The Octopus vulgaris Estrogen Receptor Is aConstitutive Transcriptional Activator: Evolutionary andFunctional ImplicationsJune Keay, Jamie T. Bridgham, and Joseph W. ThorntonCenter for Ecology and Evolutionary Biology, University of Oregon, Eugene, Oregon 97403Steroid hormones such as estrogens and androgens are im-portant regulators of reproduction, physiology, and develop-ment in a variety of animal taxa, including vertebrates andmollusks. Steroid hormone receptors, which mediate the clas-sic cellular responses to these hormones, were thought to bevertebrate specific, which left the molecular mechanisms ofsteroid action in invertebrates unresolved. Recently an estro-gen receptor (ER) ortholog was isolated from the sea hareAplysia californica, but the functional significance of the re-ceptor was unclear because estrogens and other steroids arenot known to be important in that species. Furthermore, theAplysia ER was found to be a constitutive transcriptional ac-tivator, but it was unclear whether the estrogen indepen-dence of the ER was an Aplysia-specific novelty or a moreancient character general to the mollusks. Here we report onthe isolation and functional characterization of the first ERortholog from an invertebrate in which estrogens are pro-duced and play an apparent role, the cephalopod Octopusvulgaris. We show that the Octopus ER is a strong constitutivetranscriptional activator from canonical estrogen responseelements. The receptor does not bind estradiol and is unre-sponsive to estrogens and other vertebrate steroid hormones.These characteristics are similar to those observed with theAplysia ER and support the hypothesis that the evolving ERgained constitutive activity deep in the mollusk lineage. Theapparent reproductive role of estrogens in Octopus and othermollusks is unlikely to be mediated by the ER and may takeplace through an ancient, non-ER-mediated pathway. (Endo-crinology 147: 3861–3869, 2006)STEROID HORMONE RECEPTORS (SRs) play crucialroles in regulating reproduction, development, and me-tabolism in vertebrates (1). They act as high-affinity molec-ular mediators between steroid hormone ligands, specifictarget sequences in genomic DNA, and coregulator proteinsthat activate or repress transcription of nearby genes. SRshave a modular structure in which two highly conserveddomains, the DNA-binding and ligand-binding domains(DBD and LBD, respectively), are particularly important toligand-activated transcription. In the classical mode of actionof steroid hormone receptors, the receptor is in an inactiveconformation in the absence of a hormone ligand. When thehormone is present, it binds tightly and specifically to thepocket of the LBD, changing its conformation and facilitatingdimerization and nuclear transport (2). The receptor DBDbinds to a specific response element, a 15-bp sequence ofgenomic DNA in the control region of target genes (3). In thisactive conformation, stabilized by ligand, surfaces of the LBDare exposed for tight interactions with coactivator proteinsthat modify chromatin or otherwise affect transcription. Ex-pression of the target gene is then selectively up- or down-regulated (4). In this way, steroid hormones stimulate coor-dinated cascades of gene expression that underlie suchfunctions as secondary sexual differentiation, female repro-ductive cycling, long-term response to stress, and adaptationto changing osmolarity throughout the vertebrates.Steroid hormones are also found in invertebrates, includ-ing mollusks, in which there is evidence that they play anendocrine role (5–10). In the cephalopod Octopus vulgaris, forexample, 17-estradiol (E2) and progesterone are found inoviduct and ovarian tissues, and the concentration of thesehormones in females correlates with phases of the repro-ductive cycle (7, 8). In addition, specific high-affinity estra-diol binding and immunoreactivity to antihuman estrogenreceptor (ER) antibodies have been detected in O. vulgarisfemale reproductive tract (7). In the bivalve mollusk, Mytilusedulis, E2 has been detected in gonads, pedal ganglia, andhemolymph (11, 12). In explants of hemocytes and pedalganglia of this species, very low doses of estradiol producestrong and rapid cell-signaling effects (12, 13). There is ev-idence that synthetic pollutants that act as estrogens in ver-tebrates cause endocrine disruption in gastropods (14). Nu-merous other steroids have also been found in a variety ofmollusks and may have functional roles (15, 16).The molecular mechanisms of steroid action in mollusksremain unknown, however. Steroid receptors were longthought to be vertebrate-specific novelties, based on theircomplete absence from the fully sequenced genomes of in-sects and nematodes, but a recent study identified an ERortholog in the mollusk Aplysia californica (17). Functionalstudies showed that the Aplysia ER does not bind E2 and isFirst Published Online May 11, 2006Abbreviations: AF, Activation function; DBD, DNA-binding domain;E2, 17-estradiol; ER, estrogen receptor; ERE, estrogen response ele-ment; ERR, ER-related receptor; ERRE, estrogen-related response ele-ment; GRE, glucocorticoid response element; HAP, hydroxyapatite;LBD, ligand-binding domain; LRH, liver receptor-homolog; RACE,rapid amplification of cDNA ends; SF, steroidogenic factor; SR, steroidhormone receptor; TEGDK, buffer of Tris-HCl, EDTA, glycerol, dithio-threitol, and KCl; UAS, upstream activating sequence.Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.0013-7227/06/$15.00/0 Endocrinology 147(8):3861–3869Printed in U.S.A. Copyright © 2006 by The Endocrine Societydoi: 10.1210/en.2006-03633861a powerful constitutive transcriptional activator, even in theabsence of E2 or any other added hormones (17). To under-stand the evolution of this feature, the ancestor of all SRs wasreconstructed, synthesized, and functionally characterized.This receptor was found to specifically bind E2 and activatetranscription only when estrogens were added, a result con-sistent with the ancestor’s high sequence similarity to ver-tebrate ERs, particularly at critical sites in the ligand-bindingpocket (17, 18). The constitutive activity of the Aplysia ER wastherefore inferred to be a derived state. Several other nuclearreceptors are also constitutively active, including the liverreceptor-homolog (LRH)-1 of rodents and the ER-relatedreceptors (ERRs), the
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