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Drosophila Hormone Receptor 38 Proc. Natl. Acad. Sci. USA Vol. 92, pp. 7966-7970, August 1995 Developmental Biology Drosophila hormone receptor 38: A second partner for Drosophila USP suggests an unexpected role for nuclear receptors of the nerve growth factor-induced protein B type (ecdysone action/receptor interaction/retinoid X receptor) JAMES D. SUTHERL-AND*t, TATYANA KoZLOVA*tt, GEORGE TZERTZINIS*t, AND FOTIS C. KAFAToS*t *Department of Molecular and Cell Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138; tEuropean Molecular Biology Laboratory, Postfach 10.2209, 69012 Heidelberg, Germany; and tInstitute of Cytology and Genetics, Novosibirsk 630090, Russia Contributed by Fotis C. Kafatos, May 11, 1995 ABSTRACT In Drosophila the response to the hormone endocrinology is whether ecdysone action solely involves these ecdysone is mediated in part by Ultraspiracle (USP) and three components (ecdysteroid, EcR, and USP) or whether ecdysone receptor (EcR), which are members of the nuclear other components, such as additional members of the hormone receptor superfamily. Heterodimers of these proteins bind to receptor superfamily, might be directly implicated. Thus far ecdysone response elements (EcREs) and ecdysone to modu- USP has only a single known partner, EcR (3). In contrast, late transcription. Herein we describe Drosophila hormone vertebrate retinoid X receptors (RXRs), which are the USP receptor 38 (DHR38) and Bombyx hormone receptor 38 homologues, function as heterodimers with multiple partners (BHR38), two insect homologues of rat nerve growth factor- such as the retinoic acid, thyroid hormone, and vitamin D induced protein B (NGFI-B). Although members of the receptors (13). NGFI-B family are thought to function exclusively as mono- We describe here two insect nuclear receptors§ Drosophila mers, we show that DHR38 and BHR38 in fact interact hormone receptor 38, DHR38, and Bombyx hormone receptor strongly with USP and that this interaction is evolutionarily 38, BHR38, that are most closely related to a group of conserved. DHR38 can compete in vtro against EcR for vertebrate orphan receptors typified by nerve growth factor- dimerization with USP and consequently disrupt EcR-USP induced protein B (NGFI-B). The vertebrate members of this binding to an EcRE. Moreover, transfection experiments in group are notable in being able to bind DNA as monomers and Schneider cells show that DHR38 can affect ecdysone- are also of interest because of their rapid and transient dependent transcription. This suggests that DHR38 plays a activation by serum, growth factors, and mitogens (14-16). role in the ecdysone response and that more generally NGFI-B DHR38 shows the unexpected property of interacting strongly type receptors may be able to function as heterodimers with with USP and altering its DNA binding properties. DHR38 can retinoid X receptor type receptors in regulating transcription. disrupt EcR-USP heterodimers in vitro and in cell culture, apparently because of its interaction with USP. This interac- The nuclear hormone receptor superfamily contains a large tion is highly conserved in evolution. Therefore, we propose number of evolutionarily