Cloning and identification of a novel thyroid hormone receptor β isoform expressed in the pituitary gland

We have previously identified a novel Tr beta isoform (Tr beta I") in the rat, in which a novel exon N (108 bps) was found between exon 3 and exon 4 of Tr beta I", which represents the only difference between Tr beta I" and Tr beta 1. In this study, we searched for an elongated Tr beta 2-like subtype with one additional exon N. We successfully isolated the entire mRNA/cDNA of a novel elongated Tr beta 2 isoform via PCR in the rat pituitary gland. The mRNA/cDNA was only 108 bps (exon N) longer than that Tr beta 2, and the extension of the sequence was between exon 3 and 4 of Tr beta. The whole sequence of this novel Tr beta isoform has been published in NCBI GenBank (HM043807.1); it is named TRbeta2Delta (Tr beta 2 Delta). In adult rat pituitary tissue, quantitative real-time RT-PCR analysis showed that the mRNA levels of Tr beta 2 Delta and Tr beta 2 were roughly equal (P > 0.05). We cloned, expressed, and purified the His-Tr beta 2 Delta protein [recombinant TR beta 2 Delta (rTR beta 2 Delta)]. SDS-PAGE and western blotting revealed that the molecular weight of rTR beta 2 Delta was 58.2 kDa. Using a radioligand binding assay and an electrophoretic mobility shift assay, rTR beta 2 Delta-bound T3 with high affinity and recognized thyroid hormone response element (TRE) binding sites. Finally, in vitro transfection experiments further confirmed that rTR beta 2 Delta binding T3 significantly promotes the transcription of target genes via the TRE. Here, we have provided evidence suggesting that rTR beta 2 Delta is a novel functional TR isoform.