Study of Arnt-interacting proteins on Arnt-dependent signaling pathways

In an effort to better understand the Ah receptor nuclear translocator (Arnt)-dependent signaling mechanisms, we employed a phage display system to identify Arnt-interacting peptides. Human liver cDNA library was utilized to screen for Arnt-interacting peptides using an Arnt construct fused to thioredoxin (TH-ArntCΔ418). Two clones, namely Ainp1 and Ainp2 (Arnt-interacting peptide), were identified and subsequently characterized. Ainp2 interacted with TH-ArntCΔ418 in the GST pull-down, TALON co-precipitation, and mammalian two-hybrid assays. Northern blot results revealed that Ainp2 is predominantly expressed in human liver. The putative full-length Ainp2 cDNA sequence was subsequently cloned using RACE PCR. Endogenous expression of Ainp2 was found in Jurkat cells and human fetal/adult liver medleys. Results from the transient transfection studies using a DRE- or ERE-driven reporter plasmid and the real-time QPCR experiments examining the endogenous CYP1A1 or GREB-1 expression demonstrated that Ainp2 enhances the 3MC-induced AhR signaling pathway in HepG2 cells, while suppresses the E2-induced ER signaling pathway in MCF-7 cells. These results suggested that Ainp2 plays a role in the Arnt-dependent signaling pathways. The suppressive effect of Ainp2 in the ER signaling pathway was not observed in Arnt-knockdown cells. Additionally, co-precipitation data showed that Ainp2 did not interact with ER α and ER β, suggesting that Ainp2 suppresses the ER signaling via an Arnt-mediated mechanism. The phage display technique also revealed another potential Arnt-interacting peptide Ainp1, which contains an open reading frame of 58 amino acids. The GST pull-down and mammalian two-hybrid assays showed that Ainp1 interacts with TH-ArntCΔ418. Northern blot results demonstrated that Ainp1 is ubiquitously present in all the tested tissues, including brain, placenta, skeletal muscle, heart, kidney, pancreas, liver, lung, spleen, and colon