Structural basis of neuropeptide Y signaling through Y₁ and Y₂ receptors

Abstract

Neuropeptide Y (NPY), a 36-amino-acid peptide, functions as a neurotransmitter in both the central and peripheral nervous systems by activating the NPY receptor subfamily. Notably, NPY analogs display varying selectivity and exert diverse physiological effects through their interactions with this receptor family. [Pro³⁴]–NPY and [Leu³¹, Pro³⁴]–NPY, mainly acting on Y₁R, reportedly increases blood pressure and postsynaptically potentiates the effect of other vasoactive substances above all, while N-terminal cleaved NPY variants in human body primary mediates angiogenesis and neurotransmitter release inhibition through Y₂R. However, the recognition mechanisms of Y₁R and Y₂R with specific agonists remain elusive, thereby hindering subtype receptor-selective drug development. In this study, we report three cryo-electron microscopy (cryo-EM) structures of Gi2-coupled Y₁R and Y₂R in complexes with NPY, as well as Y₁R bound to a selective agonist [Leu³¹, Pro³⁴]–NPY. Combined with cell-based assays, our study not only reveals the conserved peptide-binding mode of NPY receptors but also identifies an additional sub-pocket that confers ligand selectivity. Moreover, our analysis of Y₁R evolutionary dynamics suggests that this sub-pocket has undergone functional adaptive evolution across different species. Collectively, our findings shed light on the molecular underpinnings of neuropeptide recognition and receptor activation, and they present a promising avenue for the design of selective drugs targeting the NPY receptor family.

Keywords

G protein coupled receptor / ligand selectivity / neuropeptide Y / neuropeptide Y receptors

