AN ALTERNATIVE SPLICE VARIANT OF THE MOUSE TRH RECEPTOR MESSENGER-RNAIS THE MAJOR FORM EXPRESSED IN THE MOUSE PITUITARY-GLAND

The sequences of the mouse and rat TRH receptors (TRH-Rs) show 94% sim ilarity at the protein level. However, they differ significantly at th eir 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 addition al 19 amino acids are added before the first stop codon. In the mouse cDNA, the sequence encoding these additional amino acids is located 22 4 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 f rom lactotrope-derived GH cell lines, we have investigated whether thi s difference at the carboxy terminus 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 GH(3) ce lls. Reverse transcription PCR analysis was performed using primers th at 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 GH, cDNA, PCR gen erated the expected 234 bp product but not a band representing the mou se sequence. In both mouse pituitary and TtT97 cDNA, neither the expec ted 441 bp nor the 234 bp fragments were amplified; instead a larger, 829 bp, product was generated. Sequence analysis revealed a 388 bp ins ertion at position 1663 in the 3'UT compared with the published mouse TRH-R sequence. Ribonuclease protection analysis using this 829 bp fra gment as a probe showed that this sequence represented the major TRH-R mRNA species in mouse pituitary and TtT97 RNA. A genomic clone contai ning 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 mous e TRH-R could be generated by alternative splicing. In summary, we hav e shown that the carboxyterminal differences between the mouse and rat TRH-Rs are species-specific rather than cell type-specific, and that the major TRH-R mRNA expressed in mouse pituitary contains an addition al 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 import ant for stability of this mRNA, this additional 3'UT sequence could ha ve major effects on the regulation and stability of the mouse TRH-R mR NA.