A single point mutation in ecdysone receptor leads to ... - PNAS

A single point mutation in ecdysone receptor leads to increased ligand specificity: Implications for gene switch applications M. B. Kumar*, T. Fujimoto*, D. W. Potter*, Q. Deng*, and S. R. Palli†‡ *RheoGene LLC, P.O. Box 949, 727 Norristown Road, Spring House, PA 19477; and †Department of Entomology, College of Agriculture, University of Kentucky, Lexington, KY 40546 Edited by Bruce D. Hammock, University of California, Davis, CA, and approved September 9, 2002 (received for review May 9, 2002)

The ecdysone receptor (EcR), a member of the nuclear receptor superfamily, plays an important role in regulating development and reproduction in insects. The EcR binds to ecdysteroids and regulates transcription of genes that contain ecdysone response elements. The EcR has been used to develop inducible gene switches for efficient regulation of foreign genes in applications such as gene therapy, protein production, and functional genomics. An EcR [Choristoneura fumiferana EcR (CfEcR)] homology model was constructed, and 17 amino acid residues were identified as critical for 20-hydroxyecdysone binding. Mutation of these amino acids followed by analysis of these mutants in transactivation (in insect and mammalian cells and in vivo in mice) and ligand-binding assays identified one particular mutant (A110P) that failed to respond to steroids, but its response to the diacylhydrazine nonsteroidal ligands RG-102240 (GSTME) and RG-102317 was unaffected. This steroid-insensitive EcR mutant has potential gene switch applications in insects and plants that have endogenous ecdysteroids. In addition, this mutant would be also useful for developing orthogonal EcR-ligand pairs for simultaneous regulation of multiple genes in the same cell.

E

cdysteroids, in concert with the ecdysone receptor (EcR) complex, play an important role in regulating development and reproduction in insects. The EcR gene is a member of the nuclear receptor superfamily and has five modular domains, A兾B (transactivation), C (DNA binding, heterodimerization), D (hinge, heterodimerization), E (ligand binding, heterodimerization, and transactivation), and F (transactivation) (1, 2). EcR heterodimerizes with ultraspiracle (USP), the insect homologue of retinoid X receptor (RXR), and on binding steroids regulates transcription of genes containing ecdysone response elements (3, 4). Molecular chaperones, such as Hsp90 and Hsc70, are required for the functioning of the EcR兾USP complex (5). The EcR gene Drosophila melanogaster encodes three receptor isoforms, EcR-A, EcR-B1, and EcR-B2 (6), that differ in their N-terminal A兾B domain but contain identical DNA- and ligand-binding domains (LBD). The EcR-A and EcR-B1 isoforms may contribute to the tissue specificity of 20hydroxyecdysone (20E) action, as the expression of these isoforms correlates with the different developmental fates of larval and adult tissues (6, 7). Several plant steroids, like ponasterone A (PonA) and muristerone A (MurA), along with select synthetic nonsteroidal ligands, bind to EcR with high affinity (8). The binding of ligand to EcR is greatly stimulated by the presence of USP (3) and also stabilizes the EcR兾USP heterodimer and increases its affinity to ecdysone response elements (EcRE; ref. 3). The crystal structure of USP has been elucidated by two groups (9, 10). The structure of USP is similar to its mammalian homologue RXR, except that USP structures show a long H1-H3 loop and an insert between H5 and H6. These structures appear to lock USP in an inactive conformation by displacing helix 12 from agonist conformation. In addition, in both crystal structures, USP has a large hydrophobic cavity, which contains phospholipid ligands (9, 10). The crystal structure of the EcR has yet to be determined; however, homology models for EcR (EcR from Chironomus ten14710 –14715 兩 PNAS 兩 November 12, 2002 兩 vol. 99 兩 no. 23

