Locust retinoid X receptors: 9-Cis-retinoic acid in embryos from a primitive insect Shaun M. Nowickyj*, James V. Chithalen†, Don Cameron†, Michael G. Tyshenko*, Martin Petkovich†‡, Gerard R. Wyatt*, Glenville Jones†§, and Virginia K. Walker*¶ Departments of *Biology, †Biochemistry, ‡Pathology, and §Medicine, Queen’s University, Kingston, ON, Canada K7L 3N6 Edited by Walter S. Leal, University of California, Davis, CA, and accepted by the Editorial Board April 10, 2008 (received for review December 21, 2007) The retinoid X receptor (RXR) is activated by its often elusive cognate high-affinity JH receptor, Methoprene-tolerant (Met; Kd ϭ 5.3 nM) ligand, 9-cis-retinoic acid (9-cis-RA). In flies and moths, molting is has been recently identified in Dm (18). mediated by a heterodimer ecdysone receptor consisting of the Remarkably, the LBD of the protein corresponding to USP from ecdysone monomer (EcR) and an RXR homolog, ultraspiracle (USP); the primitive insect, Locusta migratoria (Lm), shows greater identity the latter is believed to have diverged from its RXR origin. In the more to the vertebrate RXR than to USPs of more advanced insects (19). primitive insect, Locusta migratoria (Lm), RXR is more similar to In silico modeling of the LmRXR-L ligand binding pocket also human RXRs than to USPs. LmRXR was detected in early embryos emphasizes amino acid and tertiary structural similarity to the when EcR transcripts were absent, suggesting another role apart from human RXR␥ (hRXR␥) (19). In Lm and the German cockroach, ecdysone signaling. Recombinant LmRXRs bound 9-cis-RA and all- Blattella germanica, RXR transcripts have been detected during ؍ ؍ trans-RA with high affinity (IC50 61.2–107.7 nM; Kd 3 nM), similar early embryonic development, even before the appearance of EcR to human RXR. To determine whether specific binding had functional transcripts (20, 21). Thus at least in these two insects it is possible significance, the presence of endogenous retinoids was assessed. that this EcR binding partner could have a second function. To Embryos were extracted by using modified Bligh and Dyer and explore this possibility, two RXR cDNAs were isolated from solid-phase protocols to avoid the oily precipitate that makes this Locusta. Long and short isoforms, LmRXR-L (GenBank accession material unsuitable for assay. These extracts contained retinoids (5.4 no. AY348873) and LmRXR-S (GenBank accession no. nM) as assessed by RA-inducible Cyp26A1-promoter luciferase re- AF136372), respectively, differ only by the presence/absence of 22 porter cell lines. Furthermore, the use of HPLC and MS confirmed the aa in their LBDs. presence of retinoids and identified in any embryo, 9-cis-RA, in addition to all-trans-RA. We estimate that whole embryos contain 3 Results nM RA, including 9-cis-RA at a concentration of 1.6 nM. These findings Recombinant LmRXR-S was expressed by using the phage T5 strongly argue for a functional role for retinoids in primitive insects promoter in pQE-32 after transfer to Escherichia coli M15[pREP4] and favor a model where signaling through the binding of 9-cis-RA to cells. Under similar conditions, LmRXR-L expression was disap- its RXR is established relatively early in evolution and embryonic development. pointing (results not shown). Therefore the hinge and ligand binding domains of LmRXR-L were subcloned into pET-15b all-trans-retinoic acid ͉ Locusta migratoria ͉ ultraspiracle [LmRXR-L(DE)] and expressed in BL21(DE3) cells by using the T7lac promoter. As a control for this shorter (Ϸ28 kDa) sequence, the Ϸ29-kDa human-derived sequence, hRXR␣(DE), was similarly nsect development and metamorphosis are directed by two expressed. Typically, the purification protocol (a total of 10 puri- Iprincipal lipophilic hormones: 20-hydroxyecdysone (20-OH-Ec), fications were done for the long and short isoforms) yielded 0.8 the active molting hormone, and juvenile hormone (JH), whose mg/ml LmRXR-S and 1.7 mg/ml LmRXR-L(DE) protein [see titer determines the nature of the molt (1, 2). As demonstrated in supporting information (SI) Fig. S1 A and B; lanes 7 and 8). the fruitfly, Drosophila melanogaster (Dm), 20-OH-Ec binds to the Expression and purification of hRXR␣(DE) normally yielded 1.0 ecdysone receptor (EcR), which in turn is bound to its obligate mg/ml. These preparations were used for assays and antibody heterodimerization partner ultraspiracle (USP), a homologue of production. the vertebrate retinoid X receptor (RXR) (3–6). As members of the ␣ nuclear receptor superfamily, EcR and USP/RXR share a common