Plasmids expressing different Gal4 fusion proteins were used in mating. localization of AhR (Kazlauskas role for XAP2. Results and Discussion XAP2 shows TR1-specific functional interactions To identify new TR1 partners, we used a yeast two-hybrid system. Full-length mouse TR1, fused to the GAL4 DNA-binding domain in pGBKT7, was used to screen a mouse PVN cDNA library fused to the GAL4 activation domain; the library was screened with and without T3 (1 M). XAP2 was isolated in the presence of T3. TR1 and XAP2 interactions in yeast Rabbit Polyclonal to CSGLCAT were T3 dependent and TR isoform specific (Fig 1A). No other TR isoform tested (TR2 and TR1) interacted with XAP2 with or without T3, but all TRs tested interacted with a known partner, RXR (Fig 1A). Open in a separate window Figure 1 Functional interactions between hepatitis virus B X-associated protein 2 and thyroid hormone receptor in yeast. (A) XAP2: two-hybrid experiments in yeast show T3-dependent specific interactions of XAP2 with TR1 but not with TR1 or TR2. Cterm: when the carboxy-terminal region of XAP2 is deleted, the interaction between TR1 and XAP2 is lost. RXR: positive control of interaction with each TR. (B) XAP2 and Befetupitant TR1 interactions in yeast depend on T3 concentration. Plasmids expressing different Gal4 fusion proteins were used in mating. Diploids were grown with increasing doses of T3 in selection medium. -Galactosidase (-Gal) activity was measured by densitometry. Each bar represents the average of two different transformants. T3, triiodothyronine; TR, thyroid hormone receptor; TR1, TR1, TR2, TR subtypes; XAP2, hepatitis virus B X-associated protein 2. To further analyse TR1 and XAP2 interactions, we deleted three conserved domains in the carboxy-terminal end of XAP2, the so-called tetratricopeptide repeats (TPR; CtermXAP2 construct). These domains show high homologies with those of the steroid hormone receptor-interacting immunophilin FKBP52 (Carver & Bradfield, 1997; Ma & Whitlock, 1997; Meyer in mammalian cells was described by Carver & Bradfield (1997) for XAP2 and AhR interactions. Open in a separate window Figure 2 Hepatitis virus B X-associated protein 2CTR1 interactions in mammalian cells. Befetupitant (A) XAP2 interacts with TR1 (but not with TR) in mammalian cells. P19 cells were incubated in the absence or presence of 1 1 M of T3 for 2 h. Cells were then collected and whole-cell extracts (WCEs; lane 1) prepared. Extracts were used for immunoprecipitation using IgG (lane 2) or no antibody (lane 3) as controls, and TR1 (lanes 4,5) and TR antibodies (lanes 6,7). The presence of XAP2 was examined by western blot analysis using XAP2 antibody. (B, C): Both TR1 (B) and TR (C) are present in the P19 WCE. Samples (100 g) of the WCE of the P19 cells used for the above experiment were fractionated on a Befetupitant 10% SDSCpolyacrylamide gel electrophoresis gel. The levels of TR1 and TR were detected by western blot analysis using TR1 and TR antibodies, showing that both receptors were expressed in these cells. -Actin was used as loading control. T3, triiodothyronine; TR, TR1, thyroid hormone receptor subtypes; XAP2, hepatitis virus B X-associated protein 2. XAP2 colocalizes with TRH and TR1 in the PVN We next determined whether XAP2 was expressed in the PVN and whether it colocalized with TRH and TR1, as colocalization of TRH and TR1 had already been documented by Lechan (1994). Using hybridization (supplementary information and supplementary Fig S1A online), we found that TRH and XAP2 transcripts were expressed in the same regions of the PVN, and XAP2 seemed to be more.
