C-terminus of OX2R significantly affects downstream signaling pathways

Wang,Chunmei; Xu,Chao; Liu,Minghui; Pan,Yanyou; Bai,Bo; Chen,Jing

doi:10.3892/mmr.2017.6557

C-terminus of OX2R significantly affects downstream signaling pathways

Authors:
- Chunmei Wang
- Chao Xu
- Minghui Liu
- Yanyou Pan
- Bo Bai
- Jing Chen
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Affiliations: Neurobiology Institute, Jining Medical University, Jining, Shandong 272067, P.R. China
Published online on: May 9, 2017 https://doi.org/10.3892/mmr.2017.6557
Pages: 159-166
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The human orexin 2 receptor (OX2R) is a G-protein‑coupled receptor (GPCR) that has been implicated in a number of diverse physiological functions. Recent studies have identified a number of functions of the C‑termini of GPCRs. However, the importance of the OX2R C‑terminus in regulating signaling and surface expression remains unclear. In the present study, the function of the OX2R C‑terminus was investigated using three C‑terminal mutants, which were truncated at residues 368, 384 and 414, respectively, and the wild‑type control, which expressed the full‑length OX2R. HEK‑293 cells were transfected with the mutated and control OX2R constructs. ELISA, western blot analysis and calcium assays were used to investigate the effects of the mutations on OX2R function. The present results demonstrated that residues 385‑414 and 415‑444 exhibited a cumulative effect on the surface expression of OX2R. Residues 369‑384 exhibited a significant influence on inositol phosphate production and extracellular signal‑regulated kinase 1/2 phosphorylation. Residues 385‑414 significantly influenced agonist‑induced internalization, whereas residues 369‑384 and 385‑414 significantly influenced Ca2+ release. The results of the present study suggest that the C‑terminus of OX2R is important for its role in various physiological and pathological processes, and may therefore be associated with such disorders as depression and anorexia.

