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), ERand ER. Among cartilaginous fish (sharks, rays, skates), which are jawless vertebrates that evolved about 525 million years ago, only activation by steroids of ER orthologs has been characterized. To remedy this gap in understanding estrogen signaling in jawless vertebrates, we studied estrogen activation of orthologs of human ER and ER from elephant shark (Callorhynchus milii). Un-expectedly, we found that C. milii contained three estrogen-responsive ER genes: ER1 (596 amino acids), ER2 (600 amino acids), and ER3 (599 amino acids) with strong sequence simi-larity to each other. We also found an estrogen-unresponsive gene, ER4 (561 amino acids), with a 39 amino acid deletion in the DNA-binding domain. An estrogen-responsive ER ortholog (580 amino acids) also was present in C. milii. The three active C. milii ERs have a similar length to human ER (595 amino acids); however, C. milii ER is longer than human ER (530 amino acids). We determined the half-maximal response (EC50) and fold-activation to estradiol (E2), estrone (E1), and estriol (E3) of C. milii ER1, ER2, ER3, and ER. Among these es-trogens, E2 had the lowest EC50 for all four ERs. Fold-activation by E2 and E3 was similar for ER1, ER2, ER3, and ER. Overall, estrogen activation of C. milii ER and ER was simi-lar to that for human ER and ER, indicating substantial conservation of the vertebrate ER in the 525 million years since the divergence of cartilaginous fish and humans from a common ancestor.