title: Determination of the androgenicity of ligands using a single-chain probe carrying androgen receptor N-terminal peptides.
abstract: The present study demonstrates a single-molecular bioluminescent probe carrying functional peptides in the N-terminal domain of the androgen receptor (AR NTD) with an improved sensorial property to androgens. The N-terminal peptides in AR were genetically fused to the ligand binding domain of AR (AR LBD) with a flexible linker, and then sandwiched between the N- and C-terminal fragments of split-firefly luciferase (FLuc) dissected at D415. We found that the proline-rich region in AR NTD efficiently interacts with AR LBD and exerts (i) an enhanced signal-to-background ratio and (ii) discrimination between agonists and antagonists with (iii) a 100-times improved sensitivity to androgens, upon comparison with previous references. A deletion mutation to the proline-rich region in AR revealed that this region is critical for the transcriptional activities. The quantum yields of these single-chain probes were estimated to be 37.8 +/- 0.6%. This monomeric AR LBD-peptide binding is necessary, and sufficient for discriminating an agonist and an antagonist, where the dimerization of AR LBD is not involved. The present study guides a fundamental methodology on how to discriminate weak protein-peptide binding, and provides a new insight into the contribution of functional peptides in AR NTD to the initial activation of monomeric AR.
authors.affiliation: Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan.
authors.forename: Sung Bae
authors.initials: SB
authors.lastname: Kim
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Umezawa
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tao
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000726:Androgen Antagonists / Q000494:pharmacology; D000728:Androgens / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D013196:Dihydrotestosterone / Q000494:pharmacology; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008024:Ligands; D057230:Limit of Detection; D049408:Luminescent Agents / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011944:Receptors, Androgen / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D000726:Androgen Antagonists; D000728:Androgens; D008024:Ligands; D049408:Luminescent Agents; D010446:Peptide Fragments; D011944:Receptors, Androgen; D013196:Dihydrotestosterone