References

1	AG Blomqvist, H Herzog. Y-receptor subtypes–how many more? Trends Neurosci. 1997;20(7):294-298.
2	D Larhammar, E Salaneck. Molecular evolution of NPY receptor subtypes. Neuropeptides. 2004;38(4):141-151.
3	X Pedragosa-Badia, J Stichel, AG Beck-Sickinger. Neuropeptide Y receptors: how to get subtype selectivity. Front Endocrinol (Lausanne). 2013;4:5.
4	D Lindner, J Stichel, AG Beck-Sickinger. Molecular recognition of the NPY hormone family by their receptors. Nutrition. 2008;24(9):907-917.
5	D Larhammar. Evolution of neuropeptide Y, peptide YY and pancreatic polypeptide. Regul Pept. 1996;62(1):1-11.
6	AG Blomqvist, C S?derberg, I Lundell, RJ Milner, D Larhammar. Strong evolutionary conservation of neuropeptide Y: sequences of chicken, goldfish, and Torpedo marmorata DNA clones. Proc Natl Acad Sci USA. 1992;89(6):2350-2354.
7	K Tatemoto. Neuropeptide Y: complete amino acid sequence of the brain peptide. Proc Natl Acad Sci USA. 1982;79(18):5485-5489.
8	MA Raghanti, EN Miller, DN Jones, et al. Hedonic eating, obesity, and addiction result from increased neuropeptide Y in the nucleus accumbens during human brain evolution. Proc Natl Acad Sci USA. 2023;120(38):e2311118120.
9	AF Budnik, D Aryaeva, P Vyshnyakova, PM Masliukov. Age related changes of neuropeptide Y-ergic system in the rat duodenum. Neuropeptides. 2020;80:101982.
10	K Abid, B Rochat, PG Lassahn, et al. Kinetic study of neuropeptide Y (NPY) proteolysis in blood and identification of NPY3-35: a new peptide generated by plasma kallikrein. J Biol Chem. 2009;284(37):24715-24724.
11	SA Hubers, JR Wilson, C Yu, et al. DPP (dipeptidyl peptidase)-4 inhibition potentiates the vasoconstrictor response to NPY (Neuropeptide Y) in humans during renin-angiotensin-aldosterone system inhibition. Hypertension (Dallas, Tex : 1979). 2018;72(3):712-719.
12	N Frerker, L Wagner, R Wolf, et al. Neuropeptide Y (NPY) cleaving enzymes: structural and functional homologues of dipeptidyl peptidase 4. Peptides. 2007;28(2):257-268.
13	R Mentlein, T Roos. Proteases involved in the metabolism of angiotensin II, bradykinin, calcitonin gene-related peptide (CGRP), and neuropeptide Y by vascular smooth muscle cells. Peptides. 1996;17(4):709-720.
14	D Grandt, M Schimiczek, W Rascher, et al. Neuropeptide Y 3–36 is an endogenous ligand selective for Y2 receptors. Regul Pept. 1996;67(1):33-37.
15	MM Berglund, SK Holmberg, H Eriksson, et al. The cloned guinea pig neuropeptide Y receptor Y1 conforms to other mammalian Y1 receptors. Peptides. 1999;20(9):1043-1053.
16	J Fuhlendorff, U Gether, L Aakerlund, et al. [Leu31, Pro34]neuropeptide Y: a specific Y1 receptor agonist. Proc Natl Acad Sci USA. 1990;87(1):182-186.
17	R Abounader, JG Villemure, E Hamel. Characterization of neuropeptide Y (NPY) receptors in human cerebral arteries with selective agonists and the new Y1 antagonist BIBP 3226. Br J Pharmacol. 1995;116(4):2245-2250.
18	JL Krstenansky, TJ Owen, MH Payne, SA Shatzer, SH Buck. C-terminal modifications of neuropeptide Y and its analogs leading to selectivity for the mouse brain receptor over the porcine spleen receptor. Neuropeptides. 1990;17(3):117-120.
19	EK Potter, MJ McCloskey. [Leu31, Pro34] NPY, a selective functional postjunctional agonist at neuropeptide-Y receptors in anaesthetised rats. Neurosci Lett. 1992;134(2):183-186.
20	C Wahlestedt, L Grundemar, R Hakanson, et al. Neuropeptide Y receptor subtypes, Y1 and Y2. Ann N Y Acad Sci. 1990;611:7-26.
21	EK Potter, L Mitchell, MJD McCloskey, et al. Pre- and postjunctional actions of neuropeptide Y and related peptides. Regul Pept. 1989;25(2):167-177.
22	C Wahlestedt, N Yanaihara, R Hakanson. Evidence for different pre-and post-junctional receptors for neuropeptide Y and related peptides. Regul Pept. 1986;13(3-4):307-318.
23	Z Zukowska, DS Grant, EW Lee. Neuropeptide Y: a novel mechanism for ischemic angiogenesis. Trends Cardiovasc Med. 2003;13(2):86-92.
24	EW Lee, M Michalkiewicz, J Kitlinska, et al. Neuropeptide Y induces ischemic angiogenesis and restores function of ischemic skeletal muscles. J Clin Invest. 2003;111(12):1853-1862.
