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

Srini C. Perera, Tim R. Ladd, T. S. Dhadialla, Peter J. Krell, Sardar S. Sohi, Arthur Retnakaran, Subba R. Palli

Research output: Contribution to journalArticlepeer-review

43 Scopus citations

Abstract

A full-length cDNA clone corresponding to the Choristoneura fumiferana ecdysone receptor-A isoform (CfEcR-A) was isolated. The deduced amino acid sequence 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 EcR-A isoforms of Manduca sexta, Bombyx mori, Drosophila melanogaster and Tenebrio molitot. Isoform-specific probes were used to study the expression of 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, the increase in EcR-A and EcR-B mRNA levels were more pronounced in the midgut than in epidermis and fat body. Both EcR-A and EcR-B mRNAs were induced in CF-203 cells (a cell line developed from C. fumiferana midgut) grown in the 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 culture medium. RH-5992, a stable ecdysone agonist, caused a similar induction pattern of EcR-A and EcR-B mRNAs in the midgut, epidermis and fat body of sixth 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/USP and EcR-B/USP protein complexes. The K(d) values indicated that both complexes have similar binding affinities for ecdysone response elements and ponasterone A.

Original languageEnglish
Pages (from-to)73-84
Number of pages12
JournalMolecular and Cellular Endocrinology
Volume152
Issue number1-2
DOIs
StatePublished - Jun 25 1999

Keywords

  • 20E induction
  • DNA binding
  • Developmental expression
  • Ligand binding

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Endocrinology

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