tans, CtEcR) LBD based on retinoic acid and vitamin D receptor structures have been generated (11). Gene switches are inducible gene regulation systems that are used to control the expression of transgenes. Precise regulation of expression of transgenes is very important for various applications such as gene therapy, large-scale production of proteins in cells, cell-based high-throughput screening assays, functional genomics, and regulation of traits in transgenic plants and animals. Several gene switches based on the tetracycline repressor, immunosuppressive molecules, as well as steroid hormone receptors, including progesterone and ecdysone receptors, have been developed (see ref. 12 for a recent review). Initial studies showed that an EcR-based gene switch system consisting of an EcR from D. melanogaster (DmEcR), an RXR from Homo sapiens, and the steroid, PonA, transactivated reporter genes in mammalian cell lines and transgenic mice (13, 14). Subsequent studies showed that a nonsteroidal ecdysone agonist, tebufenozide, induced high levels of transactivation of reporter genes in mammalian cells through an EcR from Bombyx mori (15) and endogenous RXR (16). Recently, we have developed a Choristoneura fumiferana EcR (CfEcR)-based gene switch that showed improved sensitivity to nonsteroidal ligands as well as an increase in ligand-induced reporter levels when compared with earlier versions. The EcR-based gene switch has also been observed to function in yeast, albeit in the presence of an ectopically expressed coactivator, glucocorticoid receptor-interacting protein 1 [GRIP1 (17)]. EcR-based gene switches are attractive for mammalian applications because both the receptor and ligand are foreign to mammalian cells. Synthetic ligands such as diacylhydrazines, which are capable of binding to EcR and activating ecdysone responsive genes, were introduced by Rohm & Haas (8). These compounds bind EcR and initiate a premature molt that leads to mortality of larvae (18) but are harmless to vertebrates (19). Several other classes of synthetic ligands that show different affinities to EcR have been subsequently discovered (8). These nonsteroidal ligands are being used to develop novel EcR-based gene switches. For in vivo applications such as gene therapy, it is desirable to have a gene switch that responds well to synthetic nonsteroidal ligands but not to endogenous steroids. In the case of insects and some plants, the application of an EcR-based gene switch could be limited by the presence of endogenous ecdysteroids that could potentially activate this gene switch. Thus, an EcR-based gene This paper was submitted directly (Track II) to the PNAS office. Abbreviations: EcR, ecdysone receptor; USP, ultraspiracle; RXR, retinoid X receptor; 20E, 20-hydroxyecdysone; PonA, ponasterone A; MurA, muristerone A; SEAP, secreted alkaline phosphatase; EcRE, ecdysone response element; DmEcR, EcR from Drosophila melanogaster; CfEcR, EcR from Choristoneura fumiferana; GRIP1, glucocorticoid receptorinteracting protein 1; RLU, relative light units; GST:cfE(DEF), GST:CfEcR(DEF); G:CfE(DEF), GAL4:CfEcR(DEF); V:CfE(CDEF), VP16:CfEcR(CDEF); GST:CfU(A-F), GST:CfUSP(A兾BCDEF); V:Hs-LmR(EF), VP16:Hs␤RXR (helices 1– 8) and LmRXR (helices 9 –12 ⫹F); LBD, ligandbinding domain; DBD, DNA-binding domain. ‡To

whom correspondence should be addressed. E-mail: [email protected].

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Materials and Methods Ligands. MurA, PonA, ␣-ecdysone, and 20-hydroxyecdysone were purchased from Alexis (San Diego). Nonsteroidal ligands RG102240, also known as GSTME [N-(1,1-dimethylethyl)-N⬘-(2-ethyl3-methoxybenzoyl)-3,5-dimethylbenzohydrazide], RG-100915 [N-(1,1-dimethylethyl)-N⬘-(2-methyl-3-nitrobenzoyl)-3,5-dimethylbenzohydrazide], and RG-102317 [N-(1,1-dimethylethyl)-N⬘-(5methyl-2,3-dihydro-benzo[1,4]dioxane-6-carbonyl)-3,5-dimethylbenzohydrazide] were synthesized at Rohm & Haas. All ligands were applied in DMSO, and the final concentration of DMSO was maintained at 0.01%. [3H]-PonA and [3H]-␣-ecdysone was purchased from NEN. [3H]-RH-2485 [N-(1,1-dimethylethyl)-N⬘-(2methyl-3-methoxybenzoyl)-3,5-dimethylbenzohydrazide] was synthesized at NEN by tritiating RH-2485 at the methoxy group. Plasmids. The EcR and RXR constructs were prepared by clon-

ing various domains of CfEcR (20) or Locusta migratoria RXR (LmRXR; ref. 21) into pM or pVP16 vectors (CLONTECH). Details on the construction of various plasmids are published as Supporting Materials and Methods in supporting information on the PNAS web site, www.pnas.org.

Homology Modeling. A homology model for CfEcR was generated

by using the Composer module from SYBYL, Ver. 6.5, from Tripos Associates (St. Louis). Three protein structures of the estrogen receptor (ER, 1ERD), the thyroid hormone receptor (1BSX), and the vitamin D receptor (1DB1) were used in creating this model. The protein termini were capped, and the structure was minimized by using the all-atom Amber force field implemented in SYBYL. After annealing to 350 K, molecular dynamics was run at that temperature for 70 ps. Examination of the radius of gyration for the protein showed general convergence of the structure expansion after 40 ps, and the last 10 ps were averaged to generate a new structure that was again minimized. Basic structure validation was done by using PROCHECK (European Bioinformatics Institute, Cambridge, U.K.). The structure contained no disallowed regions, and 98% of the residues were in conservatively allowed regions. The location of the identified binding site from the thyroid hormone and estrogen receptors was emulated in the placement of 20-hydroxyecdysone within the CfEcR model. Several Flexidock (Tripose, St. Louis) runs were conducted to identify optimum binding modes. Two general binding modes were identified as differentiated by the ligand being rotated at 180° in the binding cavity. Site-Directed Mutagenesis. Site-directed mutagenesis was carried out by using the QuikChange site-directed mutagenesis kit (Stratagene). Mutations were verified by sequencing. Transient Transfections. The 3T3 cells were transfected with Super-

Fect (Qiagen, Valencia, CA), and L57 cells were transfected with lipofectamine reagent (Life Technologies, Rockville, MD). Luciferase was measured by using the dual-luciferase reporter assay system from Promega. ␤-Galactosidase was measured by using the Gal-Screen system from Applied Biosystems. All transactivation assays were performed in triplicate and repeated three times. Detailed transfection methods are published as Supporting Materials and Methods in supporting information on the PNAS web site.

