RAR-related orphan receptor alpha explained

RAR-related orphan receptor alpha (RORα), also known as NR1F1 (nuclear receptor subfamily 1, group F, member 1) is a nuclear receptor that in humans is encoded by the RORA gene.^[1] RORα participates in the transcriptional regulation of some genes involved in circadian rhythm. In mice, RORα is essential for development of cerebellum^{[2] [3]} through direct regulation of genes expressed in Purkinje cells.^[4] It also plays an essential role in the development of type 2 innate lymphoid cells (ILC2) and mutant animals are ILC2 deficient.^{[5] [6]} In addition, although present in normal numbers, the ILC3 and Th17 cells from RORα deficient mice are defective for cytokine production.^[7]

Discovery

The first three-human isoforms of RORα were initially cloned and characterized as nuclear receptors in 1994 by Giguère and colleagues, when their structure and function were first studied.^[8]

In the early 2000s, various studies demonstrated that RORα displays rhythmic patterns of expression in a circadian cycle in the liver, kidney, retina, and lung. Of interest, it was around this time that RORα abundance was found to be circadian in the mammalian suprachiasmatic nucleus. RORα is necessary for normal circadian rhythms in mice, demonstrating its importance in chronobiology.

Structure

The protein encoded by this gene is a member of the NR1 subfamily of nuclear hormone receptors. In humans, 4 isoforms of RORα have been identified, which are generated via alternative splicing and promoter usage, and exhibit differential tissue-specific expression. The protein structure of RORα consists of four canonical functional groups: an N-terminal (A/B) domain, a DNA-binding domain containing two zinc fingers, a hinge domain, and a C-terminal ligand-binding domain. Within the ROR family, the DNA-binding domain is highly conserved, and the ligand-binding domain is only moderately conserved. Different isoforms of RORα have different binding specificities and strengths of transcriptional activity.

Regulation of circadian rhythm

The core mammalian circadian clock is a negative feedback loop which consists of Per1/Per2, Cry1/Cry2, Bmal1, and Clock.^[9] This feedback loop is stabilized through another loop involving the transcriptional regulation of Bmal1.^[10] Transactivation of Bmal1 is regulated through the upstream ROR/REV-ERB Response Element (RRE) in the Bmal1 promoter, to which RORα and REV-ERBα bind. This stabilizing regulatory loop itself is induced by the Bmal1/Clock heterodimer, which induces transcription of RORα and REV-ERBα. RORα, which activates transcription of Bmal1, and REV-ERBα, which represses transcription of Bmal1, compete to bind to the RRE. This feedback loop regulating the expression of Bmal1 is thought to stabilize the core clock mechanism, helping to buffer it against changes in the environment.

Mechanism

Specific association with ROR elements (RORE) in regulatory regions is necessary for RORα's function as a transcriptional activator.^[11] RORα achieves this by specific binding to a consensus core motif in RORE, RGGTCA. This interaction is possible through the association of RORα's first zinc finger with the core motif in the major groove, the P-box, and the association of its C-terminal extension with the AT-rich region in the 5’ region of RORE.

Homology

RORα, RORβ, and RORγ are all transcriptional activators recognizing ROR-response elements.^[12] ROR-alpha is expressed in a variety of cell types and is involved in regulating several aspects of development, inflammatory responses, and lymphocyte development.^[13] The RORα isoforms (RORα1 through RORα3) arise via alternative RNA processing, with RORα2 and RORα3 sharing an amino-terminal region different from RORα1. In contrast to RORα, RORβ is expressed in Central Nervous System (CNS) tissues involved in processing sensory information and in generating circadian rhythms while RORγ is critical in lymph node organogenesis and thymopoeisis.

The DNA-binding domains of the DHR3 orphan receptor in Drosophila shows especially close homology within amino and carboxy regions adjacent to the second zinc finger region in RORα, suggesting that this group of residues is important for the proteins' functionalities.

PDP1 and VRI in Drosophila regulate circadian rhythm's by competing for the same binding site, the VP box, similarly to how ROR and REV-ERB competitively bind to RRE. PDP1 and VRI constitute a feedback loop and are functional homologs of ROR and REV-ERB in mammals.

Direct orthologs of this gene have been identified in mice and humans.

Human cytochrome c pseudogene HC2 and RORα share overlapping genomic organization with the HC2 pseudogene located within the RORα2 transcription unit. The nucleotide and deduced amino acid sequences of cytochrome c-processed pseudogene are on the sense strand while those of the RORα2 amino-terminal exon are on the antisense strand.

