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Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay
Published on: December 19, 2018
Juvenile hormone receptor relies on conserved domain interfaces for dimerization-dependent activity
Lenka Bittova1, Sarka Tumova1, Mykola Yatsenko1,2
1Department of Molecular Physiology and Genetics, Institute of Entomology, Biology Center of the Czech Academy of Sciences, Ceske Budejovice 37005, Czech Republic.
Summary
Charged residues in the PAS-A domain of germ cell-expressed (GCE) protein are crucial for juvenile hormone (JH) signaling in Drosophila. Mutations impair JH receptor (JHR) complex activation, nuclear import, and transcriptional activity.
Area of Science:
- Molecular biology
- Insect endocrinology
- Protein structure-function relationships
Background:
- Juvenile hormone (JH) is essential for insect development and reproduction.
- JH signaling in Drosophila involves bHLH-PAS proteins MET and GCE, forming JH receptor (JHR) complexes with TAI.
- The precise activation mechanism of the JHR complex remains incompletely understood.
Purpose of the Study:
- To investigate the role of the PAS-A domain of the Drosophila GCE protein in JH signaling.
- To elucidate the molecular mechanisms underlying JHR complex activation.
Main Methods:
- Site-directed mutagenesis of conserved charged residues in the GCE PAS-A domain.
- In vitro and in vivo functional assays to assess JHR signaling.
- Biochemical analyses including nuclear import, protein dissociation, and dimerization assays.
- Molecular modeling to predict structural consequences of mutations.
Main Results:
- Mutations in conserved charged residues of the GCE PAS-A domain abolish JHR signaling in vitro and in vivo.
- These mutations do not affect JH binding to the PAS-B domain but impair JH-induced nuclear import of GCE.
- Mutations disrupt GCE dissociation from HSP83 and dimerization with TAI, preventing DNA binding and transcriptional activity.
- Molecular modeling suggests altered inter- and intradomain interactions within the GCE protein.
Conclusions:
- The PAS-A domain plays a critical, previously unrecognized role in JHR signaling.
- Interactions between the PAS-A and PAS-B domains are essential for JHR complex activation and are allosterically coupled.
- Conserved charged residues in the PAS-A domain are vital for regulating GCE function in response to JH.
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