Characterization of the locust retinoid X receptors
Nowickyj, Shaun M.
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The retinoid X receptor (RXR) participates in a multitude of nuclear receptor signaling pathways and is induced by its highly sought-after cognate ligand, 9-cis-retinoic acid (9-cis-RA). In flies and moths, molting is mediated by the ecdysone receptor that consists of a heterodimer comprising the ecdysone receptor monomer (EcR) and the invertebrate RXR homolog ultraspiracle (USP); the latter, however, is believed to have diverged from its RXR origin. From the more evolutionarily-primitive insect Locusta migratoria (Lm), long and short RXR transcripts (LmRXR-L and LmRXR-S, respectively) were detected during embryogenesis. This thesis reports the immunochemical detection of RXR cross-reactive material throughout Locusta embryogenesis, suggesting that the protein may have another role besides ecdysone signaling. Thus, the RXR isoforms were cloned for recombinant expression and purification in order to demonstrate retinoid specificity. Both isoforms bound 9-cis-RA and all-trans-RA with high affinity. Binding was further corroborated by the identification of endogenous retinoids during embryogenesis. Embryos were first subjected to modified “Bligh and Dyer” as well as solid phase extractions to circumvent oil precipitation that rendered whole homogenates unsuitable for retinoid assay and detection. The RA-inducible Cyp26A1-promoter reporter cell line identified the presence of endogenous RAs (5.4 nM) from insect embryo extracts. Finally, high pressure liquid chromatography followed by mass spectroscopy (HPLC/MS) confirmed the identity of all-trans-RA and the more abundant 9-cis-RA (1.3 nM). These findings suggest a functional role for 9-cis-RA in the invertebrate embryo and favour signaling through the combination of 9-cis-RA and RXR in evolutionarily early RA-driven animal development.