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Poult Sci
2020 Mar 01;993:1643-1654. doi: 10.1016/j.psj.2019.10.062.
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Characterization of a novel thyrotropin-releasing hormone receptor, TRHR3, in chickens.
Li X
,
Li Z
,
Deng Y
,
Zhang J
,
Li J
,
Wang Y
.
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The physiological roles of thyrotropin-releasing hormone (TRH) are proposed to be mediated by TRH receptors (TRHR), which have been divided into 3 subtypes, namely, TRHR1, TRHR2, and TRHR3, in vertebrates. Although 2 TRH receptors (TRHR1 and TRHR3) have been predicted to exist in birds, it remains unclear whether TRHR3 is a functional TRH receptor similar to TRHR1. Here, we reported the functionality and tissue expression of TRHR3 in chickens. The cloned chicken TRHR3 (cTRHR3) encodes a receptor of 387 amino acids, which shares high-amino-acid identities (63-80%) to TRHR3 of parrots, lizards, Xenopus tropicalis, and tilapia and comparatively lower sequence identities to chicken TRHR1 or mouse TRHR2. Using cell-based luciferase reporter assays and Western blot, we demonstrated that similar to chicken TRHR1 (cTRHR1), cTRHR3 expressed in HEK 293 cells can be potently activated by TRH and that its activation stimulates multiple signaling pathways, indicating both TRH receptors are functional. Quantitative real-time PCR revealed that cTRHR1 and cTRHR3 are widely, but differentially, expressed in chicken tissues, and their expression is likely controlled by promoters located upstream of exon 1, which display strong promoter activities in cultured DF-1 cells. cTRHR1 is highly expressed in the anteriorpituitary and testes, while cTRHR3 is highly expressed in the muscle, testes, fat, pituitary, spinal cord, and many brain regions (including hypothalamus). These findings indicate that TRH actions are likely mediated by 2 TRH receptors in chickens. In conclusion, our data provide the first piece of evidence that both cTRHR3 and cTRHR1 are functional TRH receptors, which helps to elucidate the physiological roles of TRH in birds.
Figure 1. (A) The full-length cDNA and amino acid sequences of chicken TRHR3. (B) Exon organization of chicken TRHR1 and TRHR3 genes. Numbers in the boxes indicate the size (bp) of the coding region (shaded) and noncoding region. Numbers under the broken lines indicate the size (bp) of the introns.
Figure 2. Amino acid alignment of chicken TRHR3 (MK138989) with parrot TRHR3 (XP_005146871.1), lizard TRHR3 (XP_003220685.1), Xenopus tropicalis TRHR3 (XP_002933212.1), tilapia TRHR3 (XP_003439144.1), mouse TRHR2 (NP_573465.1), chicken TRHR1 (NP_990261.1), and human TRHR1 (NP_003292). The seven transmembrane domains (TMD1-7) are shaded. The ERY motif is boxed, and the 4 conserved amino acid residues (Tyr108, Asn112, Tyr277, Arg301) associated with ligand binding are marked with black arrows. Note: dots indicate the amino acids identical to chicken TRHR3, and dashes denote the gaps in the sequence.
Figure 3. (A) Detection of promoter activities of the 5′-flanking region of chicken TRHR1 and TRHR3 in cultured DF-1 cells. Various stretches of the 5′-flanking regions of TRHR1 and TRHR3 were cloned into pGL3-Basic vector for the generation of multiple promoter–luciferase constructs. The promoter–luciferase vector and pRL-TK vector were cotransfected into DF-1 cells, and the promoter activities were detected by the dual-luciferase reporter system. All data represent the mean ± SEM of 4 replicates (N = 4). ***P < 0.001 vs. pGL3-Basic vector. The transcriptional start site (A/G, boxed) identified by rapid amplification of 5′ cDNA ends (5′-RACE) was designated as '+1'. (B) Predicted transcription factor binding sites in the promoter regions of cTRHR1 and cTRHR3. The corresponding binding sites are shaded. Sp1, specificity protein 1; SRY, sex-determining region; AP4, activating enhancer–binding protein 4; MZF1, myeloid zinc finger 1; STAT3, signal transducer and activator of transcription 3; CEBPA, CCAAT/enhancer-binding protein alpha.
