A Four-Amino-Acid Insertion in the Ligand-Binding Domain Inactivates hRXRβ and Renders Dominant Negative Activity

Abstract

Retinoid X receptors (RXRs) are members of the steroid and thyroid hormone receptor superfamily of hormone-dependent transcription factors that mediate the pleiotropic effect of retinoids. Here, we report the initial characterization of an isoform of hRXRβ, termed hRXRβ3, which was previously identified as an H-2RI-IBP isoform (Epplen and Epplen, 1992). The hRXRβ3 isoform cotains an in-frame insertion of four amino acids (SLSR) in the ligand binding domain at codon 419. The isoform is generated by alternate use of a 3′ splice acceptor site and was detectable by reverse transcription polymerase chain reaction (RT-PCR) in all human tumor cell lines and mouse tissues examined. Chimeric receptors, in which the ligand-binding domain of hRXRα was substituted by the corresponding domain from hRXRβ3, were used to investigate the consequences of the SLSR insertion on the transactivation and DNA-binding functions of the chimeric receptor. Co-transfection assays revealed that a chimera RXRα/β3 receptor failed to transactivate the RXR-specific CRBPII promoter, whereas the identical chimera lacking the SLSR insertion was active. The RXRα/β3 receptor exhibited dominant negative activity against active retinoid X and retinoic acid receptors on retinoidresponsive promoters. Moreover, the RXRα/β3 protein failed to interact physically with the retinoic acid receptor (RAR) to form heterodimers as detected by physical association assays, and failed to bind DNA containing an RAR-responsive element. Therefore, this suggests that the SLSR insertion in the ligand-binding domain of the RXRα/β3 receptor is responsible for the altered behavior of the chimera. Our findings raise the possibility that RXRα/β3, and perhaps hRXRβ3 isoform, function by titrating a limiting adaptor molecule that is involved in mediating retinoid function.