Molecular determinants of differential ligand sensitivities of insect ecdysteroid receptors

Abstract

The functional receptor for insect ecdysteroid hormones is a heterodimer consisting of two nuclear hormone receptors, ecdysteroid receptor (EcR) and the retinoid X receptor homologue Ultraspiracle (USP). Although ecdysone is commonly thought to be a hormone precursor and 20-hydroxyecdysone (20E), the physiologically active steroid, little is known about the relative activity of ecdysteroids in various arthropods. As a step toward characterization of potential differential ligand recognition, we have analyzed the activities of various ecdysteroids using gel mobility shift assays and transfection assays in Schneider-2 (S2) cells. Ecdysone showed little activation of the Drosophila melanogaster receptor complex (DmEcR-USP). In contrast, this steroid functioned as a potent ligand for the mosquito Aedes aegypti receptor complex (AaEcR-USP), significantly enhancing DNA binding and transactivating a reporter gene in S2 cells. The mosquito receptor also displayed higher hormone-independent DNA binding activity than the Drosophila receptor. Subunit-swapping experiments indicated that the EcR protein, not the USP protein, was responsible for ligand specificity. Using domain-swapping techniques, we made a series of Aedes and Drosophila EcR chimeric constructs. Differential ligand responsiveness was mapped near the C terminus of the ligand binding domain, within the identity box previously implicated in the dimerization specificity of nuclear receptors. This region includes helices 9 and 10, as determined by comparison with available crystal structures obtained from other nuclear receptors. Site-directed mutagenesis revealed that Phe529 in Aedes EcR, corresponding to Tyr611 in Drosophila EcR, was most critical for ligand specificity and hormone-independent DNA binding activity. These results demonstrated that ecdysone could function as a bona fide ligand in a species-specific manner.

FIG. 1

Differential effects of ecdysteroids on receptor DNA binding activities. (A) In vitro-translated AaEcR and AaUSP proteins were incubated with ³²P-labeled IR^hsp-1 EcRE in the absence of ligand (lane 1) or in the presence of 5 × 10⁻⁵ M 20E (lane 2), ecdysone (lane 3), 2DE (lane 4), 22A (lane 5), PolB (lane 6), PonA (lane 7), or MurA (lane 8). The reaction mixtures were subjected to EMSA and autoradiography. (B) Same as panel A except that DmEcR and DmUSP were used as receptor proteins. The molar amount of DmEcR and DmUSP proteins was 50 times more than that of AaEcR and AaUSP so that any trace DNA binding activity of DmEcR-DmUSP could be detected.

FIG. 2

AaEcR conferred specific response to ecdysone. In vitro-translated proteins AaEcR and AaUSP (lanes 1 to 3), DmEcR and DmUSP (lanes 4 to 6), AaEcR and DmUSP (lanes 7 to 9), or DmEcR and AaUSP (lanes 10 to 12) were incubated with 50 fmol of ³²P-labeled IR^hsp-1 EcRE probe either in the absence of hormone (lanes 1, 4, 7, and 10) or in the presence of 5 × 10⁻⁵ M 20E (lanes 2, 5, 8, and 11) or ecdysone (lanes 3, 6, 9, and 12). The reaction mixtures were subjected to EMSA and autoradiography. The molar amount of DmEcR and DmUSP proteins was 50 times more than that of AaEcR and AaUSP so that any trace DNA binding activity of DmEcR-DmUSP could be detected. Free probe is indicated by an asterisk. Complexes containing AaEcR and DmEcR proteins are indicated by solid and open arrowheads, respectively.

FIG. 3

(A) Ecdysone (10⁻⁵ M) more potently activated AaEcR than DmEcR in S2 cells. S2 cells were transfected with 25 ng of reporter pAc5-LacZ and 100 ng of reporter plasmid Eip-Luc with no expression plasmid (column 1) or with 12.5 ng each of AaEcR, AaUSP, DmEcR, and DmUSP expression vectors in pairwise combinations: AaEcR and AaUSP (column 2), AaEcR and DmUSP (column 3), DmEcR and AaUSP (column 4), and DmEcR and DmUSP (column 5). After transfection, cells were incubated either in the absence of hormone or in the presence of 5 × 10⁻⁵ M 20E or ecdysone for 36 h and harvested for β-galactosidase and luciferase activities. (B) Ecdysone (10⁻⁶ M) highly activated only the Aedes receptor, not the Drosophila receptor. S2 cells (2.5 × 10⁵) were transfected with 12.5 ng of reporter pAc5-LacZ and 50 ng of reporter plasmid Eip-Luc (columns 1 to 3) or Hsp-Luc (columns 4 to 6) together with no expression plasmid (columns 1 and 4) or with 6.5 ng each of AaEcR and DmUSP (columns 2 and 5) or DmEcR and DmUSP (columns 3 and 6) expression vectors. After transfection, cells were incubated in the absence of hormone or in the presence of 10⁻⁶ M ecdysone (Ecd) or 20E for 24 h and harvested for β-galactosidase and luciferase activities. Luciferase activity was normalized with β-galactosidase activity. The results are expressed as fold induction of the luciferase activity from cells treated with hormone over that from cells treated with control vehicle ethanol.

