An Hsp27-related, dominant-negative-acting intracellular estradiol-binding protein.
New World primates (NWPs) exhibit a compensated form of resistance to gonadal steroid hormones. We demonstrated recently that estrogen resistance in NWP cells was associated with the overexpression of two proteins, a nonreceptor-related, dominant-negative-acting estrogen response element (ERE)-binding protein (ERE-BP) and an intracellular estradiol-binding protein (IEBP). Based on the N-terminal sequences of tryptic fragments of IEBP isolated from a 17beta-estradiol (E2) affinity column we cloned a full-length cDNA for IEBP from the estrogen-resistant NWP cell line, B95-8. Subsequent sequence analysis revealed 87% sequence identity between the deduced peptide for IEBP and human Hsp27. When hormone-responsive, wild-type Old World primate (OWP) cells were transiently transfected with IEBP cDNA, E2-directed ERE reporter luciferase activity was reduced by 50% compared with vector only-transfected OWP cells (p < 0.0018). When IEBP and ERE-BP were cotransfected, ERE promoter-reporter activity was reduced by a further 60% (p < 0.0001). Electrophoresis mobility shift analyses showed that IEBP neither bound to ERE nor competed with the estrogen receptor (ER) for binding to ERE. However, there was evidence of protein-protein interaction of IEBP and ERalpha; IEBP was coimmunoprecipitated with anti-ERalpha antibody in wild-type cells stably transfected with IEBP. A specific interaction between ERalpha and IEBP was confirmed in glutathione S-transferase pull-down and yeast two-hybrid assays. Data indicate that the Hsp27-related IEBP interacts with the ligand binding domain of the ERalpha. In summary, by inhibiting the ERalpha-E2 interaction, IEBP acts to squelch ERalpha-directed ERE-regulated transactivation and promote estrogen resistance in NWP cells.
Chen
Hong
H
Division of Endocrinology, Diabetes, and Metabolism, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, California 90048, USA.
Hewison
Martin
M
Hu
Bing
B
Sharma
Manju
M
Sun
Zijie
Z
Adams
John S
JS
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002144:Callithrix; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000378:metabolism*; D047628:Estrogen Receptor alpha; D005799:Genes, Dominant; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008253:Macaca mulatta; D008954:Models, Biological; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D020798:Two-Hybrid System Techniques; D038201:alpha-Crystallins / Q000737:chemistry; D000099190:MAP-Kinase-Activated Kinase 2
D002352:Carrier Proteins; D018076:DNA, Complementary; D004958:Estradiol; D047628:Estrogen Receptor alpha; D005982:Glutathione Transferase; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D010455:Peptides; D017346:Protein Serine-Threonine Kinases; D011960:Receptors, Estrogen; D038201:alpha-Crystallins; D000099190:MAP-Kinase-Activated Kinase 2