Expression of GST-Fusion Proteins. GST:CfE(DEF) [GST:

CfEcR(DEF)] and GST:CfUSP(A兾BCDEF) [GST:CfU(A-F)]

Kumar et al.

were expressed in pLysS兾BL-21 Escherichia coli cells. The fusion proteins were extracted by following a protocol outlined by Amersham Pharmacia Biotech. Ligand-Binding Assay. The ligand-binding assays were performed in

T-buffer (90 mM Tris, pH 8.0兾10 ␮M DTT兾protease inhibitors) containing 8 ␮l of GAL4:CfEcR(DEF) [G:CfE(DEF)] protein (prepared by using the TNT kit from Promega) and 2.5 ␮l of GST:CfU(A-F), 0.04 ␮Ci of [3H]-PonA (200 Ci兾mmol), and 200 ␮M unlabeled PonA as a competitor in a 100-␮l reaction. The reaction was carried out at room temperature for 1 h. Dextrancoated activated charcoal was added to the mixture, centrifuged at 10,000 ⫻ g for 10 min, and the amount of [3H]-PonA in the supernatant was quantified. The assays were done in triplicate. In addition, the translation efficiency of proteins was checked by analyzing 5 ␮l of proteins on SDS兾PAGE. For determination of IC50 values, 0.65 pmol of [3H]-RH-2485 was bound to CfE(DEF) and GST:CfU(A-F) proteins and competed with increasing concentrations of unlabeled ligands. GST Pull-Down Assays. The GST protein, GST:CfE(DEF), its A110P

mutant, and兾or GST:CfU(A-F) were incubated with 40 ␮l of glutathione Sepharose 4B (Amersham Pharmacia Biotech) for 20 min in PD buffer (20 mM Hepes兾150 mM KCl兾10 mM MgCl2兾 10% glycerol兾1 mM DTT兾0.1% Nonidet P-40兾0.1% Triton X-100兾 protease inhibitor mixture兾20 ␮g/ml BSA). The resin was washed three times with PD buffer, and 35S-labeled GRIP1 was added to the resin in PD buffer and incubated at 4°C for 1.5 h. The resin was washed five times with PD buffer, and the associated proteins were separated by SDS兾PAGE and analyzed by fluorography. RT-PCR Analysis of Expression of Endogenous DHR3 Gene. L57 cells were transfected with 1 ␮g of VP16:CfEcR(CDEF) [V:CfE (CDEF)] or its A110P mutant EcR by using lipofectamine reagent, and total RNA was isolated by using the RNeasy midi kit (Qiagen). Twenty-five picograms of total RNA was used for RT-PCR reactions by using Superscript One-step RT-PCR with Platinum Taq (Life Technologies). Two DHR3 gene-specific primers (5⬘cgccggcgggacaaaacaact-3⬘ and 5⬘-actgcgcggctcgtaggtggtg-3⬘) were used for RT-PCR. Comparison of PonA- and RG-102240-Mediated Induction of Reporter in Vivo. The gene switch plasmids were electroporated into the

quadriceps of C57BL兾6 mice. Briefly, the animals were anesthetized, shaved, and DNA vector injected into the muscle. Electrode conductivity gel was applied; an electrode (1 ⫻ 1 cm) was placed on the hind leg and electroporated with 200 V兾cm, eight times for 20 msec兾pulse at 1-sec time intervals. The transverse electrical field direction was reversed after the animals received half of the pulses. Animals were treated with PonA or RG-102240 in DMSO by i.p. injection 3 days after electroporation. The secreted alkaline phosphatase (SEAP) activity in mouse sera was evaluated 3 days after ligand administration by using the Phospha-Light System from Applied Biosystems. Results Identification of Residues Critical for Ligand Response. To identify critical residues of EcR that are involved in ligand binding, we built a homology model of CfEcR and docked 20E into the binding pocket (Fig. 1). Amino acid residues located within 3.6 Å contact radius of the docked 20E are E20, T58, R95, and A110, which interact with the side chain portion of 20E, and T55, M92, F109, and Y120, which interact with the ring system of ecdysteroids. Amino acids I51, L223, and L230 are near the steroid A ring region, M125 is near the AB ring junction, and L234 is near the steroid B ring. The amino acid residue, M93, is over the steroid C ring but rather distant, and F48 and A123 are at the end of the steroid A ring. The amino acid residue numbers shown here reflect the numbering of PNAS 兩 November 12, 2002 兩 vol. 99 兩 no. 23 兩 14711

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switch that does not respond to steroids would be useful for various applications in humans, animals, insects, and plants. To develop such a gene switch, we generated several mutant EcRs by changing amino acid residues in the LBD of CfEcR. These mutant EcRs were evaluated in ligand-binding and transactivation assays. Mutation of a critical alanine residue, A110, in the CfEcR LBD led to a selective disruption of both binding and transactivation with steroids but not with nonsteroidal ligands.