Figure 4. (A–C) Effects of chicken thyrotropin-releasing hormone (TRH) on activating chicken TRHR3, as monitored by the (A) pGL3-NFAT-RE-luciferase reporter system, (B) pGL4-SRE-luciferase reporter system, and (C) pGL3-CRE-luciferase reporter system. All data represent the mean ± SEM of 3 replicates (N = 3). (D) Effects of 2-aminoethoxydiphenyl borate (2-APB) on TRH-induced luciferase activities of HEK 293 cells expressing chicken TRHR3, as monitored by the pGL3-NFAT-RE-luciferase reporter system. 2-APB (100 μM) was added 0.5 h before TRH treatment. T represents TRH treatment (10 nM, 6 h), and C represents control group without TRH treatment. All data represent the mean ± SEM of 3 replicates (N = 3). **P < 0.01 vs. control; ##P < 0.01 between 2 treatment groups. (E) Western blot detection of ERK1/2 phosphorylation (pERK1/2) and total ERK1/2 in HEK 293 cells expressing cTRHR3 treated by cTRH (10 nM) and without TRH (control) for 10 min.
Figure 5. (A–C) Effects of chicken thyrotropin-releasing hormone (TRH) on activating chicken TRHR1, as monitored by the (A) pGL3-NFAT-RE-luciferase reporter system, (B) pGL4-SRE-luciferase reporter system, and (C) pGL3-CRE-luciferase reporter system. All data represent the mean ± SEM of 3 replicates (N = 3). (D) Effects of 2-aminoethoxydiphenyl borate (2-APB), H89, and MDL-12330A (MDL) on TRH-induced luciferase activities of HEK 293 cells expressing chicken TRHR1, as monitored by the pGL3-NFAT-RE-luciferase reporter system and pGL3-CRE-luciferase reporter system. 2-APB (100 μM), H89 (10 μM) or MDL (20 μM) was added 0.5 h before TRH treatment. T represents TRH treatment (10 nM, 6 h), and C represents control group without TRH treatment. All data represent the mean ± SEM of 3 replicates (N = 3). **P < 0.01 vs. control; ##P < 0.01 between 2 groups. (E) Western blot detection of ERK1/2 phosphorylation (pERK1/2) and total ERK1/2 in HEK 293 cells expressing cTRHR1 treated by TRH (10 nM) and without TRH (control) for 10 min.
Figure 6. Schematic diagram showing the similarities and differences of the downstream signaling pathways of cTRHR1 and cTRHR3 when activated by thyrotropin-releasing hormone (TRH). Both receptors can couple to Gq protein, and their activation stimulates intracellular calcium mobilization and MAPK/ERK signaling pathway. Unlike cTRHR3, cTRHR1 can also couple to Gs protein and stimulate adenylate cyclase/cAMP/PKA signaling pathway.
Figure 7. Quantitative real-time PCR assay of cTRH, cTRHR3, and cTRHR1 mRNA expression in adult chicken tissues, including the telencephalon (Tc), midbrain (Mb), cerebellum (Cb), hindbrain (Hb), hypothalamus (Hp), heart (He), kidneys (Ki), liver (Li), lung (Lu), muscle (Mu), ovary (Ov), testes (Te), anteriorpituitary (Pi), spleen (Sp), pancreas (Pa), subcutaneous fat (Fat), spinal cord (Sc), skin (Sk), duodenum (Du), crop (Cp), proventriculus (Pr), gizzard (Gi), jejunum (Je), ileum (Ie), cecum (Ce), and colon (Co). The mRNA level of each gene was normalized with the mRNA level of β-actin as an internal control and expressed as the fold difference compared with that of telencephalon (Tc). All data represent the mean ± SEM of 6 individual adult chickens (3 males and 3 females) (N = 6).
Aoki,
Molecular evolution of prepro-thyrotropin-releasing hormone in the chicken (Gallus gallus) and its expression in the brain.
