GPR171 is a hypothalamic G protein-coupled receptor for BigLEN, a neuropeptide involved in feeding Ivone Gomesa, Dipendra K. Aryalb,1, Jonathan H. Wardmana,c,1, Achla Guptaa, Khatuna Gagnidzea,2, Ramona M. Rodriguizb,d, Sanjai Kumare, William C. Wetselb,d,f,g,h, John E. Pintari, Lloyd D. Frickerc, and Lakshmi A. Devia,3 aDepartment of Pharmacology and Systems Therapeutics, Ichan School of Medicine at Mount Sinai, New York, NY 10029; Departments of bPsychiatry and Behavioral Sciences, fCell Biology, gNeurobiology, and hMedicine (Endocrinology) and dMouse Behavioral and Neuroendocrine Analysis Core Facility, Duke University Medical Center, Durham, NC 27710; cDepartment of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461; eDepartment of Chemistry and Biochemistry, Queens College and the Graduate Center of The City University of New York, Queens, NY 11367; and iDepartment of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 00854 Edited by Huda Akil, University of Michigan, Ann Arbor, MI, and approved August 23, 2013 (received for review July 9, 2013) Multiple peptide systems, including neuropeptide Y, leptin, ghre- as an orphan G protein-coupled receptor (GPCR) activated by lin, and others, are involved with the control of food intake and BigLEN that plays a functional role in feeding and metabolism. body weight. The peptide LENSSPQAPARRLLPP (BigLEN) has been proposed to act through an unknown receptor to regulate body Results and Discussion weight. In the present study, we used a combination of ligand- Characterization of a Functional Receptor for BigLEN in the Hypothalamus binding and receptor-activity assays to characterize a Gαi/o protein- and in Neuro2A Cells. To characterize the BigLEN receptor, we coupled receptor activated by BigLEN in the mouse hypothalamus carried out binding and signaling studies with hypothalamic 125 and Neuro2A cells. We then selected orphan G protein-coupled membranes. Binding assays with [ I]Tyr-BigLEN revealed a fi fi K ∼ B receptors expressed in the hypothalamus and Neuro2A cells and speci c and high-af nity site ( d 0.5 nM) for BigLEN (Fig. 1 C 125 tested each for activation by BigLEN. G protein-coupled receptor and ). Comparison of [ I]Tyr-BigLEN binding in mouse brain 171 (GPR171) is activated by BigLEN, but not by the C terminally regions, pituitary, and peripheral tissues showed highest binding truncated peptide LittleLEN. The four C-terminal amino acids of in the hypothalamus, moderate binding in the cortex, and low NEUROSCIENCE BigLEN are sufficient to bind and activate GPR171. Overexpression binding in the peripheral tissues (Table S1). G protein activity of GPR171 leads to an increase, and knockdown leads to a de- assays with rat hypothalamic membranes revealed that BigLEN, but not a control peptide, produced a dose-dependent increase crease, in binding and signaling by BigLEN and the C-terminal 35 γ D peptide. In the hypothalamus GPR171 expression complements in [ S]GTP S binding (Fig. 1 ). Preincubation with pertussis toxin, a specificGαi/o inhibitor, led to a significant attenuation of the expression of BigLEN, and its level and activity are elevated 35 γ E in mice lacking BigLEN. In mice, shRNA-mediated knockdown of BigLEN-mediated [ S]GTP S binding (Fig. 1 ), suggesting that the hypothalamic BigLEN receptor is a Gα -coupled receptor. hypothalamic GPR171 leads to a decrease in BigLEN signaling and i/o Consistent with this idea, BigLEN treatment led to a significant results in changes in food intake and metabolism. The combina- decrease in adenylate cyclase activity (Fig. 1F). Neuro2A neu- tion of GPR171 shRNA together with neutralization of BigLEN roblastoma cells express a BigLEN receptor with properties peptide by antibody absorption nearly eliminates acute feeding similar to those of the hypothalamic receptor. Membranes from in food-deprived mice. Taken together, these results demonstrate – that GPR171 is the BigLEN receptor and that the BigLEN GPR171 fi system plays an important role in regulating responses associated Signi cance with feeding and metabolism in mice. The mechanism by which vertebrate animals control their body proSAAS | NPY/AgRP | neuroendocrine peptide | deorphanization | weight is a complex process involving a variety of molecules orexigenic that regulate feeding and