Interactions

• DNA: RORα binds to the P-box of the RORE.^[14]
• Co-activators:
  • SRC-1, CBP, p300, TRIP-l, TRIP-230, transcription intermediary protein-1 (TIF-1), peroxisome proliferator-binding protein (PBP), and GRIP-1 physically interact with RORα.^[15]
    • LXXLL motif: ROR interacts with SRC-1, GRIP-l, CBP, and p300 via the LXXLL (L=Leucine, X=any amino acid) motifs on these proteins.
• Ubiquitination: RORα is targeted for the proteasome by ubiquitination. A co-repressor, Hairless, stabilizes RORα by protecting it from this process, which also represses RORα.^[16]
• Sumoylation

UBE21/UBC9: Ubiquitin-conjugating enzyme I interacts with RORs, but its effect is not yet known.

• Phosphorylation

• • Phosphorylation of RORα1, which inhibits its transcriptional activity, is induced by Protein Kinase C.
  • ERK2

Extracellular signal-regulated kinase-2 also phosphorylates RORα.^[17]

• ATXN1

ATXN1 and RORα form part of a protein complex in Purkinje cells.

• FOXP3

FOXP3 directly represses the transcriptional activity of RORs.

• NME1

ROR has been shown to specifically interact with NME1.^[18]

• NM23-2

NM23-2 is a nucleoside diphosphate kinase involved in organogenesis and differentiation.^[19]

• NM23-1

NM23-1 is the product of a tumor metastasis suppressor candidate gene.^[19]

As a drug target

Because RORα and REV-ERBα are nuclear receptors that share the same target genes and are involved in processes that regulate metabolism, development, immunity, and circadian rhythm, they show potential as drug targets. Synthetic ligands have a variety of potential therapeutic uses, and can be used to treat diseases such as diabetes, atherosclerosis, autoimmunity, and cancer. T0901317 and SR1001, two synthetic ligands, have been found to be RORα and RORγ inverse agonists that suppress reporter activity and have been shown to delay onset and clinical severity of multiple sclerosis and other Th17 cell-mediated autoimmune diseases. SR1078 has been discovered as a RORα and RORγ agonist that increases the expression of G6PC and FGF21, yielding the therapeutic potential to treat obesity and diabetes as well as cancer of the breast, ovaries, and prostate. SR3335 has also been discovered as a RORα inverse agonist.

