Transcriptional Activation of Estrogen Receptor-Alpha and Estrogen Receptor-Beta from Elephant Shark (Callorhynchus milii)

Humans and other terrestrial vertebrates contain two estrogen receptors (ERs), ERa and ERb. Among cartilaginous fish (sharks, rays, skates), which are jawless vertebrates that evolved about 525 million years ago, only activation by steroids of ERb orthologs has been characterized. To remedy this gap in understanding estrogen signaling in jawless vertebrates, we studied estrogen activation of orthologs of human ERa and ERb from elephant shark (Callorhynchus milii). Unexpectedly, we found that C. milii contained three estrogen-responsive ERa genes: ERa1 (596 amino acids), ERa2 (600 amino acids), and ERa3 (599 amino acids) with strong sequence similarity to each other. We also found an estrogen-unresponsive gene, ERa4 (561 amino acids), with a 39 amino acid deletion in the DNA-binding domain. An estrogen-responsive ERb ortholog (580 amino acids) also was present in C. milii. The three active C. milii ERas have a similar length to human ERa (595 amino acids); however, C. milii ERb is longer than human ERb (530 amino acids). We determined the half-maximal response (EC50) and fold-activation to estradiol (E2), estrone (E1), and estriol (E3) of C. milii ERa1, ERa2, ERa3, and ERb. Among these estrogens, E2 had the lowest EC50 for all four ERs. Fold-activation by E2 and E3 was similar for ERa1, ERa2, ERa3, and ERb. Overall, estrogen activation of C. milii ERa and ERb was similar to that for human ERa and ERb, indicating substantial conservation of the vertebrate ER in the 525 million years since the divergence of cartilaginous fish and humans from a common ancestor.