Characterization and partial cloning of ecdysteroid receptor from a cotton boll weevil embryonic cell line

A cell line derived from the embryos of the cotton boll weevil, Anthonomus grandis (BRL-AG-2), was used to study morphological and biochemical responses to 20-hydroxyecdysone (20E). The cells respond to 10(-6) M 20E by inhibition of cell growth and enhanced production of some secreted proteins. Crude nuclear extracts containing the ecdysteroid receptor complex proteins consisting of the ecdysteroid receptor (EcR) and ultraspiracle (USP) bound ponasterone A with a Kd of 6.1 nM. Bound radiolabeled ponasterone A was displaced by both 20E and the lepidopteran-specific non-steroidal ecdysteroid agonist, RH-5992, with 41- and about 1,900-fold higher Kd values, respectively. We identified the ecdysteroid receptor components in this cell line, using monoclonal antibodies against the Drosophila ecdysteroid receptor (DmEcR) and ultraspiracle (DmUSP) proteins. A predominant band of about 70 kDa was-detected with anti-EcR, and multiple bands ranging from 50-55 kDa were detected with anti-USP in the A. grandis extracts. Using degenerate primer RT-PCR, we isolated a 450 bp cDNA fragment of the putative AgEcR. Using this fragment as a probe, we identified a large mRNA of ca. 10 kb by Northern blot analysis. These results demonstrate the usefulness of this cell line for the study of ecdysone response and the isolation of the receptor components in A. grandis.