XB-ART-57316
Poult Sci
March 1, 2020;
99
(3):
1643-1654.
Characterization of a novel thyrotropin-releasing hormone receptor, TRHR3, in chickens.
Li X
,
Li Z
,
Deng Y
,
Zhang J
,
Li J
,
Wang Y
.
Abstract
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
anterior pituitary 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.
PubMed ID:
32115036
PMC ID:
PMC7587745
Article link:
Poult Sci
Species referenced:
Xenopus laevis
Genes referenced:
cebpa
mzf1
nfatc2
prl.2
sox13
sp1
stat3.2
trh
trhr
trhr2
trhr3
Article Images:
[+] show captions
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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.
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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.
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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.
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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), anterior pituitary (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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