Studies on two ecdysone receptor isoforms of the spruce budworm, Choristoneura fumiferana

A full-length cDNA clone corresponding to the Choristoneura fumiferana ecdy sone receptor-a isoform (CfEcR-A) was isolated. The deduced amino acid sequ ence of CfEcR-A differed from CfEcR-B in the NH2-terminal region of the A/B domain. The CfEcR-A-specific region showed high amino acid identity with E cR-A isoforms of Manduca sexta, Bombyx mori, Drosophila melanogaster and Te nebrio molitor. Isoform-specific probes were used to study the expression o f EcR-A and EcR-B mRNAs. Both probes detected 6 kb mRNAs that were present in second-sixth larval instars and in the pupae. Both EcR-A and EcR-B mRNA levels increased during the molting periods. In the sixth instar larvae, th e increase in EcR-A and EcR-B mRNA levels were more pronounced in the midgu t than in epidermis and fat body. Both EcR-A and EcR-B mRNAs were induced i n CF-203 cells (a cell line developed from C. fumiferana midgut) grown in t he presence of 4 x 10(-6) M 20E. EcR-B specific mRNAs were induced within 1 h of exposure to 20E, but EcR-A specific mRNAs were induced only after 3 h of exposure to 20E. Induction of mRNAs for both isoforms was unaffected by the presence of a protein synthesis inhibitor, cyclohexamide, in the cultu re medium. RH-5992, a stable ecdysone agonist, caused a similar induction p attern of EcR-A and EcR-B mRNAs in the midgut, epidermis and fat body of si xth instar larvae. In vitro translated CfEcR-A, CfEcR-B and CfUSP proteins were used to study the DNA binding and ligand binding properties of EcR-A/U SP and EcR-B/USP protein complexes. The K-d values indicated that both comp lexes have similar binding affinities for ecdysone response elements and po nasterone A. (C) 1999 Published by Elsevier Science Ireland Ltd. All rights reserved.