Cloning of two thyrotropin-releasing hormone receptor subtypes from a lower vertebrate (Catostomus commersoni): Functional expression, gene structure, and evolution

A PCR approach was used to clone thyrotropin-releasing hormone receptors (T RH-R) from the brain and anterior pituitary of the teleost Catostomus comme rsoni (cc), the white sucker. Two distinct TRH-R, designated ccTRH-R-1 and ccTRH-R-2, were identified. ccTRH-R-1 was similar to mammalian TRH-R of the subtype 1, whereas ccTRH-R2 exhibited the highest identity (61% at the ami no acid level) with the recently discovered rat TRH-R-2. It is postulated t hat ccTRH-R-2 and rat TRH-R-2 are members of the same TRH-R subfamily 2. Fu nctional expression of ccTRH receptors in human embryonic kidney cells and in Xenopus laevis oocytes demonstrated that both ccTRH receptors were fully functional in both systems. Oocytes expressing either receptor responded t o the application of TRH by an induction of membrane chloride currents, ind icating that ccTRH-R of both subtypes are coupled to the inositol phosphate /calcium pathway. The analysis of genomic clones revealed, for the first ti me, both similarities and differences in the structure of TRH-R subtype gen es. Both ccTRH-R genes contained an intron within the coding region at the beginning of transmembrane domain (TM) 6. The position of this intron is hi ghly conserved, as it was found at an identical position in the human TRH-R -1 gene. The ccTRH-R-2 gene contained an additional intron at the end of TM 3 that was not found in any of the TRH-Ri genes identified so far. The ana lysis of the gene structure of ccTRH-R and the amino acid sequence comparis ons of mammalian and teleost TRH-R of both subtypes suggest that TRH recept ors have been highly conserved during the course of vertebrate evolution. A common ancestral TRH receptor gene that could be found much earlier in evo lution, possibly in invertebrates, might be the origin of ccTRH-R genes. (C ) 2001 Academic Press.