Determination of the androgenicity of ligands using a single-chain probe carrying androgen receptor N-terminal peptides.
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.
Kim
Sung Bae
SB
Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan.
Umezawa
Yoshio
Y
Tao
Hiroaki
H
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
D000726:Androgen Antagonists; D000728:Androgens; D008024:Ligands; D049408:Luminescent Agents; D010446:Peptide Fragments; D011944:Receptors, Androgen; D013196:Dihydrotestosterone