Progress 10/01/05 to 09/30/06
Outputs
During the third year of the funding period, several cloning projects and studies supporting the aims and objectives of the proposed project were carried out: 1. EcR (A and B isoforms) and USP cell culture vectors derived from cDNAs encoding the functional components of the Leptinotarsa decemlineata (Colorado potato beetle)were constructed and tested in the cell culture system that was previously used to test the Drosophila melanogaster EcR and USP isoforms. 2. Comparative studies revealed that the effect of ecdysteroids and nonsteroidal agonists was considerably different between beetles and flies, indicating that the cell culture assay provides a valid basis for differentiating the effects of test ligands in these two insect species. 3. The beetle receptor is not as amenable to the effects of JH potentiation as previously shown for the fly EcR/USP heterodimer. 4. The beetle USP behaves differently than the fly USP as a heterodimeric partner for EcR. Particularly, the
beetle USP shows the ability to activate transcription as an EcR partner even without a DNA-binding domain, whereas the fly USP behaves as a dominant negative (forming an inactive complex with EcR). This finding infers that there are functional differences among the USPs from different insect species. 5. Transgenic vectors were constructed for the purpose of carrying out in vivo experiments to test the dominant negative characteristics of the fly USP. It is predicted that the fly USP will behave as a dominant negative in tissues utilizing the EcR-A and EcR-B2 isoforms. However, cell culture results suggest the possibility that this form of USP will retain its ability to form an active dimer with EcR-B1.
Impacts
The current project has resulted in an assay for testing both inducers and modulators of the insect ecdysteroid receptor from the Colorado potato beetle and flies. The availability of this test offers the possibility for high-throughput screening of articificial and natural compounds for their ability to perturb insect receptor function, and this mode of action has proven to be a viable target for insecticides that disrupt feeding and growth stages of insects. Moreover, the assay can be utilized to screen for candidate insecticides based on their specificity for affecting a specific insect species' receptor, offering the possibility for developing targeted insecticides. This in turn, may offer species-specific strategies for controlling insect populations, that minimize the impact upon other nonharmful insect species.
Publications
- Nieva C, Gwozdz T, Dutko-Gwozdz J, Wiedenmann J, Spindler-Barth M, Wieczorek E, Dobrucki J, Dus D, Henrich V, Ozyhar A, Spindler KD. (2005) Ultraspiracle promotes the nuclear localization of ecdysteroid receptor in mammalian cells. Biol. Chem. 386:463-470.
- Beatty, JM, T Fauth, JC Callender, M Spindler-Barth, and VC Henrich (2006) Analysis of transcriptional activity mediated by Drosophila melanogaster ecdysone receptor isoforms in a heterologous cell culture system. Insect Mol. Biol. 15, 785-795.
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Progress 10/01/04 to 09/30/05
Outputs
The major points of progress in the last year have focused on the effect of structural modifications upon Drosophila ecdysteroid receptor activity: 1. The three natural isoforms (A, B1, and B2) of Drosophila EcR exhibit different levels of transcriptional activity, and the B2 isoform is the most strongly potentiated by juvenile hormone in cell culture experiments. Other studies have shown that B2 is the most efficient form for rescuing larval development in Drosophila mutants, suggesting a functional corollary in vivo. 2. The EcRB1 isoform mediates the highest levels of transcriptional activity and is the only isoform that can mediate ecdysteroid-induced gene expression in the presence of a USP protein that is lacking a DNA-binding domain. 3. The USP protein lacking a DNA-binding domain is unable to mediate JH potentiation of any of the three natural EcR isoforms. 4. A site-directed mutation in the shared ligand-binding domain of the three EcR isoforms causes a
strongly elevated ligand-independent transcriptional activity, but only in the B2 isoform. The constitutive transcriptional activity is associated with a high affinity for a known ecdysone responsive promoter element. 5. A site-directed mutation that has not been associated with ligand-binding through EcR computational modeling destroys ligand-induced transcriptional activity in all three isoforms. 6. In collaboration with Klaus Dieter-Spindler, we have shown that USP is localized in the nucleus, and that EcR translocates to the nucleus in the presence of ecdysteroid, revealing a potential target for insecticidal development. 7. The level and nature of EcR-mediated transcriptional activity is altered when USP is derived from different insect species, demonstrating that USP plays a role in determining the responsiveness and specificity of the receptor complex. The following results have also been obtained that are relevant to the aims presented in the initial proposal: 1. Site-directed
mutations of hypothetical phosphorylation sites exert no effect on in in vitro transcriptional capabilities. Moreover, the cell culture system does not appear to phosphorylate USP, based on Western blotting, so this aspect of transcriptional regulation will require in vivo studies. 2.So far, site-directed mutations of predicted JHIII binding sites in USP have failed to perturb the JH potentiation of transcriptional activity that we had reported previously. 3.When transfected into our cell culture system, the Drosophila MET protein did not influence JH potentiation, though a second protein may be necessary for MET action, based on recent results from Currently, we are undertaking the following activities to fulfill the goals of the USDA funding period: (1) subcloning the EcR and USP of Leptinotarsa into cell culture vectors in order to compare responsiveness with the Drosophila USP, (2) testing Drosophila transformant lines to assess the effects of site-directed EcR and USP mutations
that we had previously made.
Impacts
From the studies currently underway, the mode of action by which JH potentiation of an insect ecdysteroid receptor occurs will be explored, and the possibility of targeting insecticides through this mode of action will be assessed. Further, the utility of the assay as a basis for isolating compounds that act in a broad species or species-specific fashion will be evalauted. Ultimately, the assay can serve as a means for isolating novel compounds on the basis of their activity before undertaking the expense and energy of field trials for testing insecticidal activity.
Publications
- Bergman, T., V.C. Henrich, U. Schlattner, and M. Lezzi (2004) Ligand control of in vivo interaction between ecdysteroid receptor and Ultraspiracle ligand binding domain. Biochem. J., 378, 779-784.
- Gaziova, I, P Bonnette, V.C. Henrich, and M Jindra (2004) Cell autonomous roles of the ecdysoneless gene in Drosophila development and oogenesis. Development, 131: 2715-2725.
- Henrich, V (2005) The Ecdysteroid Receptor. In: Comprehensive Insect Physiology, Biochemistry, and Molecular Biology Series, Volume 3. Elsevier Press. pp. 243-286.
- Nieva C, Gwozdz T, Dutko-Gwozdz J, Wiedenmann J, Spindler-Barth M, Wieczorek E, Dobrucki J, Dus D, Henrich V, Ozyhar A, Spindler KD. (2005) Ultraspiracle promotes the nuclear localization of ecdysteroid receptor in mammalian cells. Biol. Chem. 386, 463-470.
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