GPR171 is a hypothalamic G protein-coupled receptor for BigLEN, a neuropeptide involved in feeding

Abstract

Multiple peptide systems, including neuropeptide Y, leptin, ghrelin, and others, are involved with the control of food intake and body weight. The peptide LENSSPQAPARRLLPP (BigLEN) has been proposed to act through an unknown receptor to regulate body weight. In the present study, we used a combination of ligand-binding and receptor-activity assays to characterize a Gαi/o protein-coupled receptor activated by BigLEN in the mouse hypothalamus and Neuro2A cells. We then selected orphan G protein-coupled receptors expressed in the hypothalamus and Neuro2A cells and tested each for activation by BigLEN. G protein-coupled receptor 171 (GPR171) is activated by BigLEN, but not by the C terminally truncated peptide LittleLEN. The four C-terminal amino acids of BigLEN are sufficient to bind and activate GPR171. Overexpression of GPR171 leads to an increase, and knockdown leads to a decrease, in binding and signaling by BigLEN and the C-terminal peptide. In the hypothalamus GPR171 expression complements the expression of BigLEN, and its level and activity are elevated in mice lacking BigLEN. In mice, shRNA-mediated knockdown of hypothalamic GPR171 leads to a decrease in BigLEN signaling and results in changes in food intake and metabolism. The combination of GPR171 shRNA together with neutralization of BigLEN peptide by antibody absorption nearly eliminates acute feeding in food-deprived mice. Taken together, these results demonstrate that GPR171 is the BigLEN receptor and that the BigLEN-GPR171 system plays an important role in regulating responses associated with feeding and metabolism in mice.

Keywords: NPY/AgRP; deorphanization; neuroendocrine peptide; orexigenic; proSAAS.

Fig. 1.

ProSAAS-derived peptides bind to and activate a Gα_i/o-coupled receptor. (A) Schematic diagram of peptides derived from proSAAS processing. R, arginine; K, lysine. (B) Binding of [¹²⁵I]Tyr-BigLEN to rat hypothalamic membranes. Nonspecific binding was determined with 10 µM Tyr-BigLEN and was <10% of the total binding. Bmax, maximal binding capacity. (C) BigLEN, but not PEN or a control peptide (Ctl. Pep.), displaces [¹²⁵I]Tyr-BigLEN binding to rat hypothalamic membranes. (D) BigLEN, but not a control peptide, dose-dependently increases [³⁵S]GTPγS binding to rat hypothalamic membranes. Emax, maximal possible effect. (E) Pretreatment with pertussis toxin (PTX) blocks the BigLEN-mediated increases in [³⁵S]GTPγS binding. *P < 0.0036 from +PTX (unpaired t test). (F) BigLEN inhibits adenylate cyclase activity in hypothalamic membranes. *P < 0.0001 vs. basal response. (G) Specific binding of [¹²⁵I]Tyr-BigLEN to Neuro2A cells in the presence of BigLEN, PEN or a control peptide. (H) BigLEN induces neurite outgrowth in Neuro2A cells; this outgrowth is blocked by PTX pretreatment. *P < 0.0036 from +PTX (unpaired t test). Data represent means ± SE of three independent experiments performed in triplicate in B, C, and E–G, six independent experiments performed in triplicate in D, and four independent experiments in performed triplicate in H. n.s., not significant.

Fig. 2.

Identification of GPR171 as the receptor activated by the proSAAS-derived peptide, BigLEN. (A) BigLEN, but not LittleLEN or a control peptide (Ctl. Pep.), increases intracellular Ca⁺² levels in CHO cells expressing GPR171 but not in cells expressing GPR19, GPR108, or GPR165. (B) Representative plots from A of intracellular Ca⁺² mobilization in CHO-GPR171 cells (along with a chimeric G_16/i3 protein) treated with buffer, LittleLEN, BigLEN, or ATP (1 µM). (C) A phylogenetic tree showing the relation of GPR171 to P2Y receptors, dicarboxylic acid receptors, free fatty acid receptors (FFARs), and other orphan GPCRs. (Scale bar, 0.1 substitutions per nucleotide.) (D) Intracellular Ca⁺² mobilization in CHO-GPR171 cells treated with or without 1 µM BigLEN or ligands including dicarboxylic acids. Data in A, B, and D represent means ± SE of three independent experiments carried out in sextuplicate.

