RESUMO
Ecdysteroid regulation of gene transcription in Drosophila melanogaster and other insects is mediated by a heterodimer comprised of Ultraspiracle (USP) and one of three ecdysone receptor (EcR) isoforms (A, B1 and B2). This study revealed that the EcR/USP heterodimer displays isoform-specific capabilities. EcRB1 is normally induced with a form of USP that is missing its DNA-binding domain (DBD), although potentiation by juvenile hormone (JH) III is reduced. The EcRA and B2 isoforms, however, display almost no response to ecdysteroids with the DBD(-) USP. A mutation, K497E, in the shared ligand-binding domain of the EcR isoforms caused elevated EcRB2-specific affinity for a canonical ecdysone response element. The effects of directed modification and mutagenesis offer a strategy for developing hypotheses and considerations for studying in vivo function.
Assuntos
Drosophila melanogaster/metabolismo , Proteínas de Insetos/metabolismo , Receptores de Esteroides/metabolismo , Transcrição Gênica , Animais , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Ecdisterona/análogos & derivados , Ecdisterona/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Immunoblotting , Proteínas de Insetos/genética , Mutagênese Sítio-Dirigida , Mutação/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Esteroides/genética , Elementos de Resposta/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
The ligand-binding domain (LBD) encompassing the C-terminal parts of the D- and the complete E-domains of the ecdysteroid receptor (EcR) fused to Gal4(AD) is present in two high molecular weight complexes (600 and 150 kDa) in yeast extracts according to size exclusion chromatography (Superdex 200 HR 10/30). Hormone binding is mainly associated with 150-kDa complexes. Complex formation is not influenced by hormone, but the ligand stabilizes the complexes at elevated salt concentrations. Mutational analysis of Gal4(AD)-EcR(LBD) revealed that formation of 600-kDa, but not 150-kDa, complexes depends on dimerization mediated by the EcR(LBD). Deletion of helix 12 is without effect. Mutation of K497 in helix 4, known to be essential for comodulator binding, abolishes 600-KDa complexes, but does not interfere with the formation of 150-kDa complexes. In contrast, the DE-domains of USP fused to Gal4(DBD) elute as monomer after elimination of the dimerization capacity of the ligand-binding domains by mutation of P463 in helix 10. The data presented here reveal that the complex formation of ligand-binding domains EcR and USP ligand is different.