Fig. 1. (A) Homology model for CfEcR. The estrogen receptor (ER), thyroid hormone receptor (TR), and vitamin D receptor protein structures were used to generate the CfEcR model. The ecdysteroid, 20E, was docked into the ligand-binding pocket emulating ligand binding in the ER and TR, as described in Materials and Methods. The ligand, 20E, is shown in yellow, and the amino acid residues involved are displayed in gray as a ball-andstick model. (B) PonA binding by WT G:CfE(DEF) and its mutants. The ligand-binding assay was performed by using WT G:CfE(DEF) or its A110 mutants, GST:CfU(A-F), [3H]PonA, and unlabeled PonA. (C) RH-2485 binding by WT G:CfE(DEF) and its mutants. The ligand-binding assay was performed by using WT G:CfE(DEF) or its A110 mutants, GST:CfU(A-F), [3H]-RH-2485, and unlabeled RG-102240.

the LBD starting at helix 1. Corresponding amino acid numbers in full-length EcR are shown in parentheses in Table 1. The amino acid residues identified in the homology model as critical for ligand binding were mutated to alanine, to proline in the case of A110, or to phenylalanine in the case of A123. The mutant EcRs were assayed in 3T3 cells in the presence of 2.5 ␮M PonA or RG-102240. Most of the mutations affected both PonA and RG102240 (Table 1). One particular mutant, A110P, displayed a drastic reduction in PonA activity (0.3% of the WT activity), but not in RG-102240 activity (70% of the WT activity). The R95A mutant also failed to respond to PonA, but unlike the A110P mutant, it showed a significant reduction in RG-102240 activity. Interestingly, Table 1. Transactivation and binding activity of CfEcR mutants % WT activity Amino acid Y120A (Y403A) W238A (W521A) L234A (L517A) L230A (L513A) I51A (I334A) M125A (M408A) F48A (F331A) A123F (A406F) L223A (L506A) F109A (F392A) T55A (T338A) R95A (R378A) M93A (M376A) T58A (T341A) A110P (A393P) M92A (M375A) E20A (E303A)

RG-102240 (2.5 ␮M)

PonA (2.5 ␮M)

% WT RH-2485 binding

0.05 0.06 0.07 0.08 0.26 0.30 0.41 7.6 9.3 22.2 25.1 33.7 38.3 34.9 69.7 70.0 149.0

0.33 0.44 0.31 0.34 0.27 0.36 0.38 0.33 0.45 0.32 1.29 0.38 3.70 55.0 0.33 39.0 108.0

6.3 2.6 1.5 8.6 10.2 5.9 0.0 0.0 40.4 52.8 33.9 105.7 0.0 58.8 105.6 84.0 83.6

CfEcR mutants were assayed in 3T3 cell transactivation assay and ligandbinding assay as described in Materials and Methods. The values presented are percent WT receptor activity assayed under identical conditions. 14712 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.222278999

both A110 and R95 are predicted to be in the vicinity of the tail end of the 20E ligand (Fig. 1 A). The ligand-binding ability of the EcR LBD mutants was examined by using a ligand-binding assay where labeled [3H]-RH-2485 bound to EcR兾USP complex was competed with unlabeled RG102240. In general, the mutants that showed reduced transactivation response (⬍20% WT activity) also showed reduction in ligand binding (⬍10% WT ligand binding, Table 1), indicating that the reduced transactivation response is directly related to the reduced ligand binding. The A110 Mutants. To further probe the role of the A110 residue in

ligand binding and transactivation, a library of A110 mutants was prepared by using degenerate primers and screened in a transactivation assay in 3T3 cells. Four different A110 residue mutants (A110M, A110S, A110P, and A110L) were identified. These four A110 mutants and WT EcR were tested in a ligand-binding assay. The G:CfE(DEF) and its A110 mutants were assayed in the presence of GST:CfU(A-F) by binding with [3H]-PonA or [3H]RH-2485 and competing with 100 times the excess of unlabeled ligands PonA or RG-102240. The PonA binding to A110P mutant was undetectable, whereas the other three A110 mutants showed 5–10% of WT CfEcR binding (Fig. 1B). On the other hand, all A110 mutants showed RH-2485 binding similar to that of WT CfEcR (Fig. 1C). Thus, of four A110 mutants tested, A110P appears to be the better mutant. The relative binding affinities of PonA, MurA, RG-102240, and RG-102317 to G:CfE(DEF) and its A110P mutant were measured by binding with [3H]-RH-2485 and competing with unlabeled ligands RG-102240, RG-102317, MurA, or PonA. The nonsteroidal ligands bound well to both G:CfE(DEF) (IC50 ⫽ 85 nM for RG-102240 and IC50 ⫽ 10 nM for RG-102317) and its A110P mutant (IC50 ⫽ 13 nM for RG-102240 and IC50 ⫽ 26 nM for RG-102317). The steroids bound G:CfE(DEF) with high affinity [IC50 ⫽ 346 nM for PonA and IC50 ⫽ 424 nM for MurA], but not to its A110P mutant (IC50 ⬎ 1 mM for both PonA and MurA). The performance of WT EcR and its A110P mutant was compared in a transactivation assay in 3T3 cells in the presence Kumar et al.

