TY - JOUR
T1 - Highly flexible ligand binding pocket of ecdysone receptor
T2 - A single amino acid change leads to discrimination between two groups of nonsteroidal ecdysone agonists
AU - Kumar, Mohan B.
AU - Potter, David W.
AU - Hormann, Robert E.
AU - Edwards, Angela
AU - Tice, Colin M.
AU - Smith, Howard C.
AU - Dipietro, Martha A.
AU - Polley, Mitch
AU - Lawless, Michael
AU - Wolohan, Philippa R.N.
AU - Kethidi, Damodhar R.
AU - Palli, Subba R.
PY - 2004/6/25
Y1 - 2004/6/25
N2 - The insect steroid hormone 20-hydroxyecdysone works through a ligand-activated nuclear receptor, the ecdysone receptor (EcR), which plays critical roles in insect development and reproduction. The EcR has been exploited to develop insecticides to control pests and gene switches for gene regulation. Recently reported crystal structures of the EcR protein show different but partially overlapping binding cavities for ecdysteroid (ECD) and diacylhydrazine (DAH) ligands, providing an explanation for the differential activity of DAH ligands in insects. 1-Aroyl-4- (arylamino)-1,2,3,4- tetrahydroquinoline (THQ) ligands were recently discovered as ecdysone agonists. Mutagenesis of the EcR (from Choristoneura fumiferana, CfEcR) ligand binding domain followed by screening in a reporter assay led to the identification of CfEcR mutants, which responded well to THQ ligands but poorly to both ECD and DAH ligands. These mutants were further improved by introducing a second mutation, A110P, which was previously reported to cause ECD insensitivity. Testing of these V128F/A110P and V128Y/A110P mutants in a C57BL/6 mouse model coactivator interaction assay and in insect cells showed that this mutant EcR is activated by THQ ligands but not by ECD or DAH ligands. The CfEcR and its V128F/A110P mutant were used to demonstrate simultaneous regulation of two reporter genes using THQ and DAH ligands.
AB - The insect steroid hormone 20-hydroxyecdysone works through a ligand-activated nuclear receptor, the ecdysone receptor (EcR), which plays critical roles in insect development and reproduction. The EcR has been exploited to develop insecticides to control pests and gene switches for gene regulation. Recently reported crystal structures of the EcR protein show different but partially overlapping binding cavities for ecdysteroid (ECD) and diacylhydrazine (DAH) ligands, providing an explanation for the differential activity of DAH ligands in insects. 1-Aroyl-4- (arylamino)-1,2,3,4- tetrahydroquinoline (THQ) ligands were recently discovered as ecdysone agonists. Mutagenesis of the EcR (from Choristoneura fumiferana, CfEcR) ligand binding domain followed by screening in a reporter assay led to the identification of CfEcR mutants, which responded well to THQ ligands but poorly to both ECD and DAH ligands. These mutants were further improved by introducing a second mutation, A110P, which was previously reported to cause ECD insensitivity. Testing of these V128F/A110P and V128Y/A110P mutants in a C57BL/6 mouse model coactivator interaction assay and in insect cells showed that this mutant EcR is activated by THQ ligands but not by ECD or DAH ligands. The CfEcR and its V128F/A110P mutant were used to demonstrate simultaneous regulation of two reporter genes using THQ and DAH ligands.
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U2 - 10.1074/jbc.M403839200
DO - 10.1074/jbc.M403839200
M3 - Article
C2 - 15107428
AN - SCOPUS:3042547057
SN - 0021-9258
VL - 279
SP - 27211
EP - 27218
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 26
ER -