CGP 52608

See also

• RAR-related orphan receptor
• REV-ERBα
• Aromatase deficiency

Further reading

• Giguère V, Beatty B, Squire J, Copeland NG, Jenkins NA . The orphan nuclear receptor ROR alpha (RORA) maps to a conserved region of homology on human chromosome 15q21-q22 and mouse chromosome 9 . Genomics . 28 . 3 . 596–8 . August 1995 . 7490103 . 10.1006/geno.1995.1197 .
• Steinhilber D, Brungs M, Werz O, Wiesenberg I, Danielsson C, Kahlen JP, Nayeri S, Schräder M, Carlberg C . The nuclear receptor for melatonin represses 5-lipoxygenase gene expression in human B lymphocytes . The Journal of Biological Chemistry . 270 . 13 . 7037–40 . March 1995 . 7706239 . 10.1074/jbc.270.13.7037 . free .
• Forman BM, Chen J, Blumberg B, Kliewer SA, Henshaw R, Ong ES, Evans RM . Cross-talk among ROR alpha 1 and the Rev-erb family of orphan nuclear receptors . Molecular Endocrinology . 8 . 9 . 1253–61 . September 1994 . 10.1210/mend.8.9.7838158 . 7838158 . free .
• Becker-André M, André E, DeLamarter JF . Identification of nuclear receptor mRNAs by RT-PCR amplification of conserved zinc-finger motif sequences . Biochemical and Biophysical Research Communications . 194 . 3 . 1371–9 . August 1993 . 7916608 . 10.1006/bbrc.1993.1976 .
• Carlberg C, Hooft van Huijsduijnen R, Staple JK, DeLamarter JF, Becker-André M . RZRs, a new family of retinoid-related orphan receptors that function as both monomers and homodimers . Molecular Endocrinology . 8 . 6 . 757–70 . June 1994 . 10.1210/mend.8.6.7935491 . 7935491 . 22342101 . free .
• Paravicini G, Steinmayr M, André E, Becker-André M . The metastasis suppressor candidate nucleotide diphosphate kinase NM23 specifically interacts with members of the ROR/RZR nuclear orphan receptor subfamily . Biochemical and Biophysical Research Communications . 227 . 1 . 82–7 . October 1996 . 8858107 . 10.1006/bbrc.1996.1471 .
• Lau P, Bailey P, Dowhan DH, Muscat GE . Exogenous expression of a dominant negative RORalpha1 vector in muscle cells impairs differentiation: RORalpha1 directly interacts with p300 and myoD . Nucleic Acids Research . 27 . 2 . 411–20 . January 1999 . 9862959 . 148194 . 10.1093/nar/27.2.411 .
• Atkins GB, Hu X, Guenther MG, Rachez C, Freedman LP, Lazar MA . Coactivators for the orphan nuclear receptor RORalpha . Molecular Endocrinology . 13 . 9 . 1550–7 . September 1999 . 10.1210/mend.13.9.0343 . 10478845 . free .
• Meyer T, Kneissel M, Mariani J, Fournier B . In vitro and in vivo evidence for orphan nuclear receptor RORalpha function in bone metabolism . Proceedings of the National Academy of Sciences of the United States of America . 97 . 16 . 9197–202 . August 2000 . 10900268 . 16845 . 10.1073/pnas.150246097 . 2000PNAS...97.9197M . free .
• Gawlas K, Stunnenberg HG . Differential binding and transcriptional behaviour of two highly related orphan receptors, ROR alpha(4) and ROR beta(1) . Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression . 1494 . 3 . 236–41 . December 2000 . 11121580 . 10.1016/s0167-4781(00)00237-2 .
• Delerive P, Chin WW, Suen CS . Identification of Reverb(alpha) as a novel ROR(alpha) target gene . The Journal of Biological Chemistry . 277 . 38 . 35013–8 . September 2002 . 12114512 . 10.1074/jbc.M202979200 . free .
• Moretti RM, Montagnani Marelli M, Motta M, Limonta P . Role of the orphan nuclear receptor ROR alpha in the control of the metastatic behavior of androgen-independent prostate cancer cells . Oncology Reports . 9 . 5 . 1139–43 . 2003 . 12168086 . 10.3892/or.9.5.1139 .
• Raspè E, Mautino G, Duval C, Fontaine C, Duez H, Barbier O, Monte D, Fruchart J, Fruchart JC, Staels B . Transcriptional regulation of human Rev-erbalpha gene expression by the orphan nuclear receptor retinoic acid-related orphan receptor alpha . The Journal of Biological Chemistry . 277 . 51 . 49275–81 . December 2002 . 12377782 . 10.1074/jbc.M206215200 . free .
• Kallen J, Schlaeppi JM, Bitsch F, Delhon I, Fournier B . Crystal structure of the human RORalpha Ligand binding domain in complex with cholesterol sulfate at 2.2 A . The Journal of Biological Chemistry . 279 . 14 . 14033–8 . April 2004 . 14722075 . 10.1074/jbc.M400302200 . free .
• Migita H, Satozawa N, Lin JH, Morser J, Kawai K . RORalpha1 and RORalpha4 suppress TNF-alpha-induced VCAM-1 and ICAM-1 expression in human endothelial cells . FEBS Letters . 557 . 1–3 . 269–74 . January 2004 . 14741380 . 10.1016/S0014-5793(03)01502-3 . 24388280 . free .
• Miki N, Ikuta M, Matsui T . Hypoxia-induced activation of the retinoic acid receptor-related orphan receptor alpha4 gene by an interaction between hypoxia-inducible factor-1 and Sp1 . The Journal of Biological Chemistry . 279 . 15 . 15025–31 . April 2004 . 14742449 . 10.1074/jbc.M313186200 . free .
• Migita H, Morser J, Kawai K . Rev-erbalpha upregulates NF-kappaB-responsive genes in vascular smooth muscle cells . FEBS Letters . 561 . 1–3 . 69–74 . March 2004 . 15013753 . 10.1016/S0014-5793(04)00118-8 . 84456190 . free .

