Neofunctionalization of Androgen Receptor by Gain-of-Function Mutations in Teleost Fish Lineage

Steroid hormone receptor family provides an example of evolution of diverse transcription factors through whole-genome duplication (WGD). However, little is known about how their functions have been evolved after the duplication. Teleosts present a good model to investigate an accurate evolutionary history of protein function after WGD, because a teleost-specific WGD (TSGD) resulted in a variety of duplicated genes in modern fishes. This study focused on the evolution of androgen receptor (AR) gene, as two distinct paralogs, AR alpha and AR beta, have evolved in teleost lineage after TSGD. AR alpha showed a unique intracellular localization with a higher transactivation response than that of AR beta. Using site-directed mutagenesis and computational prediction of protein-ligand interactions, we identified two key substitutions generating a new functionality of euteleost AR alpha. The substitution in the hinge region contributes to the unique intracellular localization of AR alpha. The substitution on helices 10/11 in the ligand-binding domain possibly modulates hydrogen bonds that stabilize the receptor-ligand complex leading to the higher transactivation response of AR alpha. These substitutions were conserved in Acanthomorpha (spiny-rayed fish) AR alpha s, but not in an earlier branching lineage among teleosts, Japanese eel. Insertion of these substitutions into ARs from Japanese eel recapitulates the evolutionary novelty of euteleost AR alpha. These findings together indicate that the substitutions generating a new functionality of teleost AR alpha were fixed in teleost genome after the divergence of the Elopomorpha lineage. Our findings provide a molecular explanation for an adaptation process leading to generation of the hyperactive AR subtype after TSGD.