Cloning and characterization of retinoid X receptor (RXR) isoforms in the rock shell, Thais clavigera.

The organotin compounds tributyltin (TBT) and triphenyltin (TPT) belong to a diverse group of widely distributed environmental pollutants that induce imposex in gastropods. These organotins have high affinity for retinoid X receptor (RXR), which is a transcription factor activated by retinoids, such as 9-cis retinoic acid (9cRA), in vertebrates. However, the molecular mechanisms underlying the regulation of RXR by retinoids and organotins have not been clarified in gastropods. We isolated two isoforms of RXR cDNAs, RXR isoform 1 (TcRXR-1) and RXR isoform 2 (TcRXR-2), in the rock shell Thais clavigera. The deduced amino acid sequences of TcRXR-1 and TcRXR-2 are highly homologous with those of other gastropods. These TcRXR isoforms displayed 9cRA-dependent activation of transcription in a reporter gene assay using COS-1 cells. The transcriptional activity of TcRXR-2, the encoded protein of which has five additional amino acids in the T-box of the C domain, was significantly lower than that of TcRXR-1. Decreases of the transcriptional activity by TcRXR-1 were observed when more than equal amount of TcRXR-2 fused expression vector was existed in a co-transfection assay. Immunoblot analysis showed several shifted bands for TcRXR isoforms resulting from phosphorylation. Mutation of potential phosphorylation sites from serine to alanine in the A/B domain of TcRXR-1 showed that, in the S89A/S103A mutant, there was a band shift and significantly higher transcriptional activity than in the controls when stimulated with 9cRA. Our findings could contribute to a better understanding of the role of interactions between RXR and retinoids and organotins, not only in the induction mechanism of imposex in gastropods but also in the endocrinology of mollusks.