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1	Bockaert J and Pin JP: Molecular tinkering of G protein-coupled receptors: An evolutionary success. Embo J. 18:1723–1729. 1999. View Article : Google Scholar : PubMed/NCBI
2	Croft W, Hill C, McCann E, Bond M, Esparza-Franco M, Bennett J, Rand D, Davey J and Ladds G: A physiologically required G protein-coupled receptor (GPCR)-regulator of G protein signaling (RGS) interaction that compartmentalizes RGS activity. J Biol Chem. 288:27327–27342. 2013. View Article : Google Scholar : PubMed/NCBI
3	Lu ZL, Saldanha JW and Hulme EC: Seven-transmembrane receptors: Crystals clarify. Trends Pharmacol Sci. 23:140–146. 2002. View Article : Google Scholar : PubMed/NCBI
4	Wistrand M, Käll L and Sonnhammer EL: A general model of G protein-coupled receptor sequences and its application to detect remote homologs. Protein Sci. 15:509–521. 2006. View Article : Google Scholar : PubMed/NCBI
5	Fortin JP, Zhu Y, Choi C, Beinborn M, Nitabach MN and Kopin AS: Membrane-tethered ligands are effective probes for exploring class B1 G protein-coupled receptor function. Proc Natl Acad Sci USA. 106:8049–8054. 2009. View Article : Google Scholar : PubMed/NCBI
6	Thompson A and Kanamarlapudi V: Distinct regions in the C-Terminus required for GLP-1R cell surface expression, activity and internalisation. Mol Cell Endocrinol. 413:66–77. 2015. View Article : Google Scholar : PubMed/NCBI
7	Feng Y, Liu T, Li XQ, Liu Y, Zhu XY, Jankovic J, Pan TH and Wu YC: Neuroprotection by Orexin-A via HIF-1α induction in a cellular model of Parkinson's disease. Neurosci Lett. 579:35–40. 2014. View Article : Google Scholar : PubMed/NCBI
8	Lowther KM, Uliasz TF, Götz KR, Nikolaev VO and Mehlmann LM: Regulation of constitutive GPR3 signaling and surface localization by GRK2 and β-arrestin-2 Overexpression in HEK293 cells. PLoS One. 8:e653652013. View Article : Google Scholar : PubMed/NCBI
9	Katsushima Y, Sato T, Yamada C, Ito M, Suzuki Y, Ogawa E, Sukegawa I, Sukegawa J, Fukunaga K and Yanagisawa T: Interaction of PICK1 with C-terminus of growth hormone- releasing hormone receptor (GHRHR) modulates trafficking and signal transduction of human GHRHR. J Pharmacol Sci. 122:193–204. 2013. View Article : Google Scholar : PubMed/NCBI
10	Shim JY, Ahn KH and Kendall DA: Molecular basis of cannabinoid CB1 receptor coupling to the G protein heterotrimer Gαiβγ: identification of key CB1 contacts with the C-terminal helix α5 of Gαi. J Biol Chem. 288:32449–32465. 2013. View Article : Google Scholar : PubMed/NCBI
11	Gandía J, Fernández-Dueñas V, Morató X, Caltabiano G, González-Muñiz R, Pardo L, Stagljar I and Ciruela F: The Parkinson's disease-associated GPR37 receptor-mediated cytotoxicity is controlled by its intracellular cysteine-rich domain. J Neurochem. 125:362–372. 2013. View Article : Google Scholar : PubMed/NCBI
12	Ammoun S, Holmqvist T, Shariatmadari R, Oonk HB, Detheux M, Parmentier M, Akerman KE and Kukkonen JP: Distinct recognition of OX1 and OX2 receptors by orexin peptides. J Pharmacol Exp Ther. 305:507–514. 2003. View Article : Google Scholar : PubMed/NCBI
13	Baccari MC: Orexins and gastrointestinal functions. Curr Protein Pept Sci. 11:148–155. 2010. View Article : Google Scholar : PubMed/NCBI
14	Mavanji V, Perez-Leighton CE, Kotz CM, Kotz CM, Billington CJ, Parthasarathy S, Sinton CM and Teske JA: Promotion of wakefulness and energy expenditure by Orexin-A in the ventrolateral preoptic area. Sleep. 38:1361–1370. 2015. View Article : Google Scholar : PubMed/NCBI
15	Kannan H, Shirasaka T, Watanabe S, Yu NS, Kuitake T and Takasaki M: Central action of orexins on sympathetic outflow and cardiovascular function with a focus on the paraventricular nucleus of the hypothalamus. Masui. 56:30–39. 2007.(In Japanese). PubMed/NCBI
16	Kaminski T and Smolinska N: Expression of orexin receptors in the pituitary. Vitam Horm. 89:61–73. 2012. View Article : Google Scholar : PubMed/NCBI
17	Tang J, Chen J, Ramanjaneya M, Punn A, Conner AC and Randeva HS: The signalling profile of recombinant human orexin-2 receptor. Cell Signal. 20:1651–1661. 2008. View Article : Google Scholar : PubMed/NCBI
18	Kara E, Crépieux P, Gauthier C, Martinat N, Piketty V, Guillou F and Reiter E: A phosphorylation cluster of five serine and threonine residues in the C-terminus of the follicle-stimulating hormone receptor is important for desensitization but not for beta-arrestin-mediated ERK activation. Mol Endocrinol. 20:3014–3026. 2006. View Article : Google Scholar : PubMed/NCBI