PonA, MurA, RG-102317, and RG-102240. As shown in Fig. 2A, RG-102317 and RG-102240 induced reporter gene activity in a dose-dependent manner with both WT CfEcR and its A110P mutant. In contrast, PonA and MurA induced dose-dependent reporter gene activity only with WT EcR but not with its A110P mutant. Thus, the A110P mutant is insensitive to steroids, but its nonsteroidal ligand activity is not affected. The other three A110 mutants, A110M, A110L, and A110S, were also tested in transactivation assay and showed similar results, in that their nonsteroidal ligand response was similar to WT EcR and steroids failed to induce reporter gene activity with these mutants as well (data not shown). In 3T3 and other mammalian cells, G:CfE(DEF) is more sensitive to nonsteroidal ligands such as RG-102240 and RG-102317 than to steroids such as PonA and MurA. Among steroids, PonA works better than MurA through G:CfE(DEF) switch (data not shown). To dispel the possibility that the selective responsiveness of the A110 mutants to steroids may be an artifact of fusion of the EcR DEF domains to the GAL4 DNA-binding domain (DBD), the A110P mutation was introduced into the full-length EcR [CfE(AF)] and assayed for EcRE-driven reporter gene activity in 3T3 cells. The WT CfE(A-F) induced EcRE-driven reporter activity with both PonA and RG-102240 (Fig. 5, which is published as supporting information on the PNAS web site). On the other hand, the A110P mutant induced reporter gene activity only in the presence of RG-102240 but not in the presence of PonA, indicating that the change in ligand specificity is indeed due to the A110P mutation. The natural ligand of EcR, 20E, does not support transactivation through the CfEcR-based gene switch in mammalian cells. To Kumar et al.

determine whether the A110P mutant responds to 20E, we tested this mutant EcR in an EcRE-driven reporter assay in insect (L57) cells. In these cells, WT V:CfE(CDEF) induced reporter gene activity with 20E and RG-102240, whereas its A110P mutant induced reporter gene activity with RG-102240 but not with 20E (Fig. 2B). WT V:CfE(CDEF) induced reporter gene activity at 1 nM or higher concentration of RG-102240 and 100 nM or higher concentration of 20E. In insect cells, CfEcR is more sensitive to RG-102240 and related diacylhydrazines than to 20E and other ecdysteroids (data not shown). The relative light units (RLUs) differ by several orders of magnitude with the different constructs used in Fig. 2, which may be attributed to different RE used to control the expression of the reporter genes. In mammalian cells, GAL4 RE-GAL4 DBD combination performs better than EcRE-EcR DBD combination (data not shown). In Fig. 2 A, the GAL4 RE-GAL4 DBD combination was used, therefore the RLUs were higher. To test the full length EcR A110P mutant, the EcRE-EcR DBD combination was used, therefore the RLUs were lower. In Fig. 2B, the EcRE-EcR DBD combination was used in insect cells; this combination performs well in insect cells and showed higher RLUs. The alanine residue at position 110 is highly conserved in EcRs from insects and tick (Fig. 6, which is published as supporting information the PNAS web site), except in the case of EcRs from Tenebrio molitor (22) and Uca pugilator (23). To determine whether the function of the alanine residue is conserved in other EcRs as well, we mutated the A107 residue in DmEcR (1) to proline and PNAS 兩 November 12, 2002 兩 vol. 99 兩 no. 23 兩 14713

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Fig. 2. (A) 3T3 cells were transfected with WT G:CfE(DEF) or its mutants, V:LmR(DEF) and pFRLUC, for 4 h. The cells were treated with PonA, MurA, RG-102240, or RG-102314 for 40 h, harvested, and assayed. The values presented are mean ⫾ SD (n ⫽ 3). (B) Transactivation of reporter gene by CfEcR and its mutants in L57 cells. L57 cells were transfected with WT V:CfE(CDEF), its A110P mutant, or empty vector (mock transfection), along with pMK43.2 and p1E1LUC for 4 h. The cells were induced with either 20E or RG-102240 and harvested 40 h after transfection. The extracts were assayed for ␤-galtosidase and luciferase activity. The RLUs from mock-transfected cells were subtracted from the RLUs from V:CfE(CDEF)-transfected cells at each dose of ligand. The values presented are mean ⫾ SD (n ⫽ 3).