related transcription factors that that NGFI-B type receptors can participate in and interfere mediate the action of small molecules such as steroid hor- with RXR-mediated signaling. In insects DHR38 may play an mones. Members of this superfamily function by binding to important role in modulating the ecdysone response. short DNA sequences within gene promoters called hormone response elements, which usually consist of variably spaced MATERIALS AND METHODS repeats of 5 or 6 nt. Most receptors bind to the hormone response elements as homo- or heterodimers, reflecting the Cloning of BHR38 and DHR38. The DNA binding domain repetitive substructure of hormone response elements (1). of BHR38 was isolated by PCR by using degenerate oligonu- Several members have been identified, however, that bind cleotides designed to amplify nuclear receptors (17). This DNA as monomers (2). fragment was used to isolate the BHR38 clone from an ovarian Insect genomes include several genes that encode members cDNA library. The same fragment was used to isolate DHR38 of the nuclear receptor superfamily (3). Most of them are Drosophila genomic clones (unpublished data), which in turn so-called orphan receptors, since no ligands have been iden- were used to screen a cDNA library made from ecdysone- and tified that directly use them for signaling. Notable exceptions cycloheximide-treated larval organs (a gift from C. Thummel, are the ecdysone receptor (EcR) and Ultraspiracle (USP) University of Utah). The predicted open reading frame from proteins, which mediate the action of the steroid hormone one of the isolated cDNAs, cTK11, is shown in Fig. LA. The ecdysone. EcR and USP form functional heterodimers as a amino acid sequences of DHR38, BHR38, and NGFI-B were prerequisite for hormone and DNA binding (4-7). Ecdysone aligned by the CLUSTALW program (18) and manually adjusted. plays a key role in insect metamorphosis by triggering a cascade A multiple alignment of NGFI-B type sequences (Fig. 1B) and of gene expression that is thought to be modulated by changing FTZ-F1 (GenBank accession no. M63711) was generated by hormone titers and regulated expression of receptors. Ecdy- using CLUSTALW. The A/B domains and residues 477-577 of sone titers show complex dynamics that are related to the FTZ-F1 were removed to avoid excessive gaps, and a neighbor- developmental program (8). Moreover, there are several iso- joining tree was calculated. forms of EcR that are developmentally modulated and may have distinct functions (9-11). USP is expressed in many Abbreviations: USP, Ultraspiracle; EcR, ecdysone receptor; DHR38, tissues throughout development with fluctuations in mRNA Drosophila hormone receptor 38; BHR38, Bombyx hormone receptor and protein levels (12). An important question in insect 38; NGFI-B, nerve growth factor-induced protein B; RXR, retinoid X receptor; GST, glutathionine S-transferase; EMSA, electrophoretic mobility shift assay; CAT, chloramphenicol acetyltransferase. The publication costs of this article were defrayed in part by page charge §The sequences reported in this paper have been deposited in the payment. This article must therefore be hereby marked "advertisement" in GenBank data base [accession