Notes and References

1. Giguère V, Tini M, Flock G, Ong E, Evans RM, Otulakowski G . Isoform-specific amino-terminal domains dictate DNA-binding properties of ROR alpha, a novel family of orphan hormone nuclear receptors . Genes & Development . 8 . 5 . 538–53 . March 1994 . 7926749 . 10.1101/gad.8.5.538 . free .
2. Sidman RL, Lane PW, Dickie MM . Staggerer, a new mutation in the mouse affecting the cerebellum . Science . 137 . 3530 . 610–2 . August 1962 . 13912552 . 10.1126/science.137.3530.610. 1962Sci...137..610S . 30733570 .
3. Hamilton BA, Frankel WN, Kerrebrock AW, Hawkins TL, FitzHugh W, Kusumi K, Russell LB, Mueller KL, van Berkel V, Birren BW, Kruglyak L, Lander ES . Disruption of the nuclear hormone receptor RORalpha in staggerer mice . Nature . 379 . 6567 . 736–9 . February 1996 . 8602221 . 10.1038/379736a0 . 1996Natur.379..736H . 4318427 .
4. Gold DA, Baek SH, Schork NJ, Rose DW, Larsen DD, Sachs BD, Rosenfeld MG, Hamilton BA . RORalpha coordinates reciprocal signaling in cerebellar development through sonic hedgehog and calcium-dependent pathways . Neuron . 40 . 6 . 1119–31 . December 2003 . 14687547 . 2717708 . 10.1016/s0896-6273(03)00769-4.
5. Halim TY, MacLaren A, Romanish MT, Gold MJ, McNagny KM, Takei F . Retinoic-acid-receptor-related orphan nuclear receptor alpha is required for natural helper cell development and allergic inflammation . Immunity . 37 . 3 . 463–74 . September 2012 . 22981535 . 10.1016/j.immuni.2012.06.012 . free .
6. Gold MJ, Antignano F, Halim TY, Hirota JA, Blanchet MR, Zaph C, Takei F, McNagny KM . Group 2 innate lymphoid cells facilitate sensitization to local, but not systemic, TH2-inducing allergen exposures . The Journal of Allergy and Clinical Immunology . 133 . 4 . 1142–8 . April 2014 . 24679471 . 10.1016/j.jaci.2014.02.033 .
7. Lo BC, Gold MJ, Hughes MR, Antignano F, Valdez Y, Zaph C, Harder KW, McNagny KM . The orphan nuclear receptor ROR alpha and group 3 innate lymphoid cells drive fibrosis in a mouse model of Crohn's disease . Science Immunology . 1 . 3 . eaaf8864 . September 2016 . 28670633 . 5489332 . 10.1126/sciimmunol.aaf8864 .
8. Kojetin DJ, Burris TP . REV-ERB and ROR nuclear receptors as drug targets . Nature Reviews. Drug Discovery . 13 . 3 . 197–216 . March 2014 . 24577401 . 4865262 . 10.1038/nrd4100 .
9. Ko CH, Takahashi JS . Molecular components of the mammalian circadian clock . Human Molecular Genetics . 15 Spec No 2 . 2 . R271-7 . October 2006 . 16987893 . 10.1093/hmg/ddl207 . free . 3762864 .
10. Emery P, Reppert SM . A rhythmic Ror . Neuron . 43 . 4 . 443–6 . August 2004 . 15312644 . 10.1016/j.neuron.2004.08.009 . free .
11. Laitinen S, Staels B . Potential roles of ROR-alpha in cardiovascular endocrinology . Nuclear Receptor Signaling . 1 . e011 . 2003 . 16604183 . 1402228 . 10.1621/nrs.01011 .
12. Zhao X, Cho H, Yu RT, Atkins AR, Downes M, Evans RM . Nuclear receptors rock around the clock . EMBO Reports . 15 . 5 . 518–28 . May 2014 . 24737872 . 4210094 . 10.1002/embr.201338271 .
13. Du J, Huang C, Zhou B, Ziegler SF . Isoform-specific inhibition of ROR alpha-mediated transcriptional activation by human FOXP3 . Journal of Immunology . 180 . 7 . 4785–92 . April 2008 . 18354202 . 10.4049/jimmunol.180.7.4785 . free .
14. Jetten AM . Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular metabolism . Nuclear Receptor Signaling . 7 . e003 . 19381306 . 10.1621/nrs.07003 . 2670432. 2009 .
15. Jetten AM, Kurebayashi S, Ueda E . The ROR nuclear orphan receptor subfamily: critical regulators of multiple biological processes . Progress in Nucleic Acid Research and Molecular Biology . 69 . 205–47 . 2001 . 11550795 . 10.1016/S0079-6603(01)69048-2 . 978-0-12-540069-5 .
16. Jetten AM, Joo JH . Retinoid-related Orphan Receptors (RORs): Roles in Cellular Differentiation and Development . Advances in Developmental Biology . 16 . 313–355 . 18418469 . 2312092 . 10.1016/S1574-3349(06)16010-X . 2006 . 978-0-444-52873-5 .
17. Xiong G, Wang C, Evers BM, Zhou BP, Xu R . RORα suppresses breast tumor invasion by inducing SEMA3F expression . Cancer Research . 72 . 7 . 1728–39 . April 2012 . 22350413 . 3319846 . 10.1158/0008-5472.CAN-11-2762 .
18. Paravicini G, Steinmayr M, André E, Becker-André M . The metastasis suppressor candidate nucleotide diphosphate kinase NM23 specifically interacts with members of the ROR/RZR nuclear orphan receptor subfamily . Biochemical and Biophysical Research Communications . 227 . 1 . 82–7 . October 1996 . 8858107 . 10.1006/bbrc.1996.1471 .
19. Web site: Entrez Gene: RORA RAR-related orphan receptor A.