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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).
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