2007, Pubmed
Aoki,
Molecular evolution of prepro-thyrotropin-releasing hormone in the chicken (Gallus gallus) and its expression in the brain.
2007,
Pubmed
Bidaud,
Characterization and functional expression of cDNAs encoding thyrotropin-releasing hormone receptor from Xenopus laevis.
2002,
Pubmed
,
Xenbase
Boler,
The identity of chemical and hormonal properties of the thyrotropin releasing hormone and pyroglutamyl-histidyl-proline amide.
1969,
Pubmed
Burgus,
Characterization of ovine hypothalamic hypophysiotropic TSH-releasing factor.
1970,
Pubmed
Cai,
Characterization of the Two CART Genes (CART1 and CART2) in Chickens (Gallus gallus).
2015,
Pubmed
De Groef,
Corticotropin-releasing hormone (CRH)-induced thyrotropin release is directly mediated through CRH receptor type 2 on thyrotropes.
2003,
Pubmed
Duthie,
Cloning and functional characterisation of the human TRH receptor.
1993,
Pubmed
Engel,
Thyrotropin-releasing hormone and its receptors--a hypothesis for binding and receptor activation.
2007,
Pubmed
Geris,
Distribution of somatostatin in the brain and of somatostatin and thyrotropin-releasing hormone in peripheral tissues of the chicken.
2000,
Pubmed
Gershengorn,
Molecular and cellular biology of thyrotropin-releasing hormone receptors.
1996,
Pubmed
Gáspár,
Thyrotropin-releasing hormone selectively stimulates human hair follicle pigmentation.
2011,
Pubmed
Gáspár,
Thyrotropin releasing hormone (TRH): a new player in human hair-growth control.
2010,
Pubmed
Harder,
Cloning of two thyrotropin-releasing hormone receptor subtypes from a lower vertebrate (Catostomus commersoni): functional expression, gene structure, and evolution.
2001,
Pubmed
,
Xenbase
Harvey,
The effect of thyrotropin-releasing hormone (TRH) and somatostatin (GHRIH) on growth hormone and prolactin secretion in vitro and in vivo in the domestic fowl (Gallus domesticus).
1978,
Pubmed
Harvey,
Thyrotrophin-releasing hormone: a growth hormone-releasing factor.
1990,
Pubmed
He,
Molecular characterization of three NPY receptors (Y2, Y5 and Y7) in chickens: Gene structure, tissue expression, promoter identification, and functional analysis.
2016,
Pubmed
Heuer,
Expression of thyrotropin-releasing hormone receptor 2 (TRH-R2) in the central nervous system of rats.
2000,
Pubmed
Huang,
Glucagon-like peptide (GCGL) is a novel potential TSH-releasing factor (TRF) in Chickens: I) Evidence for its potent and specific action on stimulating TSH mRNA expression and secretion in the pituitary.
2014,
Pubmed
International Chicken Genome Sequencing Consortium,
Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution.
2004,
Pubmed
Itadani,
Cloning and characterization of a new subtype of thyrotropin-releasing hormone receptors.
1998,
Pubmed
Kanasaki,
Mitogen-activated protein kinase activation by stimulation with thyrotropin-releasing hormone in rat pituitary GH3 cells.
1999,
Pubmed
Kumar,
A molecular timescale for vertebrate evolution.
1998,
Pubmed
Lamacz,
Three distinct thyrotropin-releasing hormone-immunoreactive axonal systems project in the median eminence-pituitary complex of the frog Rana ridibunda. Immunocytochemical evidence for co-localization of thyrotropin-releasing hormone and mesotocin in fibers innervating pars intermedia cells.
1989,
Pubmed
Lechan,
Thyrotropin-releasing hormone precursor: characterization in rat brain.
1986,
Pubmed
Liu,
Genome-wide association and replication studies identified TRHR as an important gene for lean body mass.
2009,
Pubmed
Lu,
Pharmacological studies of thyrotropin-releasing hormone (TRH) receptors from Xenopus laevis: is xTRHR3 a TRH receptor?
2003,
Pubmed
,
Xenbase
Marangell,
Effects of intrathecal thyrotropin-releasing hormone (protirelin) in refractory depressed patients.