Polyclonal antibodies made against purified hRXR (DE) and modular structure (7) comprised of a N-terminal variable domain LmRXR-L(DE) were used for immunological detection by West- (A/B), a DNA binding domain (C), hinge (D), and C-terminal ern blotting (see Fig. S1 C—E). The high similarity between the ligand-binding domain (LBD; or domain E/F). expressed human and locust RXR LBDs is demonstrated by the ␣ The vertebrate RXRs are known heterodimeric partners of observation that the hRXR (DE) antibody cross-reacted with several members of the nuclear receptor superfamily, including the purified LmRXR-L(DE) and the LmRXR-L(DE) antibody cross- ␣ retinoid, thyroid, and vitamin D receptors (8). As demonstrated in reacted with the purified hRXR (DE). Western blots further vivo, these RXRs can also form homodimers and conceivably demonstrated the presence of cross-reacting material to the anti- mediate an independent retinoid signaling pathway (9, 10). Indeed, LmRXR-L(DE) in whole embryo extracts (see Fig. S1E). the vertebrate RXRs are known ligand-activated transcription factors that bind 9-cis-retinoic acid (9-cis-RA), a stereoisomer of the Author contributions: S.M.N., J.V.C., G.R.W., G.J., and V.K.W. designed research; S.M.N., vitamin A derivative, all-trans-RA (11, 12). RA receptors (RARs), J.V.C., D.C., M.G.T., G.R.W., and V.K.W. performed research; J.V.C., M.G.T., M.P., G.R.W., reported only in vertebrates, are distinct in that they bind both G.J., and V.K.W. contributed new reagents/analytic tools; S.M.N., J.V.C., D.C., G.R.W., G.J., all-trans-RA and 9-cis-RA with high affinity (13, 6). In contrast to and V.K.W. analyzed data; and S.M.N. and V.K.W. wrote the paper. the vertebrate RXRs, crystal structures reveal that DmUSP and the The authors declare no conflict of interest. USPs from the moth Heliothis virescens and the flour beetle This article is a PNAS Direct Submission. W.S.L. is a guest editor invited by the Editorial Tribolium castaneum probably adopt an inactive conformation Board. (14–16) and are unlikely to have an activating ligand. Nevertheless, ¶To whom correspondence should be addressed. E-mail: [email protected]. Jones et al. (17) have suggested that USP could be a JH receptor This article contains supporting information online at www.pnas.org/cgi/content/full/ because it binds methyl farnesoate, an unepoxidated derivative of 0712132105/DCSupplemental. JH III (Kd ϭ 44 nM) and JH to a lesser extent (Kd ϭ 6700 nM). A © 2008 by The National Academy of Sciences of the USA 9540–9545 ͉ PNAS ͉ July 18, 2008 ͉ vol. 105 ͉ no. 28 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712132105 Downloaded by guest on September 28, 2021 A 100 B 100 75 0.6 75 50 0.4 50 25 Bound/Free -RA Bound (%)-RA Bound -RA Bound (%)-RA Bound 0.2 cis cis 25 9- 9- 0 0.0 0.0 0.5 1.0 1.5 Bound (fmol/µg) -25 0 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 log [9-cis-RA] (M) log [all- trans-RA] (M) C 100 D 100 75 75 50 50 -RA Bound (%)-RA Bound -RA Bound (%)-RA Bound cis cis 25 25 9- 9- 0 0 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 log [DHA] (M) log [Methoprene Acid] (M) Fig. 1. Retinoid binding analysis. Competitive binding of 9-cis-RA (A), all-trans-RA (B), DHA (C), and methoprene acid (D) with [3H]-9-cis-RA to the purified receptors LmRXR-Short (Œ), LmRXR-L(DE) (F), and hRXR␣(DE) (■). (A Inset) A Scatchard analysis of [3H]-9-cis-RA binding to LmRXR-L(DE). Competition binding studies were done three to seven times on a total of two to seven independently prepared recombinant protein purifications. Ligand Binding Assays. Although control insect protein did not bind respectively, than 9-cis-RA for LmRXR-S (Fig. 1 C and D and to [3H]-9-cis-RA (data not shown), when recombinant LmRXR-S, Table 1). The two nonretinoids were also not as effective compet- 3 LmRXR-L(DE), and hRXR␣(DE) were incubated with [ H]-9- itors for 9-cis-RA binding to LmRXR-L(DE). Overall, the IC50 for cis-RA, there was evidence of binding (Fig. 1). Competition with DHA was similar for both RXR isoforms but the IC50 of metho- unlabeled 9-cis-RA showed that for all three protein preparations, prene acid for LmRXR-S was 30% that of LmRXR-L(DE). binding was specific and very similar. The IC50 for 9-cis-RA by hRXR␣(DE) was 74.2 nM, in reasonable agreement with a previ- Retinoids in Locust Embryos. The analysis of early locust embryos ously published value (5.6 nM) (22). LmRXR-S showed 1.8 times was difficult because of the presence of yolk and a tough chorion or the affinity (IC50 ϭ 61.2 nM) for 9-cis-RA than did LmRXR-L(DE) egg shell.