Meeting Abstract
10.3 Thursday, Jan. 3 Retinoid-X-receptor (RXR) isoforms in the fiddler crab, Uca pugilator HOPKINS, P.*; DURICA, D.; WASHINGTON, T.; Univ. of Oklahoma, Norman; Univ. of Oklahoma, Norman; Univ. of Oklahoma, Norman phopkins@ou.edu
Ecdysteroids exert their effects through a heterodimer formed by the ecdysteroid receptor(EcR)and another member of the nuclear receptor superfamily. The partner is usually a homologue of the retinoid-X-receptor(RXR). In the fiddler crab, there are at least two isoforms of RXR that differ from one another by a 33 amino acid insert in the ligand binding domain. Here we look at the functional differences of these two isoforms. Both isoforms are expressed in limb bud tissues of the crab in specific patterns and heterodimerize with UpEcR in the absence of ligand. In vitro binding of the functional UpEcR to ecdysteroids is, however, affected by the UpRXRs. When partnered with the smaller isoform, UpEcR binds with three fold greater affinity than when paired with the larger isoform (Equilibrium Kd�s = 7.7 nM versus 21.8 nM). UpEcR (with either of the isoforms) binds to Ponasterone A (Pon A) with greatest affinity, but Pon A has no effect on dimerization. UpEcR dimerization to the larger isoform is significantly increased in the presence of high levels of 20-hydroxyecdysone. Endogenous ligands for the UpRXRs are present in the regenerating limb bud blastema. Retinoids and their metabolites have been isolated from blastemas and identified using HPLC, UV absorption, and mass spectrometry. The apparent Kd for the putative RXR ligands is very high (0.2- 0.4 mM). Putative RXR ligands at high concentrations have little or no effect on in vitro homodimerization. At low concentrations, however, 9-cis-retinoic acid does inhibit the ability of UpEcR to bind PonA, perhaps by reducing heterodimerization (through inducing homodimerization). These variations in dimer function will be discussed in light of variations in circulating steroids in crab hemolymph during the regeneration and molting cycles.
