A New And Er¦Â Interacting Protein Erip2 Functional Studies

Estrogen receptor (ER) is a member of the nuclear receptor superfamily that regulates the transcription of estrogen-responsive genes and plays an important role in the development of breast cancer. Recently ER is thought to act as a target of the therapy of breast cancer and a good index of prognosis. ER contains two subtypes: ERαand ERβ. They are both subdivided into six parts (A-F), which contain two transactive regions, called AF1 and AF2, respectively. AF1 locates in the A/B region, which has ligand-independent transactive function. And AF2 locates in the E/F region, with ligand-dependent transactive function. The effective transactivation of AF1 and AF2 depends on their interaction with other proteins. Until now, the endocrine therapy of mammary carcinoma is acquired mainly by decreasing ER transcriptional activity. Therefore, discovering and characterizing the new proteins that modulate ER activity is of great significance in developing the drugs of estrogen-related diseases.In order to deeply research the way that ERβacts in the the transcriptional regulation of estrogen-responsive genes, a novel ERβ-interacting protein named ERIP2 was isolated by yeast two-hybrid screen from a human mammary library with ERβAF2 as bait. As a new gene, ERIP2 encodes 334 amino acids, most of which are arginine and serine. However, its function is unknown. Bioinformatics analysis showed that amino acid residues 180-224 of ERIP2 can form a coiled coil structure, which may interact with other proteins. Additionally, ERIP2 might have three sumoylation sites. So we identified several sumoylation-related proteins-SUMO1, UBC9 and PIAS, from the human mammary library with ERIP2 as bait.To further confirm the specific interaction both in vitro and in vivo between ERIP2 and sumoylation-related proteins as well as ERβ, we constructed some related expression vectors, and performed GST pull-down and co-immunoprecipitation(Co-IP) assays. The results verified their interaction, consistent with that of yeast two-hybrid screens. Furthermore, the domain in ERIP2 binding to ERβwas mapped to the region of amino acid residues 121-224 by co-immunoprecipitation assay. In addition, we found that ERIP2 interacted only with ERβin an estrogen–independent manner, but did not bind to ERα. That is very interesting, so the research will become very significant to estrogen-related diseases such as breast cancer.To investigate the sumoylation about ERIP2 and ER, Western blotting was done. The result further indicated that both ERIP2 and ER may be sumoylated in the cell. Furthermore, Co-IP assay showed that sumoylation severely weakened the binding between ERIP2 and ERβ. We presumed that it was possibly caused by the competition for SUMO between ERIP2 and ERβ.Since ER plays an important role in the estrogenic signal pathway, ERIP2 possibly participates in regulation of the way by binding to ER. So ERE-containing luciferase reporter assay was used to determine the effects of ERIP2 on the transcription activity of ER in this study. We found that ERIP2 could enhance the transcriptional activity in 293T cells while inhibiting the activity of breast cancer cells in a dose-dependent manner. That is to say, the effect of ERIP2 is cell-specific. Meanwhile the changing trend was found different with diverse luciferase reporter genes. In addition, we also observed that sumoylation could enhance the transcriptional activity of ER, which surpassed the effect of ERIP2.To further understand the function of ERIP2, we made the polyclonal antibody of ERIP2 by immuning Balb/c mouse. Western blotting analysis indicated that the antibody is characteristic of high specificity and high titer. Then we detected the distribution of ERIP2 in rat tissues and various breast cancer cells using the high specific polyclonal anti-ERIP2 antibody. The result suggested ERIP2 might have a universal effect in diverse conditions.Taken together, these results identify a new co-regulator of ER, ERIP2, which might be sumoylated in the cell. ERIP2 possibly plays an important role in estrogen signaling pathway through the interaction with ERβand sumoylation-related proteins. Therefore further research will help us understand its specific regulating mechanism in estrogen signal pathway and expand the proteomics study on sumoylation.