19	Lehmann A, Kliewer A, Schutz D, Nagel F, Stumm R and Schulz S: Carboxyl-terminal multi-site phosphorylation regulates internalization and desensitization of the human sst2 somatostatin receptor. Mol Cell Endocrinol. 387:44–51. 2014. View Article : Google Scholar : PubMed/NCBI
20	Venkatesan S, Petrovic A, Locati M, Kim YO, Weissman D and Murphy PM: A membrane-proximal basic domain and cysteine cluster in the C-terminal tail of CCR5 constitute a bipartite motif critical for cell surface expression. J Biol Chem. 276:40133–40145. 2001. View Article : Google Scholar : PubMed/NCBI
21	Tetsuka M, Saito Y, Imai K, Doi H and Maruyama K: The basic residues in the membrane-proximal C-terminal tail of the rat melanin-concentrating hormone receptor 1 are required for receptor function. Endocrinology. 145:3712–3723. 2004. View Article : Google Scholar : PubMed/NCBI
22	Blomenröhr M, Heding A, Sellar R, Leurs R, Bogerd J, Eidne KA and Willars GB: Pivotal role for the cytoplasmic carboxyl-terminal tail of a nonmammalian gonadotropin-releasing hormone receptor in cell surface expression, ligand binding, and receptor phosphorylation and internalization. Mol Pharmacol. 56:1229–1237. 1999.PubMed/NCBI
23	Estall JL, Koehler JA, Yusta B and Drucker DJ: The glucagon-like peptide-2 receptor C terminus modulates beta-arrestin-2 association but is dispensable for ligand-induced desensitization, endocytosis, and G-protein-dependent effector activation. J Biol Chem. 280:22124–22134. 2005. View Article : Google Scholar : PubMed/NCBI
24	Innamorati G, Giannone F, Guzzi F, Rovati GE, Accomazzo MR, Chini B, Bianchi E, Schiaffino MV, Tridente G and Parenti M: Heterotrimeric G proteins demonstrate differential sensitivity to beta-arrestin dependent desensitization. Cell Signal. 21:1135–1142. 2009. View Article : Google Scholar : PubMed/NCBI
25	Golan M, Schreiber G and Avissar S: Antidepressants, beta-arrestins and GRKs: From regulation of signal desensitization to intracellular multifunctional adaptor functions. Curr Pharm Des. 15:1699–1708. 2009. View Article : Google Scholar : PubMed/NCBI
26	Chaki S, Guo DF, Yamano Y, Ohyama K, Tani M, Mizukoshi M, Shirai H and Inagami T: Role of carboxyl tail of the rat angiotensin II type 1A receptor in agonist-induced internalization of the receptor. Kidney Int. 46:1492–1495. 1994. View Article : Google Scholar : PubMed/NCBI
27	Willars GB, Heding A, Vrecl M, Sellar R, Blomenröhr M, Nahorski SR and Eidne KA: Lack of a C-terminal tail in the mammalian gonadotropin-releasing hormone receptor confers resistance to agonist-dependent phosphorylation and rapid desensitization. J Biol Chem. 274:30146–30153. 1999. View Article : Google Scholar : PubMed/NCBI
28	Neuschäfer-Rube F, Hermosilla R, Rehwald M, Rönnstrand L, Schülein R, Wernstedt C and Püschel GP: Identification of a Ser/Thr cluster in the C-terminal domain of the human prostaglandin receptor EP4 that is essential for agonist-induced beta-arrestin1 recruitment but differs from the apparent principal phosphorylation site. Biochem J. 379:573–585. 2004. View Article : Google Scholar : PubMed/NCBI
29	Qiu Y, Loh HH and Law PY: Phosphorylation of the delta-opioid receptor regulates its beta-arrestins selectivity and subsequent receptor internalization and adenylyl cyclase desensitization. J Biol Chem. 282:22315–22323. 2007. View Article : Google Scholar : PubMed/NCBI
30	Liu Q, Dewi DA, Liu W, Bee MS and Schonbrunn A: Distinct phosphorylation sites in the SST2A somatostatin receptor control internalization, desensitization, and arrestin binding. Mol Pharmacol. 73:292–304. 2008. View Article : Google Scholar : PubMed/NCBI
31	Conchon S, Barrault MB, Miserey S, Corvol P and Clauser E: The C-terminal third intracellular loop of the rat AT1A angiotensin receptor plays a key role in G protein coupling specificity and transduction of the mitogenic signal. J Biol Chem. 272:25566–25572. 1997. View Article : Google Scholar : PubMed/NCBI
32	Conner M, Hicks MR, Dafforn T, Knowles TJ, Ludwig C, Staddon S, Overduin M, Günther UL, Thome J and Wheatley M: Functional and biophysical analysis of the C-terminus of the CGRP-receptor; a family B GPCR. Biochemistry. 47:8434–8444. 2008. View Article : Google Scholar : PubMed/NCBI