Fig. 3. In vivo comparison of WT G:CfE(DEF) and its A110P mutant-based gene switches. The plasmids containing G:CfE(DEF) or its A110P mutant, VP16:Hs␤RXR (helices 1– 8) and LmRXR (helices 9 –12 ⫹ F) [V:Hs-LmR(EF)] chimera, and 6XGAL4RE-TTR-SEAP, were electroporated into the quadriceps of C57BL兾6 mice. Animals were treated with RG-102240 (A) or PonA (B) in DMSO by i.p. injection at 3 days after electroporation. Ligand equivalence is based on the molecular weight of RG-102240. The SEAP in mouse sera was evaluated at 3 days after ligand administration. Values are the average from four animals ⫾ SD. Asterisks indicate treatments with the A110P mutant are significantly different from corresponding treatments with the WT EcR on Student’s t test (P ⬍ 0.01).

assayed both WT DmEcR and its mutant in L57 cells. RG-102240 induced reporter gene activity with both WT and mutant EcRs (Fig. 7, which is published as supporting information on the PNAS web site). On the other hand, 20E induced reporter gene activity with WT EcR but not with mutant EcR. Similar results were observed when corresponding alanine residues in EcRs from Aedes aegypti (24) and an EcR from B. mori (15) were mutated to proline (data not shown). These data suggest that the alanine residue and its function are conserved among EcRs. In Vivo Comparison of WT CfEcR and Its A110P Mutant. The G:CfE(DEF) and its A110P mutant were tested in vivo in C57BL兾6 mice by electroporating the gene switch plasmids into the muscle tissue, as described in Materials and Methods. The G:CfE(DEF)-based gene switch induced SEAP activity in response to both RG-102240 (Fig. 3A) and PonA (Fig. 3B). The A110P mutant-based gene switch induced SEAP activity only in response to RG-102240 (Fig. 3A) but not in response to PonA (Fig. 3B). The SEAP activity induced by RG-102240 through the A110P mutant-based gene switch was lower when compared with the SEAP activity induced by RG-102240 through the WT EcR-based gene switch. However, the reductions were not statistically significant at any of the doses tested (Fig. 3A). These results confirm the lack of response of A110P mutant-based gene switch to the steroid, PonA. Activation of Endogenous DHR3 Gene in Insect Cells by CfEcR and Its A110P Mutant. To further test whether the A110P mutant EcR is

sensitive to the steroid, 20E, we used V:CfE(CDEF) or its A110P

mutant to study the expression of endogenous ecdysone-inducible DHR3 gene in EcR-deficient insect cells. As shown in Fig. 4A, V:CfE(CDEF) induced DHR3 mRNA in the presence of 20E. The A110P mutant of V:CfE(CDEF) did not induce DHR3 mRNA in the presence of 20E (Fig. 4A), suggesting that the A110P mutant EcR is insensitive to 20E and therefore does not induce expression of the endogenous DHR3 gene. Impaired Recruitment of Coactivator GRIP1 by the A110P Mutant. In yeast, ligand-inducible activity of the EcR兾USP heterodimer requires the presence of the coactivator, GRIP1 (17). We hypothesized that steroid-dependent coactivator recruitment by EcR would be impaired in the case of A110P mutant. To test this hypothesis, GST fusions of WT CfEcR or its A110P mutant were assayed for interaction with [35S]-GRIP1 in the presence or absence of MurA. GRIP1 did not bind to GST alone (Fig. 4B, lane 1) but was able to bind GST:CfE(DEF) in the presence of MurA, and this interaction was impaired somewhat in the case of the A110P mutant (compare lanes 2 and 6). When GST:CfU(A-F) was added to the WT GST:CfE(DEF), there was a slight increase in the amount of GRIP1 recruited (lane 3), implying that USP may contribute to GRIP1 binding to EcR. This interaction was greatly strengthened by the addition of MurA (lane 4). However, in the case of the A110P mutant, the addition of GST:CfU(A-F) or GST:CfU(A-F) plus MurA did not increase the amount of GRIP1 recruited (lanes 7 and 8). The GST:CfU(A-F) by itself failed to interact with GRIP1 (lane

Fig. 4. (A) Induction of endogenous DHR3 by WT V:CfE(CDEF) or its A110P mutant in response to 20E. L57 cells were transfected with 1 ␮g of WT V:CfE (CDEF) or its A110P mutant. At 40 h after transfection, the cells were treated with 20E for 6 h. Total RNA was isolated, and RT-PCR was performed by using DHR3 gene-specific primers. (B) Interaction of WT GST:CfE(DEF) or its A110P mutant with GRIP1. The WT GST:CfE(DEF) or its A110P mutant and兾or GST:CfU(A-F) were mixed and purified by using glutathione Sepharose. 35S-labeled GRIP1 was added to the beads and incubated at 4°C for 1.5 h in the presence or absence of 3 ␮M MurA. The resin was washed three times, and the proteins were separated by using SDS兾PAGE and analyzed by fluorography. (C) Transactivation of reporter gene by A110P G:CfE(DEF) and G:DmE(DEF) in response to nonsteroidal ligand, RG-100915. 3T3 cells were transfected with V:Hs-LmR(EF) chimera, pFRLUC, and WT G:DmE(DEF) or A110P G:CfE(DEF) and treated with RG-100915. Extracts were assayed for luciferase activity. The RLUs shown are mean ⫾ SD (n ⫽ 3). 14714 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.222278999

Kumar et al.

