The sequences of the mouse and rat TRH receptors (TRH-Rs) show 94% similarity at the protein level. However, they differ significantly at their carboxy terminals, i.e. the mouse TRH-R ends with an asparagine at position 393 while, in the rat, residue 393 is lysine and an additional 19 amino acids are added before the first stop codon. In the mouse cDNA, the sequence encoding these additional amino acids is located 224 bp downstream in the 3′ untranslated region (3′UT). As the mouse TRH-R was cloned from thyrotrope-derived TtT97 tissue and the rat TRH-R from lactotrope-derived GH cell lines, we have investigated whether this difference at the carboxyterminus represents a species-specific or cell type-specific pattern of TRH-R expression. Total RNA was isolated from mouse pituitary and TtT97 tissue, and rat pituitary and GH3 cells. Reverse transcription PCR analysis was performed using primers that would generate DNA fragments including the stop codon in either the mouse or the rat TRH-R and, in the mouse form, the extra 224 bp of 3′UT. This would generate a product of 234 bp from the rat sequence and 441 bp from the mouse sequence. In rat pituitary and GH3 cDNA, PCR generated the expected 234 bp product but not a band representing the mouse sequence. In both mouse pituitary and TtT97 cDNA, neither the expected 441 bp nor the 234 bp fragments were amplified; instead a larger, 829 bp, product was generated. Sequence analysis revealed a 388 bp insertion at position 1663 in the 3′UT compared with the published mouse TRH-R sequence. Ribonuclease protection analysis using this 829 bp fragment as a probe showed that this sequence represented the major TRH-R mRNA species in mouse pituitary and TtT97 RNA. A genomic clone containing this region of the mouse TRH-R gene was isolated and analysis of the sequence in this region revealed that this longer form of the mouse TRH-R could be generated by alternative splicing.
In summary, we have shown that the carboxyterminal differences between the mouse and rat TRH-Rs are species-specific rather than cell typespecific, and that the major TRH-R mRNA expressed in mouse pituitary contains an additional 388 bp of 3′UT compared with the published sequence. As a region in the 3′UT of the published mTRH-R sequence has been shown to be important for stability of this mRNA, this additional 3′UT sequence could have major effects on the regulation and stability of the mouse TRH-R mRNA.