FIG. 4

Dose-dependent transactivation by 20E and ecdysone in the presence of AaEcR or DmEcR. S2 cells (5 × 10⁵ cells/well) were transfected with 25 ng of reporter pAc5-LacZ and 100 ng of reporter plasmid Eip-Luc with 12.5 ng each of DmEcR and DmUSP (A) or AaEcR and DmUSP (B) expression vectors. After transfection, cells were incubated in the absence of hormone or in the presence of increasing concentrations (from 10⁻¹⁰ to 10⁻⁵ M) of 20E or ecdysone for 24 h, and harvested for β-galactosidase and luciferase activities. Luciferase activity was normalized with β-galactosidase activity. The results are expressed as fold induction of the luciferase activity from cells treated with hormone over that from cells treated with control vehicle ethanol. Error bars for some points are too small to be visible on the graph.

FIG. 5

(A) Localization of the ecdysone-specific region to the LBD: schematic diagram shows domain-swapping chimeric proteins and their responsiveness to ecdysone. ³²P-labeled probe IR^hsp-1 EcRE was incubated with in vitro-synthesized DmUSP protein paired with chimeric protein AE^BsrG1, DE^BsrG1, DE^Xma3, DE^Kpn1, or DE^Bgl2 in the absence of hormone or in the presence of 5 × 10⁻⁵ M 20E or ecdysone. Bound and free probes were resolved by EMSA followed by autoradiography. (B) C-terminus of EcR LBD determined ecdysone binding specificity: schematic diagram of subdomain-swapping (within LBD) chimeric EcR proteins and their responsiveness to 20E and ecdysone. ³²P-labeled probe IR^hsp-1 was incubated with in vitro-synthesized DmUSP protein paired with AE^Sac1, AE^Nru1, DE^Bbs1, DE^TthIII1, DE^Csp1, DE^Spe1, or DE^BsiW1 in the absence of hormone or in the presence of 5 × 10⁻⁵ M 20E or ecdysone. Bound and free probes were resolved by EMSA followed by autoradiography. Constructs whose EMSA results are shown in panel D are in bold. (C) Transferable ligand specificity subdomains in AaEcR and DmEcR. ³²P-labeled probe IR^hsp-1 was incubated with in vitro-synthesized DmUSP protein paired with in vitro-translated AE^NB or DE^SS in the absence of hormone or in the presence of 5 × 10⁻⁵ M 20E or ecdysone. Bound and free probes were resolved by EMSA followed by autoradiography. Responsiveness to 20E and ecdysone (Ecd) is indicated by a plus sign, while lack of responsiveness is indicated by a minus sign in the schematic diagrams. Solid bars denote DmEcR sequence, and open bars denote AaEcR sequences. Domains A/B, C (DBD), D, E (LBD), and F are pointed out above AaEcR and below DmEcR sequences. (D) Ecdysone responsiveness of critical chimeric proteins revealed by EMSA. The wild-type proteins AaEcR (lanes 1 to 3) and DmEcR (lanes 16 to 18) and chimeric proteins AE^Sac1 (lanes 4 to 6), AE^Nru1 (lanes 7 to 9), DE^Spe1 (lanes 10 to 12), and DE^BsiW1 (lanes 13 to 15) were paired with in vitro-synthesized DmUSP protein and then incubated with ³²P-labeled probe IR^hsp-1 in the absence of hormone (lanes 1, 4, 7, 10, 13, and 16) or in the presence of 5 × 10⁻⁵ M 20E (lane 2, 5, 8, 11, 14, and 17) or ecdysone (lanes 3, 6, 9, 12, 15, and 18). The reaction mixtures were resolved by EMSA followed by autoradiography.

