Accession Number:

ADA525216

Title:

The Genomic Actions and Functional Implications of Nuclear PRLr in Human Breast Carcinoma

Descriptive Note:

Annual summary rept. 1 Mar 2009-28 Feb 2010

Corporate Author:

NORTHWESTERN UNIV EVANSTON IL

Report Date:

2010-03-01

Pagination or Media Count:

41.0

Abstract:

The prolactin receptor PRLr is a cytokine receptor that binds prolactin PRL via its extracellular domain and consequently signals through the Jak2Stat5 pathway. The canonical function of PRLr in this pathway is to serve as a membrane bound intermediary between PRL and downstream secondary messengers. However, recent data from our lab has revealed that PRLr localizes to the nucleus of breast cancer cells tissue. Preliminary experiments also demonstrated that PRL initiates nuclear localization of PRLr and the interaction between nuclear PRLr and the transcription factor, Stat5a. We therefore hypothesized that PRLr functions as a Stat5a coactivator. Investigation of this hypothesis via ChIP revealed that PRLr binds to the Stat5 responsive CISH promoter and reporter assays demonstrated that PRLr promoter engagement results in CISH activation. As these results pointed to a role for the PRLr in transactivation, we utilized the Gal4 assay, to determine that residues 404-448 of PRLr intracellular domain were necessary and sufficient for transactivation. Consequentially, mutation of two conserved residues Y406, and D411 disrupted transactivation, demonstrating the necessity of these residues for PRLr specific gene activation. In addition, immunoprecipitation analysis led us to reveal a novel finding that the PRLr transactivation domain is necessary for recruitment of the chromatin modifying protein, HMGN2. Subsequent studies demonstrated that HMGN2 is also recruited to the Stat5a-driven CISH promoter in a PRL dependant manner. In addition, overexpression of HMGN2 enhances Stat5a driven CISH expression, while stable knockdown of HMGN2 impairs this expression. HMGN2 knockdown also prevents binding of the PRLr to the CISH promoter suggesting that HMGN2 is required to PRLr promoter binding. Since HMGN2 possesses no sequence specificity, our findings suggest that nuclear PRLr may serve as the adaptor mresulting in full transcri

Subject Categories:

  • Biochemistry
  • Medicine and Medical Research

Distribution Statement:

APPROVED FOR PUBLIC RELEASE