Meeting Abstract

P2.13  Tuesday, Jan. 5  EcR/RXR LBD Isoforms in Crustacea DURICA, DS*; PHILLIPS, B; HOPKINS, PM; University of Oklahoma; University of Oklahoma; University of Oklahoma ddurica@ou.edu

The primary amino acid sequence encoded by the U. pugilator EcR and RXR genes was previously elucidated by cDNA sequencing. These studies indicated isoforms within the hinge and ligand binding domain (LBD) of RXR and the hinge region of EcR. An isoform missing the C-terminal region of EcR LBD was observed, but N-terminal isoforms were not detected, suggesting the LBD variants might have different physiological functions. We describe here the intron-exon organization of the UpEcR gene determined from overlapping genomic clones. A contig covering a total of 9320 bases was assembled identifying seven exons and six introns. An intron was observed within the A/B region of the EcR gene, leaving the possibility for alternative A/B splicing. Hinge region isoforms arising through alternative splicing were identified; in addition, a cryptic exon in the ligand binding domain was detected and evidence for the expression of this isoform was obtained from RT-PCR. To examine whether the LBD isoforms of Uca RXR might have different physiological properties in vitro, EMSA analyses were performed with different isoforms of the Drosophila receptors. The cognate crab and fly receptors differ in EMSA assays in their responses to hormone in that neither 20E nor PA increases DNA binding for the Uca heterodimers, while ecdysteroids stimulate binding for the Drosophila EcR/USP heterodimers. In mixing experiments, UpEcR/DmUSP heterodimers did not show ligand responsive differences in DNA binding; both Uca LBD isoforms, however, conferred ligand responsive increases in DNA binding with DmEcRs, with differing effectiveness relative to different DmEcR isoforms. These data indicate that both UpRXR LBD isoforms can heterodimerize with the heterologous DmEcR receptors and promote ligand and DNA binding, but suggest each may promote isoform-specific differences in EcR conformation.