Protein Evolution by Molecular Tinkering: Diversification of the Nuclear Receptor Superfamily from a Ligand-Dependent Ancestor

Howard Hughes Medical Institute, Eugene, Oregon, United States of America.
PLoS Biology (Impact Factor: 9.34). 10/2010; 8(10). DOI: 10.1371/journal.pbio.1000497
Source: PubMed


Author Summary
Many protein families are so diverse that it is hard to determine their ancestral functions and to understand how their derived functions evolved. The existence of so many different functions within protein families often creates the impression that complex, novel functions must have evolved repeatedly and independently. Nuclear receptors (NRs) are a large family of related proteins that regulate key biological processes in animals by binding to specific DNA sequences and triggering expression of nearby target genes. Many NRs are activated by a specific hormone or other small molecule, but some do not require a ligand, and still others are incapable of activating gene expression and so act primarily as repressors of transcription. To understand how the functional diversity of NRs evolved, we reconstructed the structural and functional characteristics of the ancient protein from which the entire family evolved, using genomic, biochemical, functional, and structural analyses in a phylogenetic framework. We show, contrary to current belief, that the ancestral NR was a ligand-activated transcriptional activator that existed in the earliest period of animal evolution. Our analysis reveals how the extraordinary functional diversity of modern receptors was generated by subtle tinkering with this ancestral template—slightly reshaping the ligand cavity, stabilizing the protein's active conformation so it no longer required a ligand, or disabling the protein's capacity to activate transcription without affecting its other properties. We predict that, when sufficient data are gathered to allow detailed evolutionary reconstructions in other protein families, it will become apparent that most protein functional diversity evolved by tinkering with ancient functions; invoking the evolution of wholesale “novelty” will seldom be necessary.

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    • "In humans and other vertebrates, glucocorticoids regulate many physiological responses including carbohydrate and protein metabolism, immune function and the body's anti-inflammatory processes via transcriptional activation of the glucocorticoid receptor (GR) [1] [2] [3] [4]. The GR belongs to the nuclear receptor family of transcription factors, which also contains receptors for sex steroids and other adrenal steroids, thyroid hormone and retinoids [5] [6] [7] [8] [9] [10]. The GR and other steroid receptors have a characteristic modular structure with three functional domains [8,11–14] (Fig. 1), an N-terminal domain (NTD) containing domains A and B, a central DNA-binding domain (DBD) (domain C), a hinge domain (domain D) and a C-terminal ligand-binding domain (LBD) (domain E). "
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    ABSTRACT: We studied the role of the A/B domain at the amino terminus of gar (Atractosterus tropicus) and human glucocorticoid receptors (GRs) on transcriptional activation by various glucocorticoids. In transient transfection assays, dexamethasone [DEX] and cortisol had a lower half-maximal response (EC50) for transcriptional activation of full length gar GR than of human GR. Both GRs had similar responses to corticosterone, while 11-deoxycortisol had a lower EC50 for gar GR than for human GR. In contrast, constructs of gar GR and human GR consisting of their hinge (D domain), ligand binding domain (LBD) (E domain) fused to a GAL4 DNA-binding domain (DBD) had a higher EC50 (weaker response) for all glucocorticoids. To study the role of the A/B domain, which contains an intrinsically disordered region, we investigated steroid activation of chimeric gar GR and human GR, in which their A/B domains were exchanged. Replacement of human A/B domains with the gar A/B domains yielded a chimeric GR with a lower EC50 for DEX and cortisol, while the EC50 increased for these steroids for the human A/B-gar C/E chimera, indicating that gar A/B domains contributes to the lower EC50 of gar GR for glucocorticoids. Our data suggests that allosteric signaling between the A/B domains and LBD influences transcriptional activation of human and gar GR by different steroids, and this allosteric mechanism evolved over 400 million years before gar and mammals separated from a common ancestor. Copyright © 2015. Published by Elsevier Ltd.
    The Journal of steroid biochemistry and molecular biology 08/2015; 154. DOI:10.1016/j.jsbmb.2015.07.025 · 3.63 Impact Factor
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    • "Today, there are seven subfamilies (NR0-NR6) 66 of NRs known (Thomson et al., 2009). The recent use of multiple biochemical and phylogenetic 67 analyses has shown that modern NRs evolved through subtle tinkering of an ancestral ligand- 68 dependent receptor related to HNF4 (Bridgham et al., 2010). Studies on NR phylogenetics are 69 crucial to understanding metazoan evolution, as NRs have gone through numerous duplication 70 events during evolution. "
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    ABSTRACT: The recently sequenced Daphnia pulex genome revealed the NR1L nuclear receptor group consisting of three novel receptors. Phylogenetic studies show that this group is related to the NR1I group (CAR/PXR/VDR) and the NR1J group (HR96), and were subsequently named HR97a/b/g. Each of the HR97 paralogs from Daphnia magna, a commonly used crustacean in toxicity testing, was cloned, sequenced, and partially characterized. Phylogenetic analysis indicates that the HR97 receptors are present in primitive arthropods such as the chelicerates but lost in insects. qPCR and immunohistochemistry demonstrate that each of the receptors is expressed near or at reproductive maturity, and that HR97g, the most ancient of the HR97 receptors, is primarily expressed in the gastrointestinal tract, mandibular region, and ovaries, consistent with a role in reproduction. Transactivation assays using an HR97a/b/g-GAL4 chimera indicate that unlike Daphnia HR96 that is promiscuous with respect to ligand recognition, the HR97 receptors do not respond to many of the ligands that activate CAR/PXR/HR96 nuclear receptors. Only three putative ligands of HR97 receptors were identified in this study: pyriproxyfen, methyl farnesoate, and arachidonic acid. Only arachidonic acid, which acts as an inverse agonist, alters HR97g activity at concentrations that would be considered within physiologically relevant ranges. Overall, this study demonstrates that, although closely related to the promiscuous receptors in the NR1I and NR1J groups, the HR97 receptors are mostly likely not multi-xenobiotic sensors, but rather may perform physiological functions, potentially in reproduction, unique to crustaceans and other non-insect arthropod groups.
    General and Comparative Endocrinology 08/2014; 206. DOI:10.1016/j.ygcen.2014.07.022 · 2.47 Impact Factor
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    • "Nuclear receptors are exclusive to multicellular metazoans. Their numbers in animals range from a few receptors in sponges and Trichoplax[4–7], to approximately 21 NRs in Drosophila melanogaster[8] and 48 NRs in humans. The nematode Caenorhabditis elegans possesses the highest number of NRs identified in a species with over 270 NRs [9]. "
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    BMC Genomics 05/2014; 15(1):369. DOI:10.1186/1471-2164-15-369 · 3.99 Impact Factor
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