title: Selection of estrogen receptor beta- and thyroid hormone receptor beta-specific coactivator-mimetic peptides using recombinant peptide libraries.
abstract: Steroid and thyroid hormone receptors are members of the superfamily of nuclear receptors (NR) that participate in developmental and homeostatic mechanisms by changes in the transcription of specific genes. These activities are governed by the receptors' cognate ligands and through interaction with the components of the transcriptional machinery. A number of coactivator molecules of the steroid receptor coactivator (SRC)/nuclear receptor coactivator (NCoA) family interact with activation functions within NRs through a conserved region containing helical domains of a core LXXLL sequence and, thereby, participate in transcriptional regulation. Using a mammalian-two-hybrid assay, we show that the thyroid hormone receptor beta (TRbeta) and estrogen receptor beta (ERbeta) have different LXXLL motif preferences for interactions with SRC-1. Using large random and focused (centered on the LXXLL motif) recombinant peptide diversity libraries, we have obtained novel peptide sequences that interact specifically with ERbeta or with TRbeta in a ligand-dependent manner. Random sequence libraries yielded LXXLL-containing peptides, and sequence analysis of selected clones revealed that the preferred residues within and around the LXXLL motif vary significantly between these two receptors. We compared the receptor binding of library-selected peptides to that of peptides derived from natural coactivators. The affinities of selected peptides for the ligand binding domains of ERbeta and TRbeta were similar to the best natural LXXLL motifs tested, but showed a higher degree of receptor selectivity. These selected peptides also display receptor-selective dominant inhibitory activities when introduced into mammalian cells. Finally, by directed mutations in specific residues, we were able to alter the receptor binding preference of these peptides.
authors.affiliation: Affymax Research Institute, Santa Clara, California 95051, USA. Jeff_Northrop@affymax.com
authors.forename: J P
authors.initials: JP
authors.lastname: Northrop
authors.forename: D
authors.initials: D
authors.lastname: Nguyen
authors.forename: S
authors.initials: S
authors.lastname: Piplani
authors.forename: S E
authors.initials: SE
authors.lastname: Olivan
authors.forename: S T
authors.initials: ST
authors.lastname: Kwan
authors.forename: N F
authors.initials: NF
authors.lastname: Go
authors.forename: C P
authors.initials: CP
authors.lastname: Hart
authors.forename: P J
authors.initials: PJ
authors.lastname: Schatz
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D004357:Drug Synergism; D004958:Estradiol / Q000494:pharmacology; D047629:Estrogen Receptor beta; D051548:Histone Acetyltransferases; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D056920:Nuclear Receptor Coactivator 1; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011960:Receptors, Estrogen / Q000819:agonists* / Q000737:chemistry; D011988:Receptors, Thyroid Hormone / Q000819:agonists* / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*; D020798:Two-Hybrid System Techniques
chemical_list: D047629:Estrogen Receptor beta; D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D011960:Receptors, Estrogen; D011988:Receptors, Thyroid Hormone; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004958:Estradiol; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1