Fig. 3.

BigLEN and the C-terminal peptide L2P2 exhibit activity at GPR171. (A) Intracellular Ca⁺² mobilization in CHO-GPR171 cells treated with trypsin, 1 μM BigLEN with or without trypsin or inactivated trypsin, or 1 μM L2P2 shows that L2P2 is sufficient to activate GPR171. (B) L2P2, but not LPP, displaces [¹²⁵I]Tyr-BigLEN binding from rat hypothalamic membranes. (C) BigLEN or L2P2 dose-dependently increases [³⁵S]GTPγS binding to hypothalamic membranes. (D) Treatment of hypothalamic membranes with BigLEN or L2P2 (0–10 μM) decreases cAMP levels. Data represent means ± SE of three independent experiments. n = 3–6.

Fig. 4.

Knockdown or blockade of GPR171 levels reduces signaling by BigLEN and L2P2. (A) Expression of GPR171 siRNA reduces GPR171 protein levels. *P < 0.0001 vs. control siRNA. (B) Expression of GPR171 siRNA reduces BigLEN- or L2P2-induced ERK1/2 phosphorylation but not that induced by LittleLEN or the δ-opioid receptor agonist, Deltorphin II (Delt II). *P < 0.0257 vs. control siRNA. (C) Expression of GPR171 siRNA reduces BigLEN- or L2P2-mediated neurite outgrowth in Neuro2A cells. *P < 0.0001 vs. control siRNA. (D) Overexpression of GPR171 increases but shRNA to GPR171 decreases specific [¹²⁵I]Tyr-BigLEN binding in Neuro2A cells. *P < 0.0001 vs. Neuro2A. (E) BigLEN and L2P2 do not signal in Neuro2A cells expressing GPR171 shRNA. (F) BigLEN- or L2P2-mediated increases in [³⁵S]GTPγS binding to rat hypothalamic membranes are blocked by pretreatment with the antibody to GPR171 (GPR171 Ab) but not by the antibody to endothelin-converting enzyme-2 (ECE2 Ab). *P < 0.05 vs. either No Ab or ECE2 Ab controls. Data represent means ± SE of three independent experiments in A or three independent experiments performed in triplicate in B–F. n.s., not significant.

Fig. 5.

Behaviors associated with shRNA-mediated knockdown of GPR171 expression. (A) RT-PCR analysis showing that intracerebroventricular (i.c.v.) administration of GPR171 lentiviral shRNA leads to a decrease in the levels of GPR171 mRNA in the mouse ventral hypothalamus. *P < 0.0001 vs. control shRNA (n = 6 per group). (B–F) Feeding-associated responses at peak response times during the late (0300–0900 h) and early (1800–2400 h) night. GPR171 shRNA mice consumed significantly more food (B), drank more water (C), and exhibited more activity (D) than controls during late night. A significant increase in RER (E) and heat production (F) were noted in the GPR171 shRNA mice relative to controls at both night time points. Data in B–F represent means ± SE of 12–16 mice per treatment condition. *P < 0.05, GPR171 shRNA vs. control for the same time point; ⁺P < 0.05, 0300–0900 h vs. 1800–2400 h for the same treatment. Statistical analyses are provided in Table S3. (G) I.c.v. administration of BigLEN antibody, but not control antibody (normal rabbit IgG), significantly reduces acute food intake up to 4 h postinjection. (H) GPR171 shRNA mice exhibit significantly less acute feeding following fasting than control shRNA mice. I.c.v. administration of BigLEN antibody causes a greater decrease in food intake in mice treated with GPR171 shRNA than in mice treated with control antibody. Data in G and H represent means ± SE of nine mice per treatment condition; **P < 0.01, shRNA treatment vs. BigLEN Ab; ^$P < 0.05, control shRNA+ BigLEN Ab vs. GRP171 shRNA + BigLEN Ab.