metabolism. Some of these mole- cules are neuropeptides that bind to specific receptors in feeding centers of the brain. One of the most abundant pep- europeptides are the largest class of chemical messengers with tides in brain, LENSSPQAPARRLLPP (named BigLEN), has been more than 100 known neuropeptides that function in cell–cell N proposed to function as a neuropeptide involved in regulating signaling. A number of neuropeptides contribute to the regulation body weight, but the receptor through which this peptide acts of feeding and body weight, including well-established peptides such had not been identified. We screened candidate receptors and as neuropeptide Y (NPY) and agouti gene-related peptide (AgRP). found one, G protein-coupled receptor 171 (GPR171), that is Ablation of hypothalamic NPY/AgRP neurons causes a dramatic activated by BigLEN. Additional studies showed that the reduction in body weight (1), and selective activation of these BigLEN–GPR171 system plays an important role in regulating neurons drives a robust feeding behavior in mice (2). However, feeding and metabolism in mice. Thus, GPR171 is a potential knockout of either NPY or AgRP does not significantly alter body target for developing antiobesity drugs. weight or feeding (3), leading to the hypothesis that additional factors in these neurons contribute to feeding/body weight regula- A Author contributions: D.K.A., R.M.R., W.C.W., J.E.P., L.D.F., and L.A.D. designed research; tion (4). ProSAAS-derived peptides (Fig. 1 ) are logical candidates I.G., D.K.A., J.H.W., A.G., K.G., and J.E.P. performed research; S.K. and J.E.P. contributed for this control because they are among the most abundant peptides new reagents/analytic tools; I.G., D.K.A., R.M.R., and L.A.D. analyzed data; and I.G., present in mouse hypothalamus (5–7) and are greatly enriched in R.M.R., W.C.W., L.D.F., and L.A.D. wrote the paper. the AgRP/NPY neurons of the arcuate nucleus (8). ProSAAS- The authors declare no conflict of interest. derived peptides exhibit activity-dependent release from hypotha- This article is a PNAS Direct Submission. lamic neurons (9). Transgenic overexpression of proSAAS leads 1D.K.A. and J.H.W. contributed equally to this work. to an increase (10) and proSAAS knockout leads to a decrease in 2Present address: Harold and Margaret Milliken Hatch Laboratory of Neuroendocrinology, body weight (11). Finally, immunoneutralization of two proSAAS- The Rockefeller University, New York, NY 10065. derived peptides, AVDQDLGPEVPPENVLGALLRV and 3To whom correspondence should be addressed. E-mail: [email protected]. LENSSPQAPARRLLPP (PEN and BigLEN), from the hypothal- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. amus results in reduced feeding (8). Here we describe GPR171 1073/pnas.1312938110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1312938110 PNAS | October 1, 2013 | vol. 110 | no. 40 | 16211–16216 Downloaded by guest on October 2, 2021 A B 1400 C PEN proSAAS 1200 100 R RR RR KR RR 1000 80 Ctl. Pep. PEN B BigSAAS 800 Kd max 60 600 (nM) (fmoles/mg) LittleSAAS BigLEN 40 400 0.5 ± 0.1 1302 ± 74 LittleLEN 200 20 BigLEN Specific binding (fmoles/mg protein) 0 0 D L2P2 012 8 10 0 10-11 10-9 10-7 10-5 Specific bound counts 3 4 5 6 7 9 (% control) EC E Ligand [M] 50 max E Radiolabeled BigLEN (nM) (nM) (% basal) F G 210 190 * 100 H 1.6 ± 1.2 185 ± 4 750 60 -PTX 190 170 -PTX 80 * +PTX 170 150 +PTX 50 BigLEN 60 500 S binding * 40 150 130 40 130 110 n.s. n.s. 20 30 (% control) 110 Ctl. Pep. levels cAMP 250 20 S]GTP S binding 90 S]GTP 0 35 n.s. n.s. 90 35 [ (% basal) [ 0 10-11 10-9 10-7 (% basal) 10 Basal 0 Ligand [M] Basal 0 BigLEN % Cells with neurites BigLEN Specific bound counts (cpm) Ctl. Pep. PEN BigLEN Control Ctl. Pep. BigLEN Ctl. Pep. Fig. 1. ProSAAS-derived peptides bind to and activate a Gαi/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)Pre- treatment 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,andE–G, six independent experiments performed in triplicate in D, and four independent experiments in performed triplicate in H. n.s., not significant. Neuro2A cells exhibited specific[125I]Tyr-BigLEN binding (Fig. (Fig. 3A); this treatment converts BigLEN into LittleLEN-Arg 1G) and signaling with BigLEN as evidenced by the dose- and and the C-terminal peptide Leu-Leu-Pro-Pro (L2P2).