Ligand control of interaction in vivo between ecdysteroid receptor and ultraspiracle ligand-binding domain.
Ecdysteroids (Ecs) enhance the formation of Ec receptor-ultraspiracle protein (EcR-USP) heterodimers which regulate gene transcription. To study EcR-USP heterodimerization, fusion proteins were constructed from the LBDs (ligand-binding domains) of Drosophila EcR or USP and the activation or DNA-binding region of GAL4 respectively. Reporter gene ( lacZ ) activation was fully dependent on the co-expression of the two fusion proteins and thus constitutes a reliable measure for the interaction in vivo between the two LBDs in the yeast cell. To identify structures involved in heterodimerization, a total of 27 point mutations were generated in the EcR and USP LBDs at selected sites. Heterodimerization and its inducibility by ligand were mainly affected by mutations in the dimerization interface and in the ligand-binding pocket of EcR respectively. However, also mutations not located in these structures or even in the LBD of USP influenced ligand-dependent heterodimerization. Together with previously reported ligand-binding studies, the existence of such local, intra- and inter-molecular mutation effects suggest that ligand-induced dimerization results from a synergistic interaction between ligand-binding and heterodimerization functions in EcR LBD, and that it depends on global features of the LBDs of EcR and USP and on their mutual surface compatibility.
Institute for Cell Biology, ETH-Hönggerberg, CH-8093 Zürich, Switzerland.
Thomas
T
Bergman
Vincent C
VC
Henrich
Uwe
U
Schlattner
Markus
M
Lezzi
D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011987:Receptors, Steroid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D008024:Ligands; D011987:Receptors, Steroid; D014157:Transcription Factors; C066030:USP protein, Drosophila; C034808:ecdysteroid receptor