WU, X*; DURICA, DAVID/S; HOPKINS, PENNY/M; University of Oklahoma; University of Oklahoma; University of Oklahoma: Physical characterization of the ecdysteroid and retinoid X receptors (UpEcR and UpRXR) in the fiddler crab, Uca pugilator and their distribution patterns in regenerating limbs during the molt cycle

Several UpRXR cDNA variants were found in regions that could potentially influence function. A 5 aa insertion/ deletion is located in the “T” box in the D domain. Another 33 aa insertion/deletion is found inside the ligand binding domain, between helix 1 and helix 3. Expression vectors for these UpRXR variants and UpEcR were constructed, and proteins expressed in E.coli and in vitro TNT expression systems. The expressed crab nuclear receptors were then characterized by EMSA and GST-pull down experiments. The pull-down results show UpEcR interactions with those UpRXR variants that have the 33 aa insertion, but not with those lacking the 33 aa insertion. Although weak UpRXR protein-protein binding is observed, only pairing between -33 and +33 UpRXR LBD domains results in a pull-down. EMSA results show that UpEcR/UpRXR heterocomplexes bind with a IRper-1 hormone responsive element only if UpRXR contains the 33 aa insertion. UpRXR lacking both the 5 aa and 33 aa insertion however, is found to bind a DR-1 HRE as a homocomplex. These in vitro protein-protein and protein-DNA interactions are hormone (20-hydroxy-ecdysone and 9-cis-retinoid acid) independent. Temporal and spatial distribution of UpEcR and UpRXR were studied by immunohistochemical analysis. Polyclonal antibodies against E.coli expressed A/B domain and common domain proteins of UpEcR and UpRXR were applied to limb sections throughout the regeneration cycle. A large group of tissues and cell populations are immuno-reactive to UpEcR and UpRXR antibodies. The immuno-reactive patterns suggest that UpEcR and UpRXR are often co-localized.