1. A highly conserved δ‐opioid receptor region determines RGS4 interaction
- Author
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Zafiroula Georgoussi, Alexandra Symeonof, Maria Marti-Solano, Tomasz Maciej Stepniewski, Christos Karoussiotis, and Jana Selent
- Subjects
Protein Conformation, alpha-Helical ,0301 basic medicine ,G protein ,medicine.drug_class ,Genetic Vectors ,Regulator ,Gene Expression ,GTP-Binding Protein alpha Subunits, Gi-Go ,Molecular Dynamics Simulation ,Crystallography, X-Ray ,Biochemistry ,RGS4 ,03 medical and health sciences ,0302 clinical medicine ,Opioid receptor ,Receptors, Opioid, delta ,Escherichia coli ,medicine ,Animals ,Humans ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,Cloning, Molecular ,Receptor ,Molecular Biology ,Ternary complex ,Conserved Sequence ,Regulation of gene expression ,Binding Sites ,Sequence Homology, Amino Acid ,biology ,Chemistry ,Cell Biology ,Recombinant Proteins ,Cell biology ,030104 developmental biology ,030220 oncology & carcinogenesis ,Mutation ,biology.protein ,Cattle ,Protein Conformation, beta-Strand ,Salt bridge ,Sequence Alignment ,RGS Proteins ,Protein Binding - Abstract
The δ-opioid receptor (δ-OR) couples to Gi/Go proteins to modulate a variety of responses in the nervous system. Τhe regulator of G protein signalling 4 (RGS4) was previously shown to directly interact within the C-terminal region of δ-OR using its N-terminal domain to negatively modulate opioid receptor signalling. Herein, using molecular dynamics simulations and in vitro pull-down experiments we delimit this interaction to 12 helix 8 residues of δ-ΟR and to the first 17 N-terminal residues (NT) of RGS4. Monitoring the complex arrangement and stabilization between RGS4 and δ-OR by molecular dynamics simulations combined with mutagenesis studies, we defined that two critical interactions are formed: one between Phe329 of helix8 of δ-ΟR and Pro9 of the NT of RGS4 and the other a salt bridge between Glu323 of δ-ΟR and Lys17 of RGS4. Our observations allow drafting for the first time a structural model of a ternary complex including the δ-opioid receptor, a G protein and a RGS protein. Furthermore, the high degree of conservation among opioid receptors of the RGS4-binding region, points to a conserved interaction mode between opioid receptors and this important regulatory protein.
- Published
- 2019