nos. X89246 (DHR38) and X89247 accordance with 18 U.S.C. §1734 solely to indicate this fact. (BHR38)]. Downloaded by guest on September 26, 2021 7966 Developmental Biology: Sutherland et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7967 A DHR3 8 MDEDCFPPLSGGWSASPPAPSQLQQ - LHTLQSQAQMSHPNSSNNSSNNAGNSHNNSGGYNYHGHFNAINASANLS 74 NGFI-B MDLASPETAPTAPATLPSFSTFMDGGYTGEFDTFLYQLPGTAQPCSSASSTSSSSSSATSPASASFKFEDFQVYGCYPGTLSG 83 DHR3 8 PSSSASSLYEYNGVSAADNFYGQQQQQQQQSYQQHNYNSHNGERYSLPTFPTISELAAATAAVEAAAAATVGGPPPVRRASLP 157 NGFI-B PLDETLSSSGSDYYGSPCSAPSPPTPNFQPSQLSPWDGSFGHFSPSQTYEGLRVWTEQLPKASGPPPPPTFFSFSPPTGPSPS 166 DHR38 VQRTVLPAGSTAQSPKLAKITLNQRHSHAHAHALQLNSAPNSAASSPASADLQAGRLLQA--PSQICAVCGDTAACQHYGVRT 238 BHR38 GSSSPGVAPADNTGPRAAPSS-PSQ CAVCGDTAACQHYGVRT NGFI-B LAQSSLKLFPAPATHQLGEGESYSVPAAFPGLAPTSPNCDTS-GILDAPVTSTKARSGSSGGSEGF CAVCGDNASCQHYGVRT 248 p D T/A ** DHR3 8 CEGCKGFFKRTVQKGSKYVCLADKNCPVDKRRRNRCQFCRFQKCLVVG VKEVVRTDSLKGRRGRLPSKPKSPQESPPSPPIS 320 BHR3 8 CEGCKGFFKRTVQKGSKYVCLAEKSCPVDKRRRNRCQFCRFQKCLAVG VKEVVRTDSLKGRRGRLPSKPKCPQESPPSPPIS NGFI -B CEGCKGFFKRIVQKSAKYICLANKDCPV2KRRRNRCQFCRFQKCLAVG VKEVVRTDSLKGRRGRLPSKPKQPPDA- -SPTN- 328 DHR38 LITALVRSHVDTTPDPSCLDYSHYEEQS- MSEADKVQQFYQLLTSSVDVIKQFAEKIPGYFDLLPEDQELLFQSASLEL 398 BHR3 8 LITALVRAHVDTSPDFANLDYSQYREPSPLEPPMSDLEVIQQFYSLLTTSIDMIKLFAEKVPGYGDLCPEDREQLFASARLEL NGFI -B LLTSLIRAHLDSGPNTAKLDYSKFQELVLPRFGKEDAGDVQQFYDLLSGSLDVIRKWAEKIPGFIELSPGDQDLLLESAFLEL 411 DHR3 8 FVLRLAYRARIDDTKLIFCNGTVLHRTQCLRSFGEWLNDIMEFSRSLHNLEIDISAFACLCALTLITERHGLREPKKVEQLQM 481 BHR3 8 FVLRLAYRTALEDTKLTFSNGSVLDKRQCQRSFGDWLHAVLDFSNTSYSMDIDISTFACLCALTLITDRHGLKEPHRVEQVQM NGFI -B FILRLAYRSKPGEGKLIFCSGLVLHRLQCARGFGDWIDNILAFSRSLHSLGVDVPAFACLSALVLITDRHGLQDPRRVEELQN 494 DHR3 8 KIIGSLRDHVTYNAEAQKKQHYFSRLLGKLPELRSLSVQGLQRIFYLKLEDLVPAPALIENMFVTTLPF 527 BHR3 8 KIIGCLRGHMPGGGGSSSGAAPLQRVLGALPELRSLSVKASQRIFYLKLEDLVPAPPLIENMFRASLPF NGFI -B RIASCLKEHMAAVAGDPQPASCLSRLLGKLPELRTLCTQGLQRIFCLKLEDLVPPPPIVDKIFMDTLSF 542 B DHR38 (fly) | AB | DBD | T/A LBD * I BHR38 (moth) 97% 100% 66% ] BHR38 B. mori NURRl (mouse) 391% 1100% 60% DHR38 D. melanogaster RNR-1 (rat) 94% 1% 56% NUR1 mouse 9. I I100%I r.,,~~~~~~~~~~~~10 rat nur77(mouse) 88% 100% 53% =RNR7 mouse _ 97.4 99.9 NGFI-B (rat) 88% 990% |53 NGFI-B rat 100 NAKi (human) 90% 100% 56% NAKI human (f-| : XLNGFI-B X laevis XLNGFI-B (frog) 92% 100%/ 54%__ _____________ FIG. 1. DHR38 and BHR38 are members of the NGFI-B family. (A) Amino acid sequence comparison of the predicted amino acid sequences of the complete DHR38 and NGFI-B and partial BHR38 proteins. Asterisks indicate conserved residues and dashes represent gaps. The strof)gly conserved DNA binding domain is outlined and the P, D, and T/A boxes (19) are overlined. (B) Domain comparison of DHR38, BHR38, RNR-1 (GenBank accession no. L08595), XLNGFI-B (GenBank accession no. X70700), NURR1 (GenBank accession no. S53744), NAK1 (Swiss-Prot accession no. P22736), nur77 (GenBank accession no. J04113), and NGFI-B (Swiss-Prot accession no. P22829). Percentages indicate identity to DHR38 amino acid sequence. A/B, N-terminal domain; DBD, DNA binding domain; LBD, ligand binding domain. (C) Molecular phylogeny of the NGFI-B family. Amino acid sequences
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