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

The native functional ecdysone receptor complex, a heterodimer of the ecdys one 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 ecd ysone response elements (EcREs). The use of different salt concentrations d uring preparation of nuclear extracts enabled us to characterize two distin ct subpopulations of the receptor complex, one of which was high salt-sensi tive and responsive to exogenous 20-hydroxyecdysone (20E), and the other of which was high salt-resistant and refractory to exogenous 20E. Salt-sensit ivity correlated with ligand responsiveness. Developmental EMSA analyses de monstrated that previtellogenic fat body nuclei and nuclei from the termina tion 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 co mplexes were unliganded. In contrast, the fat body nuclei from stages of ac tive vitellogenesis with high 20E titer contained almost exclusively high s alt-resistant, ligand refractory complexes, implying these complexes were l iganded; the nuclei from the intermediate stages, early and late phases of vitellogenesis, contained a mixture of the two subpopulations. The developm ental profile of fully activated, ligand refractory receptor complexes clos ely correlated with that of yolk protein expression, suggesting an intimate involvement of the ecdysone receptor complex in both the induction and mai ntenance of high level expression of yolk protein genes. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.