FIG. 6

Identification of the critical amino acid affecting heterodimerization and responsiveness to ecdysone. (A) I box in EcR proteins. DmEcR (34) and AaEcR (12) protein sequences are aligned by GCG Bestfit. SpeI and BsiWI sites in AaEcR and DmEcR cDNAs are indicated by arrows. Nonconserved residues which were subjected to site-directed mutagenesis between SpeI and BsiWI sites are in bold. The critical residues F529 in AaEcR and Y611 in DmEcR are indicated by an asterisk. (B) F529 in AaEcR is critical for ligand specificity and hormone-free DNA binding activity. ³²P-labeled probe IR^hsp-1 was incubated with in vitro-synthesized DmUSP protein paired with the wild-type AaEcR (lanes 1 to 3) or point mutant AE^A520C (lanes 4 to 6), AE^P523S (lanes 7 to 9), AE^K524M (lanes 10 to 12), AE^C525S (lanes 13 to 15), AE^S526L (lanes 16 to 18), AE^I528F (lanes 19 to 21), or AE^F529Y (lanes 22 to 24) in the absence of hormone (lanes 1, 4, 7, 10, 13, 16, 19, and 22) or in the presence of 5 × 10⁻⁵ M 20E (lanes 2, 5, 8, 11, 14, 17, 20, and 23) or ecdysone (lanes 3, 6, 9, 12, 15, 18, 21, and 24). (C) Tyr611 in DmEcR is critical for ligand specificity and hormone-free DNA binding activity. ³²P-labeled probe IR^hsp-1 was incubated with in vitro-synthesized DmUSP protein paired with the wild-type protein DmEcR (lanes 1 to 3) or point mutant DE^C602A (lanes 4 to 6), DE^S605P (lanes 7 to 9), DE^M606K (lanes 10 to 12), DE^S607C (lanes 13 to 15), DE^L608S (lanes 16 to 18), DE^F610I (lanes 19 to 21), or DE^Y611F (lanes 22 to 24) in the absence of hormone (lanes 1, 4, 7, 10, 13, 16, 19, and 22) or in the presence of 5 × 10⁻⁵ M 20E (lanes 2, 5, 8, 11, 14, 17, 20, and 23) or ecdysone (lanes 3, 6, 9, 12, 15, 18, 21, and 24). Bound and free probes were resolved by EMSA followed by autoradiography. The molar amount of DmEcR and DE mutants protein was 10 times more than that of the molar amount of AaEcR and AE mutants in the EMSA.

FIG. 7

Putative I boxes in EcR proteins. I boxes of EcR protein sequences from 15 arthropod species are aligned by GCG Pileup. Helices 9 and 10 in human RARγ (HsRARγ) (48) are indicated by dotted lines. Residues affecting hormone-free DNA binding activity and ecdysone responsiveness in AaEcR and DmEcR are in bold. The most critical residue, Phe/Tyr, is in bold italics. DmEcR, CfEcR, BaEcR, and BmEcR are underlined as their proteins contain a Tyr at the critical ligand specificity site. Data bank search yielded EcR protein sequences from 16 species: 6 Diptera species, the Mediterranean fruit fly Ceratitis capitata (CcEcR [67]), the sheep blowfly Lucilia cuprina (LcEcR [23]), the yellow fever mosquitoes A. aegypti (AaEcR, 12) and A. albopictus (not shown, as its EcR I box is 100% identical to AaEcR [28]), the midge Chironomus tentans (CtEcR [27]), and D. melanogaster (DmEcR [34]); 6 Lepidoptera species, the spruce budworm C. fumiferana (CfEcR [35]), squinting bush brown Bicyclus anynana (BaEcR [R. K. Reinhardt, P. Weber, and P. B. Koch, submitted to GenBank, accession no. CAB63236]), the silkworm B. mori (BmEcR [32, 60]), the tobacco budworm Heliothis virescens (HvEcR [41]), the tobacco hornworm M. sexta (MsEcR [18]), and the buckeye Junonia coenia (JcEcR [R. K. Reinhardt, P. Weber, and P. B. Koch submitted to GenBank, accession no. CAB63485]); 1 Orthoptera species, the migratory locust Locusta migratoria (LmEcR [55]); 1 Coleoptera species, the yellow mealworm Tenebrio moliter (TmEcR [42]); 1 crustacean species, the Atlantic sand fiddler crab Celuca pugilator (CpEcR [13]); and 1 Ixodidae species, the tick Amblyomma americanum (AamEcR [20]).