title: DRIP150 coactivation of estrogen receptor alpha in ZR-75 breast cancer cells is independent of LXXLL motifs.
abstract: 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 ERalpha-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 ERalpha 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 ERalpha. 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 alpha-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 alpha-helix, the NIFSEVRVYN (795-804) region was identified as the critical sequence required for the activity of DRIP150. These results demonstrate that coactivation of ERalpha by DRIP150 in ZR-75 cells is NR box-independent and requires a novel sequence with putative alpha-helical structure.
authors.affiliation: Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466, USA.
authors.forename: Jeongeun Eun
authors.initials: JE
authors.lastname: Lee
authors.forename: Kyounghyun
authors.initials: K
authors.lastname: Kim
authors.forename: James C
authors.initials: JC
authors.lastname: Sacchettini
authors.forename: Clare V
authors.initials: CV
authors.lastname: Smith
authors.forename: Stephen
authors.initials: S
authors.lastname: Safe
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001943:Breast Neoplasms; D045744:Cell Line, Tumor; D017931:DNA Primers; D047628:Estrogen Receptor alpha / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D005260:Female; D006801:Humans; D056892:Mediator Complex; D008969:Molecular Sequence Data; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D047628:Estrogen Receptor alpha; C492160:MED14 protein, human; D056892:Mediator Complex; D015534:Trans-Activators