Ecdysone and retinoid-X receptors of the blue crab, Callinectes sapidus: Cloning and their expression patterns in eyestalks and Y-organs during the molt cycle

- Isolate four putative isoforms of CasEcR and CasRXR cDNA sequences from Y-organs
- Identify the same domain structure and arrangement as other nuclear receptors
- Express both simultaneously in eyestalks and Y-organs but at different levels
- Exhibit translational regulatory elements: uORF and IRES in their 5′ UTRs

Crustacean molting is known to be regulated largely by ecdysteroids and crustacean hyperglycemic hormone (CHH) neuropeptide family including molt-inhibiting hormone (MIH) and CHH. The surge of 20-OH ecdysone and/or ponasterone A initiates the molting process through binding to its conserved heterodimeric nuclear receptor: Ecdysone Receptor (EcR) and Ultraspiracle (USP)/Retinoid-X Receptor (RXR). To better understand the role of ecdysteroids in the molt regulation, the full-length cDNAs of the blue crab, Callinectes sapidus EcR1 and RXR1 were isolated from the Y-organs and their expression levels were determined in both Y-organs and eyestalks at various molt stages. Y-organs show the expression of four putative isoforms of CasEcRs and CasRXRs which differ in the length of the open reading frame but share the same domain structures as in typical nuclear receptors: AF1, DBD, HR, LBD, and AF2. The putative CasEcR isoforms are derived from a 27-aa insert in the HR and a 49-aa residue substitution in the LBD. In contrast, an insertion of a 5-aa and/or a 45-aa in the DBD and LBD gives rise to CasRXR isoforms. The eyestalks and Y-organs show the co-expression of CasEcRs and CasRXRs but at the different levels. In the eyestalks, the expression levels of CasRXRs are 3-5 times higher than those of CasEcRs, while in Y-organs, CasRXRs are 2.5-4 times higher than CasEcRs. A tissue-specific response to the changes in the levels of hemolymphatic ecdysteroids indicates that these tissues may have differences in the sensitivity or responsiveness to ecdysteroids. The presence of upstream open reading frame and internal ribosome entry site in 5′ UTR sequences of C. sapidus and other arthropod EcR/RXR/USP analyzed by in silico indicates a plausible, strong control(s) of the translation of these receptors.