Analytical Sciences

Determination of the Androgenicity of Ligands Using a Single-chain Probe Carrying Androgen Receptor N-Terminal Peptides

Sung Bae KIM,* Yoshio UMEZAWA,** and Hiroaki TAO*

*Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan
**Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishitokyo, Tokyo 202-8585, Japan

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.

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