Selective Activation of A110P CfEcR Mutant by the Nonsteroidal Ligand, RG-100915. An effective gene switch should be activated not

by endogenous ligands but only by ligands exogenously administered. Conversely, the exogenous ligand should not activate the endogenous receptors. The A110P CfEcR mutant may be an efficient system for these applications. Several ligands were screened to identify a suitable A110P EcR–ligand combination that would activate only the CfEcR A110P mutant but not WT DmEcR. RG-100915 was identified as a suitable ligand. As shown in Fig. 4C, RG-100915 was able to induce reporter gene activity through the CfEcR A110P mutant-based gene switch but not through the DmEcR-based gene switch.

Discussion In this report, we describe a CfEcR mutant that responds to nonsteroidal ligands but not to steroids. We constructed a CfEcR homology model, docked 20E into the binding pocket, and identified critical amino acids involved in the binding to 20E. Some of the amino acid residues identified as critical for binding to 20E are the same as those identified in a previously reported homology model for CtEcR (11). Our model and the CtEcR model suggest that the ligand-binding pocket of EcRs consists of a bulky envelope and a shallow tube with a volume consistent with the size of 20E. The diacylhydrazine ligands, such as RH-5849 and RG-102240, are more compact than 20E (for a review, see ref. 20) and therefore are predicted to occupy only a part of the EcR-binding pocket (11). This leaves a few critical residues at the tube end of the binding pocket that are predicted to make contact with 20E but not with RH-5849 or RG-102240. We hypothesized that mutating the residues that are predicted to make contact with 20E only would affect the binding to steroids but not to nonsteroidal ligands. The residues that most affect RG-102240 binding and response, Y120, W238, L234, L230, I51, M125, F48, and A123, are located at the bottom half of the binding pocket (Fig. 1 and Table 1). Collectively, the homology model, the results of the reporter gene, and the binding assays lend credence to the proposed idea that the diacylhydrazine ligands occupy only the bottom part of the ligand-binding pocket. Of all of the mutants tested, the A110P mutation had the most significant reduction in PonA, MurA, and 20E binding and transactivation. However, both binding and transactivation through RG-102240 and RG-102317 were relatively unaffected for this mutant. This mutation could change the local conformation of protein backbone, because the current C␣ is nearly 180° and must be reduced significantly to allow proline into the sequence. This conformational change may obstruct the space where the Cterminal side chain extends, preventing PonA, MurA, and 20E from properly binding. Because the steric bulk of methionine and espe1. Koelle, M. R., Talbot, W. S., Segraves, W. A., Bender, M. T., Cherbas, P. & Hogness, D. S. (1991) Cell 67, 59–77. 2. Perera, S. C., Sundaram, M., Krell, P. J., Retnakaran, A., Dhadialla, T. S. & Palli, S. R. (1999) Arch. Insect Biochem. Physiol. 41, 61–70. 3. Yao, T. P., Forman, B. M., Jiang, Z., Cherbas, L., Chen, J. D., McKeown, M., Cherbas, P. & Evans, R. M. (1993) Nature 366, 476–479. 4. Yao, T. P., Segraves, W. A., Oro, A. E., McKeown, M. & Evans, R. M. (1992) Cell 71, 63–72. 5. Arbeitman, M. N. & Hogness, D. S. (2000) Cell 101, 67–77. 6. Talbot, W. S., Swyryd, E. A. & Hogness, D. S. (1993) Cell 73, 1323–1337. 7. Bender, M., Imam, F. B., Talbot, W. S., Ganetzky, B. & Hogness, D. S. (1997) Cell 91, 777–788. 8. Dhadialla, T. S., Carlson, G. R. & Le, D. P. (1998) Annu. Rev. Entomol. 43, 545–569. 9. Billas, I. M., Moulinier, L., Rochel, N. & Moras, D. (2001) J. Biol. Chem. 276, 7465–7474. 10. Clayton, G. M., Peak-Chew, S. Y., Evans, R. M. & Schwabe, J. W. (2001) Proc. Natl. Acad. Sci. USA 98, 1549–1554. 11. Wurtz, J. M., Guillot, B., Fagart, J., Moras, D., Tietjen, K. & Schindler, M. (2000) Protein Sci. 9, 1073–1084. 12. Fussenegger, M. (2001) Biotechnol. Prog. 17, 1–51. 13. Christopherson, K. S., Mark, M. R., Bajaj, V. & Godowski, P. J. (1992) Proc. Natl. Acad. Sci. USA 89, 6314–6318. 14. No, D., Yao, T. P. & Evans, R. M. (1996) Proc. Natl. Acad. Sci. USA 93, 3346–3351. 15. Swevers, L., Drevet, J. R., Lunke, M. D. & Iatrou, K. (1995) Insect Biochem. Mol. Biol. 25, 857–866.

