DRIP150 Coactivation of Estrogen Receptor α in ZR-75 Breast Cancer Cells Is Independent of LXXLL Motifs

• Published in: The Journal of biological chemistry Vol. 280; no. 10; pp. 8819 - 8830
• Main Authors: Lee, Jeongeun Eun, Kim, Kyounghyun, Sacchettini, James C., Smith, Clare V., Safe, Stephen
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 11-03-2005
• Subjects: Amino Acid Sequence; Base Sequence; Binding Sites; Breast Neoplasms; Cell Line, Tumor; DNA Primers; Estrogen Receptor alpha - chemistry; Estrogen Receptor alpha - genetics; Estrogen Receptor alpha - physiology; Female; Humans; Mediator Complex; Molecular Sequence Data; Trans-Activators - chemistry; Trans-Activators - genetics; Trans-Activators - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M413184200

Vitamin D receptor-interacting protein 150 (DRIP150) has been identified as part of mediator-like complexes that enhance transcriptional activation of the estrogen receptor (ER) and other nuclear receptors (NRs). DRIP150 coactivates ligand-dependent ERα-mediated transactivation in ZR-75 and MDA-MB-231 breast cancer cells transfected with a (luciferase) reporter construct (pERE3) regulated by three tandem estrogen-responsive elements. Coactivation of ERα by DRIP150 in ZR-75 cells was activation function 2-dependent and required an intact helix 12 that typically interacts with LXXLL motifs (NR box) in p160 steroid receptor coactivators. DRIP150 contains C- and N-terminal NR boxes (amino acids 1182–1186 and 69–73, respectively), and deletion analysis of DRIP150 showed that regions containing these sequences were not necessary for coactivation of ERα. Analysis of multiple DRIP150 deletion mutants identified a 23-amino-acid sequence (789–811) required for coactivation activity. Analysis of the protein crystal structure data base identified two regions at amino acids 789–794 and 795–804, which resembled α-helical motifs in Lanuginosa lipase/histamine N-methyltransferase and hepatocyte nuclear factor 1, respectively. By using a squelching assay and specific amino acid point mutations within each α-helix, the NIFSEVRVYN (795–804) region was identified as the critical sequence required for the activity of DRIP150. These results demonstrate that coactivation of ERα by DRIP150 in ZR-75 cells is NR box-independent and requires a novel sequence with putative α-helical structure.