Characterization of thyrotropin-releasing hormone (TRH) and its receptors (TRHRs) in Nile tilapia: Molecular identification, ligand-receptor interaction and expression profile

Abstract

Thyrotropin-releasing hormone (TRH) is a highly conserved tripeptide across vertebrates and regulates various biological processes, including energy metabolism, appetite, and reproduction. The functions of TRH are mediated by TRH receptors (TRHRs). In vertebrates, three TRHR subtypes have been identified, namely TRHR1, TRHR2, and TRHR3. However, TRHR2 and TRHR3 are often lost in some vertebrate lineages, highlighting the evolutionary divergence of the TRH-TRHR system. Although extensive research has been conducted in mammals, studies concerning the biological roles of TRH-TRHR system remain limited in fish. Therefore, using Nile tilapia (ti-) as a teleost model, we cloned the full-length cDNA of TRH and its receptors. Based on sequence alignment, synteny analysis and phylogenetic tree construction, we observed that TRHR2 has been lost in Nile tilapia. The cloned tiTRHRs were designated as tiTRHR1a, tiTRHR1b, and tiTRHR3. Using luciferase reporter assays, signal pathway inhibitors and western blot analysis, we demonstrated that tiTRH could effectively activate tiTRHR1a, tiTRHR1b, and tiTRHR3, leading to the stimulation of intracellular calcium mobilization, MAPK/ERK, and cAMP/PKA signaling cascades. However, the efficiencies in activating signaling pathways differed between tiTRHR subtypes upon tiTRH treatment. Quantitative real-time PCR revealed that tiTRH and tiTRHRs were mainly expressed in the central nervous system (CNS) including the hypothalamus. Moreover, hypothalamic mRNA levels of tiTRH and tiTRHR1b were significantly downregulated in response to short-term fasting and acute cold exposure, while tiTRHR1a expression was only responsive to acute cold stress. Collectively, our data will facilitate a better understanding of the components and functions of the TRH-TRHR system in teleost.