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cially leucine did not completely block binding, it seems that the backbone distortion negatively perturbs binding affinity much more than steric occlusion. According to our homology model, the A110 residue is located at the tube end of the binding pocket, and RG-102240 and RG-102317 are predicted to occupy only the bottom part of the binding pocket, resulting in no significant effect of this mutation on binding and response to these ligands. The A110 residue is highly conserved among the EcRs, except in the case of EcRs from T. molitor and U. pugilator, and is located in the loop between helices 5 and 6 in the predicted secondary structure. It appears to be in a conserved stretch of amino acids, several of which are hydrophobic amino acids; hydrophobic amino acids are predominant in the ligand-binding pocket of many steroid hormone receptors (25). The CtEcR homology model (11) and the model presented here identified several common amino acid residues, which are predicted to be important for 20E binding. However, the A110 residue was not one of the residues identified in the CtEcR model. The gene switch based on the A110P mutant of CfEcR responds to the nonsteroidal ligands RG-102240 and RG-102317 but not to the steroids 20E, PonA, and MurA. This gene switch would be useful for applications in Drosophila and other insects where the WT EcR would be activated by the endogenous 20E. We identified a nonsteroidal ligand, RG-100915, that can induce the reporter gene through the A110P CfEcR-based gene switch but not through the DmEcR-based gene switch. This ligand and the A110P CfEcRbased gene switch can be used for controlling gene expression in transgenic Drosophila. This system needs to be directly tested in vivo in Drosophila. Such a system would offer an ideal way of controlling exogenous genes to understand basic cellular pathways and also for generating invertebrate disease models. Drosophila models have been generated for neurodegenerative diseases like Alzheimer’s, Huntington’s, and Parkinson’s (for recent reviews, see refs. 26 and 27). Transgenic Drosophila are being engineered to express human proteins associated with these diseases. The A110P mutant EcRbased gene switch could potentially offer several advantages over existing systems for applications in plants, animals, and humans, where existing steroid receptor-based gene switches may be activated by endogenous steroids. In addition, the A110P mutant CfEcR is also useful for the regulation of multiple genes simultaneously. In combination with a mutant EcR that is activated by PonA, but not by RG-102240, the A110P mutant can be used to regulate two genes. Such a system is currently under development. We thank Dr. D. E. Cress for project management; Drs. M. Padidam, R. E. Horman, and C. Jolly-Tornetta for critical reading of the manuscript; Dr. M. Koelle (Stanford Univerity) for the gift of pMK43.2 reporter vector; Drs. P. Cherbas and L. Cherbas (Indiana University) for the gift of L57 cells; A. Raikhel (University of California) for the gift of AaEcR; K. Iatrou (NCSR ‘‘Demokritos,’’ Athens) for the gift of BmEcr; and two anonymous reviewers for helpful comments on the manuscript. 16. Suhr, S. T., Gil, E. B., Senut, M. C. & Gage, F. H. (1998) Proc. Natl. Acad. Sci. USA 95, 7999–8004. 17. Tran, H. T., Askari, H. B., Shaaban, S., Price, L., Palli, S. R., Dhadialla, T. S., Carlson, G. R. & Butt, T. R. (2001) Mol. Endocrinol. 15, 1140–1153. 18. Wing, K. D. (1988) Science 241, 467–469. 19. Carlson, G. R., Dhadialla, T. S., Hunter, R., Jansson, R. K., Jany, C. S., Lidert, Z. & Slawecki, R. A. (2001) Pest Manag. Sci. 57, 115–119. 20. Kothapalli, R., Palli, S. R., Ladd, T. R., Sohi, S. S., Cress, D., Dhadialla, T. S., Tzertzinis, G. & Retnakaran, A. (1995) Dev. Genet. 17, 319–330. 21. Hayward, D. C., Bastiani, M. J., Truman, J. W., Riddiford, L. M. & Ball, E. E. (1999) Dev. Genes Evol. 209, 564–571. 22. Mouillet, J. F., Delbecque, J. P., Quennedey, B. & Delachambre, J. (1997) Eur. J. Biochem. 248, 856–863. 23. Chung, A. C., Durica, D. S., Clifton, S. W., Roe, B. A. & Hopkins, P. M. (1998) Mol. Cell Endocrinol. 139, 209–227. 24. Cho, W. L., Kapitskaya, M. Z. & Raikhel, A. S. (1995) Insect Biochem. Mol. Biol. 25, 19 –27. 25. Darimont, B. D., Wagner, R. L., Apriletti, J. W., Stallcup, M. R., Kushner, P. J., Baxter, J. D., Fletterick, R. J. & Yamamoto, K. R. (1998) Genes Dev. 12, 3343–3356. 26. Link, C. D. (2001) Mech. Ageing Dev. 122, 1639–1649. 27. Beal, M. F. (2001) Nat. Rev. Neurosci. 2, 325–334.

PNAS 兩 November 12, 2002 兩 vol. 99 兩 no. 23 兩 14715

BIOCHEMISTRY

5). These results suggest that the GRIP1 interaction is impaired by the introduction of the A110P mutation into EcR.