1997,
Pubmed
Meier,
Thyrotropin-releasing hormone (TRH) promotes wound re-epithelialisation in frog and human skin.
2013,
Pubmed
,
Xenbase
Mekuchi,
Molecular cloning, gene structure, molecular evolution and expression analyses of thyrotropin-releasing hormone receptors from medaka (Oryzias latipes).
2011,
Pubmed
Mo,
Corticotropin-releasing hormone (CRH) stimulates cocaine- and amphetamine-regulated transcript gene (CART1) expression through CRH type 1 receptor (CRHR1) in chicken anterior pituitary.
2015,
Pubmed
Mo,
Characterization of NMB, GRP and their receptors (BRS3, NMBR and GRPR) in chickens.
2017,
Pubmed
Nakano,
Possible involvement of thyrotropin-releasing hormone receptor 3 in the release of prolactin in the metamorphosing bullfrog larvae.
2018,
Pubmed
O'Dowd,
TRH-R2 exhibits similar binding and acute signaling but distinct regulation and anatomic distribution compared with TRH-R1.
2000,
Pubmed
Paulssen,
The thyroliberin receptor interacts directly with a stimulatory guanine-nucleotide-binding protein in the activation of adenylyl cyclase in GH3 rat pituitary tumour cells. Evidence obtained by the use of antisense RNA inhibition and immunoblocking of the stimulatory guanine-nucleotide-binding protein.
1992,
Pubmed
Péczely,
Immunoreactivity to vasoactive intestinal polypeptide (VIP) and thyreotropin-releasing hormone (TRH) in hypothalamic neurons of the domesticated pigeon (Columba livia). Alterations following lactation and exposure to cold.
1988,
Pubmed
Rabeler,
Generation of thyrotropin-releasing hormone receptor 1-deficient mice as an animal model of central hypothyroidism.
2004,
Pubmed
Richter,
Biosynthesis of thyrotropin releasing hormone in the skin of Xenopus laevis: partial sequence of the precursor deduced from cloned cDNA.
1984,
Pubmed
,
Xenbase
Satoh,
Identification of thyrotropin-releasing hormone receptor in the rat testis.
1994,
Pubmed
Scanes,
Some in vitro effects of synthetic thyrotrophin releasing factor on the secretion of thyroid stimulating hormone from the anterior pituitary gland of the domestic fowl.
1974,
Pubmed
Segerson,
Localization of thyrotropin-releasing hormone prohormone messenger ribonucleic acid in rat brain in situ hybridization.
1987,
Pubmed
Straub,
Expression cloning of a cDNA encoding the mouse pituitary thyrotropin-releasing hormone receptor.
1990,
Pubmed
,
Xenbase
Sun,
Cloning and characterization of the chicken thyrotropin-releasing hormone receptor.
1998,
Pubmed
Tashjian,
Thyrotropin releasing hormone: direct evidence for stimulation of prolactin production by pituitary cells in culture.
1971,
Pubmed
Taylor,
The paraventricular nucleus of the hypothalamus has a major role in thyroid hormone feedback regulation of thyrotropin synthesis and secretion.
1990,
Pubmed
Vandenborne,
Cloning and hypothalamic distribution of the chicken thyrotropin-releasing hormone precursor cDNA.
2005,
Pubmed
Wang,
Identification of the receptors for prolactin-releasing peptide (PrRP) and Carassius RFamide peptide (C-RFa) in chickens.
2012,
Pubmed
,
Xenbase
Wang,
A novel prolactin-like protein (PRL-L) gene in chickens and zebrafish: cloning and characterization of its tissue expression.
2010,
Pubmed
,
Xenbase
Wang,
Identification of the endogenous ligands for chicken growth hormone-releasing hormone (GHRH) receptor: evidence for a separate gene encoding GHRH in submammalian vertebrates.
2007,
Pubmed
,
Xenbase
Wilber,
The thyrotropin-releasing hormone gene 1998: cloning, characterization, and transcriptional regulation in the central nervous system, heart, and testis.
1998,
Pubmed
