Use of suppressor mutants to probe the function of estrogen receptor-p160 coactivator interactions.
Estrogen-dependent recruitment of coactivators by estrogen receptor alpha (ERalpha) represents a crucial step in the transcriptional activation of target genes. However, studies of the function of individual coactivators has been hindered by the presence of endogenous coactivators, many of which are potentially recruited in the presence of agonist via a common mechanism. To circumvent this problem, we have generated second-site suppressor mutations in the nuclear receptor interaction domain of p160 coactivators which rescue their binding to a transcriptionally defective ERalpha that is refractory to wild-type coactivators. Analysis of these altered-specificity receptor-coactivator combinations, in the absence of interference from endogenous coregulators, indicated that estrogen-dependent transcription from reporter genes is critically dependent on direct recruitment of a p160 coactivator in mammalian cells and that the three p160 family members serve functionally redundant roles. Furthermore, our results suggest that such a change-of-specificity mutation may act as a transposable protein-protein interaction module which provides a novel tool with which to dissect the functional roles of other nuclear receptor coregulators at the cellular level.
Mak
H Y
HY
Molecular Endocrinology Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom.
Parker
M G
MG
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004268:DNA-Binding Proteins; D047628:Estrogen Receptor alpha; D005260:Female; D017930:Genes, Reporter / Q000235:genetics; D051548:Histone Acetyltransferases; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D056920:Nuclear Receptor Coactivator 1; D029543:Nucleocytoplasmic Transport Proteins; D011485:Protein Binding; D016601:RNA-Binding Proteins; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics*; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D047628:Estrogen Receptor alpha; C491719:MYBBP1A protein, human; C110421:Mybbp1a protein, mouse; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D016601:RNA-Binding Proteins; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; C538861:Ncoa1 protein, mouse; D056920:Nuclear Receptor Coactivator 1