Two distinct subpopulations of ecdysone receptor complex in the female mosquito during vitellogenesis

The native functional ecdysone receptor complex, a heterodimer of the ecdysone receptor (EcR) and ultraspiracle (USP) proteins, was identified in the fat body of adult female mosquitoes, Aedes aegypti, through electrophoretic mobility shift assays (EMSA) using previously characterized Drosophila ecdysone response elements (EcREs). The use of different salt concentrations during preparation of nuclear extracts enabled us to characterize two distinct subpopulations of the receptor complex, one of which was high salt-sensitive and responsive to exogenous 20-hydroxyecdysone (20E), and the other of which was high salt-resistant and refractory to exogenous 20E. Salt-sensitivity correlated with ligand responsiveness. Developmental EMSA analyses demonstrated that previtellogenic fat body nuclei and nuclei from the termination phase of vitellogenesis with low 20E titer contained solely high-salt-sensitive, ligand responsive complexes, which could be recovered in nuclear extracts (NEs) only by low salt tissue homogenization, suggesting these complexes were unliganded. In contrast, the fat body nuclei from stages of active vitellogenesis with high 20E titer contained almost exclusively high salt-resistant, ligand refractory complexes, implying these complexes were liganded; the nuclei from the intermediate stages, early and late phases of vitellogenesis, contained a mixture of the two subpopulations. The developmental profile of fully activated, ligand refractory receptor complexes closely correlated with that of yolk protein expression, suggesting an intimate involvement of the ecdysone receptor complex in both the induction and maintenance of high level expression of yolk protein genes.