DURICA, D.S.; WU, X.; HOPKINS, P.M.: Protein-Protein and DNA Binding Properties of a Crustacean Ecdysteroid Receptor.

To facilitate physical characterization of their binding properties, we have cloned into expression vectors the entire coding regions of the two components of the Uca pugilator ecdysteroid receptor homolog, UpEcR (Genbank AF034086) and UpRXR (GenBank AF32983). GST-pull down experiments tested whether radiolabeled in vitro synthesized UpEcR and UpRXR proteins are capable of interacting with GST-UpRXR. UpEcR and UpRXR are capable of protein/protein interactions, in the absence of ligand, but RXR/RXR interactions were not detected under these experimental conditions. In vitro synthesized UpEcR and UpRXR were also used in electrophoretic mobility shift assays to determine the hormone response element (HRE) binding properties of the receptor. For most response elements tested, both UpEcR and UpRXR were required for HRE binding. For some elements, weak band shifts were observed with UpEcR alone, perhaps through complexes formed with endogenous RXR in the lysate. Band shifts were observed in the absence of 20E, and the addition of hormone had no effect on DNA binding. As in insects, the receptor binds to a broad spectrum of HREs, including both perfect and imperfect inverted (IR) and direct (DR) repeats of various spacer lengths. For elements containing perfect repeats, competition experiments indicate relative affinities of IR-1>DR-4>IR-2>IR-3. Saturation binding assays indicate relative Kds comparable to those observed for characterized insect receptors (~0.44-2.3 nM).