonist and antagonist do not have nociceptive effects on their own, they oppositely regulate morphine-induced antinociception with the agonist enhancing and antagonist reducing antinociception. Lastly, we showed that the GPR171 antagonist or receptor knockdown decreases signaling by the mu-opioid receptor, but not the delta-opioid receptor. Taken together, these results suggest that antagonism of the GPR171 receptor reduces mu opioid receptor signaling and morphine-induced antinociception, whereas the GPR171 agonist enhances morphine antinociception, suggesting that GPR171 may be a novel target toward the development of pain therapeutics. SIGNIFICANCE STATEMENT: GPR171 is a recently deorphanized receptor that is expressed within the periaqueductal gray and can regulate mu opioid receptor signaling and antinociception. This research may contribute to the development of new therapeutics to treat pain.

Fig. 1.

GPR171 expression in different neuronal populations. (A) Image from mouse brain atlas indicating location of PAG adapted from (Paxinos and Franklin, 2001). (B) Immunohistochemistry shows high expression of GPR171 in the PAG. (C) Immunohistochemistry results show that GPR171 is colocalized with GAD67 (i.e., GABA neurons). There are fewer cells that show colocalization of GPR171 and vGlut2. Data are representative of two sections from four mice. Arrows indicate colocalization. Scale bars, 50 µm.

Fig. 2.

Small molecule ligands targetting GPR171 alone do not produce antinociception. GPR171 agonist (MS15203; 10 mg/kg, i.p.), GPR171 antagonist (MS21570; 5 mg/kg, i.p), or vehicle (10% DMSO in saline, i.p.) were administered to mice and then tested on the warm water (52°C) tail flick assay (A) or hot plate (50°C) assay (B) at 15, 30, 60, or 120 minute. The data reveal no change in nociception on either test. Data are the means ± S.E. of 8–12 animals/group.

Fig. 3.

GPR171 ligands alter morphine antinociception. GPR171 agonist (MS15203; 10 mg/kg, i.p.), GPR171 antagonist (MS21570; 5 mg/kg, i.p), or vehicle (10% DMSO in saline, i.p.) were administered 10 minutes prior to saline (10 ml/kg) or morphine (Mor; 2 or 5 mg/kg, s.c.). Antinociception was evaluated at 15, 30, 60, or 120 minutes on the warm water (52°C) tail flick assay (A) or hot plate (50°C) assay (B). GPR171 agonist, MS15203, does not alter morphine (5 mg)-induced antinociception while GPR171 antagonist decreases antinociception as measured on the tail flick assay. MS15203 increases antinociception following the lower dose of morphine (2 mg). (B) On the hot plate test, MS15203 increases 5 mg morphine-induced antinociception at 15, 30, and 60 minutes, while MS21570+Mor (5 mg) does not induce antinociception greater than saline controls. MS15203+Mor (2 mg) produced significant antinociception at 30 minutes, whereas Mor (2 mg) did not. Inset: Dunnett’s post hoc to evaluate main effect compared with saline. *P< 0.05; ***P < 0.001; ****P < 0.0001, compared with saline; #P<0.05, compared with morphine; @P<0.05, compared with morphine + agonist. n.s., Not significant; HP, hot plate; TF, tail flick. Data are the means ± S.E. of 8–15 animals/group.

Fig. 4.

GPR171 ligands do not bind or signal at the MOPr . Displacement binding assays were carried out with membranes (50 μg protein) from CHO cells stably expressing either MOPr (A) or GPR171 (B) using [³H]DAMGO (3 nM final concentration) in the absence or presence of either 10 μM DAMGO, MS15203, or MS21570 as described in Materials and Methods. Data shows that MS15203 and MS21570 do not bind to MOPr and DAMGO does not bind to GPR171. (C) Membranes (20 μg protein) from CHO cells stably expressing MOPr were subjected to a [³⁵S]GTPγS binding assay using DAMGO (0–10 μM) in the absence or presence of 10 μM MS21570 as described in Materials and Methods. Data shows that MS21570 does not affect signaling by DAMGO in cells only expressing MOPr. E_max was calculated at 10 μM DAMGO. Data are mean ± S.E. of three experiments in triplicate. ****P < 0.0001; one-way ANOVA; n.s., not significant.

Fig. 5.

GPR171 expression alters MOPr mediated signaling, but not DOPr signaling. Membranes (20 μg protein) from Neuro 2A cells (N2A), N2A cells stably overexpressing GPR171 (N2A-GPR171) or N2A cells with shRNA-mediated knockdown of GPR171 (N2A-GPR171 KD) were subjected to a [³⁵S]GTPγS binding assay using DAMGO (0–10 μM) (A) or Deltorphin II (0–10 μM) (B). Data shows that levels of GPR171 affect signaling by DAMGO, but not Deltorphin II. *P < 0.05; ****P < 0.0001 two-way ANOVA for graph. *P < 0.05; **P < 0.01; ****P < 0.0001, one-way ANOVA for inset (C) N2A or N2A-GPR171 KD were subjected to a [³⁵S]GTPgS binding assay using MS21570 (0–10 μM) in the absence or presence of 10 μM DAMGO. Data shows that the GPR171 antagonist, MS21570 dose dependently decreases signaling by 10 μM DAMGO in cells coexpressing MOPr and GPR171. E_max was calculated at 10 μM DAMGO or Deltorphin II. **P < 0.01, t test for inset (MS21570 vs. +DAMGO). Data are Mean ± S.E. of three experiments in triplicate.