In this study, we cloned and expressed the full length cDNAs of three ecdysone receptors including two EcR isoforms and one USP from P. xylostella. PxEcRA was obtained from the adult female, whereas, PxEcRB was readily obtainable in each and every developmental stage. We also determined the expression profiles of their respective mRNAs in the 4th instar larvae as well as the entire developmental stages. Furthermore, when co-expressed with USP, the binding affinities of these EcR isoforms were examined.
Two EcR isoforms, PxEcRA and PxEcRB, differ in their A/B domain. These two isoforms belong to the Type 2 A and Type 6 B1 isoform, respectively, according to Watanabe et al.. The N-terminal A/B region of EcRA and EcRB is isoform-specific, and might be an essential structural basis for the transcriptional activation functions[2, 30, 31]. The (K/R)RRW motif of the EcR-B1 isoform provides additional interaction sites for co-regulatory proteins and mediates the regulation of the B1 isoform-specific AF1 transactivation function. In this study, for both PxEcRA and PxEcRB, two splicing variants with a 15 bp difference in the hinge domain were identified. A 15bp difference has also been reported in the splicing variants from M. sexta, C. suppressalis, and Leptinotarsa decemlineata. However, the functional significance of these structural differences between the two variants has yet been fully characterized. In C. suppressalis, however, the lack of five amino acids (encoded by 15 nt) in the middle of D region of EcR did not affect the ligand-receptor binding. Similar to EcRs, multiple USP isoforms have been found in lepidopteran M. sexta and H. armigera[33, 34], dipteran Aedes aegypti and Chironomus tentans[35, 36], and coleopteran Tribolium castaneum. In this study, however, only one USP isoform was obtained in P. xylostella. Other insect species containing only one USP isoform includes D. melanogaster, Chilo suppressalis and Choristoneura fumiferana.
Results from the ligand-binding assay demonstrated that 1) tritiated PonA specifically bound to PxEcRB but not PxUSP and PxEcRA, and the specific binding to PxEcRB was enhanced by the addition of PxUSP. This is consistent with previous observations in C. suppressalis, L. decemlineata, C. tentans[36, 41] and D. melanogaster; and 2) the presence of PxUSP could not enhance the binding of tritiated PonA to PxEcRA. Similar result was observed in the Dwarf Wood scorpion, Liocheles australasiae, where the retinoid X receptor did not enhance the binding of tritiated PonA to L. australasiae ecdysone receptor-A. Unlike C. fumiferana, where the two isoform complexes have similar binding affinities for ponasternone A, there is a distinct binding affinity between PxEcRA/USP and PxEcRB/USP.
The dissociation constant (Kd =3.0±1.7 nM) for the PxEcRB/USP heterodimer is comparable to EcR/USP complex from other insects, such as in lepidopteran 1.0-2.0 nM[27, 44, 45], coleopteran 2.8 nM for L. decemlineata, dipsteran 0.9-2.8nM[2, 46], pentatomomor-phan 6.8 nM for Nezara viridula, and orthopteran 1.2 nM for Locusta migratoria. Comparative analysis of receptor-binding affinity among insects sheds light on the divergence of the toxicity of ecdysone agonists.
Previous studies showed that the expression profiles of different isoforms vary among developmental stages and tissues. In D. melanogaster, EcRA isoform predominately locates at the imaginal discs, imaginal rings, and in two sets of specialized larval cells (the prothoracic gland cells). In addition, the EcRA isoform is associated with the onset of new cuticle synthesis in every molt so its presence is not metamorphic-specific. In contrast, the EcRB1 isoform is found primarily in larval tissues and in the imaginal histoblasts that form the abdominal epithelium and the midgut of adult[49, 50]. Phenotypic analysis of mutants suggests that EcRA is required for pupal development and EcRB1 is necessary for normal metamorphic development. In Bombyx mori, during the larval-pupal transition, EcRB1 was broadly distributed in most tissues examined including midgut, epidermis, fat body, and the wing imaginal disc, while EcRA was found only in the anterior silk gland. In this study, mRNA expression levels of PxEcRB were consistently 6-fold higher than that of PxEcRA in P. xylostella throughout the entire developmental stages. By contrast, the expression of EcRA in L. decemlineata was significantly higher than that of EcRB1, and EcRA was predominantly found in larval tissues. The apparent spatial and temporal distribution discrepancies of EcR isoforms are likely due to the different species.