HOPKINS, P.M.; DURICA, D.S.; WU, X.; University of Oklahoma: Binding Properties of the crustacean ecdysteroid receptor UpEcR/RXR from the fiddler crab, Uca pugilator .

Growth, reproduction, and regeneration in the fiddler crab,Uca pugilator, are thought to be regulated by a suite of hormones � the ecdysteroids. These polyhydroxylated steroids exert their influence through interactions with the putative ecdysteroid receptor, UpEcR/UpRXR. We have cloned and sequenced the genes for these two nuclear receptor proteins and expressed them in a number of systems. When expressed in reticulocyte lysates, the UpEcR/UpRXR heterodimers are capable of binding to several defined hormone responsive elements (HRE�s). When the two proteins are co-expressed in SF-9 insect cell lines, binding of the UpEcR/UpRXR heterodimer to the IRper-1 HRE (inverted perfect repeat with 1 base spacer) is also observed; as in reticulocyte lysates, both proteins are required for significant binding to this element. Using saturation binding assays and specific competition assays, we have also shown that the dimer will bind to specific ligands. The binding affinity of the UpEcR/UpRXR dimer to the steroid ligands is highest for Ponasterone A (PA) when radio labeled PA is the competing ligand. Studies are currently underway to study the effects of ligands on protein-DNA interactions.