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, Anthon omus grandis (BRL-AG-2), was used to study morphological and biochemic al responses to 20-hydroxyecdysone (20E). The cells respond to 10(-6) M 20E by inhibition of cell growth and enhanced production of some sec reted proteins. Crude nuclear extracts containing the ecdysteroid rece ptor complex proteins consisting of the ecdysteroid receptor (EcR) and ultraspiracle (USP) bound ponasterone A with a K-d of 6.1 nM. Bound r adiolabeled ponasterone A was displaced by both 20E and the lepidopter an-specific non-steroidal ecdysteroid agonist, RH-5992, with 41- and a bout 1,900-fold higher K-d values, respectively. We identified the ecd ysteroid receptor components in this cell line, using monoclonal antib odies against the Drosophila ecdysteroid receptor (DmEcR) and ultraspi racle (DmUSP) proteins. A predominant band of about 70 kDa was detecte d with anti-EcR, and multiple bands ranging from 50-55 kDa were detect ed with anti-USP in the A. grandis extracts. Using degenerate primer R T-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 th is cell line for the study of ecdysone response and the isolation of t he receptor components in A. grandis. (C) 1997 Wiley-Liss, Inc.