25	C Cavadas, D Cefai, J Rosmaninho-Salgado, et al. Deletion of the neuropeptide Y (NPY) Y1 receptor gene reveals a regulatory role of NPY on catecholamine synthesis and secretion. Proc Natl Acad Sci USA. 2006;103(27):10497-10502.
26	K Abid, B Rochat, P-G Lassahn, et al. Kinetic study of neuropeptide Y (NPY) proteolysis in blood and identification of NPY3-35: a new peptide generated by plasma kallikrein. J Biol Chem. 2009;284(37):24715-24724.
27	L Grundemar, SE Jonas, N Morner, ED Hogestatt, C Wahlestedt, R Hakanson. Characterization of vascular neuropeptide Y receptors. Br J Pharmacol. 1992;105(1):45-50.
28	Z Yang, S Han, M Keller, et al. Structural basis of ligand binding modes at the neuropeptide Y Y(1) receptor. Nature. 2018;556(7702):520-524.
29	T Tang, C Hartig, Q Chen, et al. Structural basis for ligand recognition of the neuropeptide Y Y(2) receptor. Nat Commun. 2021;12(1):737.
30	C Park, J Kim, SB Ko, et al. Structural basis of neuropeptide Y signaling through Y1 receptor. Nat Commun. 2022;13(1):853.
31	Z Yang, S Han, M Keller, et al. Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor. Nature. 2018;556(7702):520-524.
32	T Tang, C Hartig, Q Chen, et al. Structural basis for ligand recognition of the neuropeptide Y Y2 receptor. Nat Commun. 2021;12(1):737.
33	W Shihoya, T Nishizawa, A Okuta, et al. Activation mechanism of endothelin ETB receptor by endothelin-1. Nature. 2016;537(7620):363-368.
34	B Langmead, SL Salzberg. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357-359.
35	C Hong, NJ Byrne, B Zamlynny, et al. Structures of active-state orexin receptor 2 rationalize peptide and small-molecule agonist recognition and receptor activation. Nat Commun. 2021;12(1):815.
36	TL Blundell, JE Pitts, IJ Tickle, SP Wood, CW Wu. X-ray analysis (1. 4-A resolution) of avian pancreatic polypeptide: small globular protein hormone. Proc Natl Acad Sci USA. 1981;78(7):4175-4179.
37	SD Jois, LM Nagarajarao, M Prabhakaran, A Balasubramaniam. Modeling of neuropeptide receptors Y1, Y4, Y5, and docking studies with neuropeptide antagonist. J Biomol Struct Dyn. 2006;23(5):497-508.
38	D Larhammar, CA Bergqvist. Ancient grandeur of the vertebrate neuropeptide Y system shown by the coelacanth Latimeria chalumnae. Front Neurosci. 2013;7:27.
39	Z Yang, M dos Reis. Statistical properties of the branch-site test of positive selection. Mol Biol Evol. 2010;28(3):1217-1228.
40	Z Yang. PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol. 2007;24(8):1586-1591.
41	Z Yang. Bayes empirical bayes inference of amino acid sites under positive selection. Mol Biol Evol. 2005;22(4):1107-1118.
42	C Cabrele, AG Beck-Sickinger. Molecular characterization of the ligand-receptor interaction of the neuropeptide Y family. J Pept Sci. 2000;6(3):97-122.
43	B Xu, S Vasile, S Ostergaard, et al. Elucidation of the binding mode of the carboxyterminal region of peptide YY to the human Y2 receptor. Mol Pharmacol. 2018;93(4):323-334.
44	S Hoffmann, B Rist, G Videnov, G Jung, AG Beck-Sickinger. Structure-affinity studies of C-terminally modified analogs of neuropeptide Y led to a novel class of peptidic Y1 receptor antagonist. Regul Pept. 1996;65(1):61-70.
45	N Merten, D Lindner, N Rabe, et al. Receptor subtype-specific docking of Asp6.59 with C-terminal arginine residues in Y receptor ligands. J Biol Chem. 2007;282(10):7543-7551.
46	L Cheng, F Xia, Z Li, et al. Structure, function and drug discovery of GPCR signaling. Mol Biomed. 2023;4(1):46. (2662-8651 (Electronic)).
47	JF White, N Noinaj, Y Shibata, et al. Structure of the agonist-bound neurotensin receptor. Nature. 2012;490(7421):508-513.
48	H Kang, C Park, YK Choi, et al. Structural basis for Y2 receptor-mediated neuropeptide Y and peptide YY signaling. Structure. 2023;31(1):44-57. e46.
49	T Tang, Q Tan, S Han, et al. Receptor-specific recognition of NPY peptides revealed by structures of NPY receptors. Sci Adv. 2022;8(18):eabm1232.
50	RA Laskowski, MB Swindells. LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J Chem Inf Model. 2011;51(10):2778-2786.