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Research ArticleArticle

Computational and Experimental Analysis of the Transmembrane Domain 4/5 Dimerization Interface of the Serotonin 5-HT1A Receptor

Nataliya Gorinski, Noga Kowalsman, Ute Renner, Alexander Wirth, Michael T. Reinartz, Roland Seifert, Andre Zeug, Evgeni Ponimaskin and Masha Y. Niv
Molecular Pharmacology September 2012, 82 (3) 448-463; DOI: https://doi.org/10.1124/mol.112.079137
Nataliya Gorinski
Cellular Neurophysiology, Medical School Hannover, Germany (N.G., A.W., A.Z., E.P.); Institute of Biochemistry, Food Science and Nutrition, the Robert H. Smith Faculty of Agriculture, Food and Environment, and the Fritz Haber Center for Molecular Dynamics, the Hebrew University of Jerusalem, Israel (N.K., M.Y.N.); DFG-Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany (U.R., E.P.); and Institute of Pharmacology, Medical School Hannover, Germany (M.T.R., R.S.)
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Noga Kowalsman
Cellular Neurophysiology, Medical School Hannover, Germany (N.G., A.W., A.Z., E.P.); Institute of Biochemistry, Food Science and Nutrition, the Robert H. Smith Faculty of Agriculture, Food and Environment, and the Fritz Haber Center for Molecular Dynamics, the Hebrew University of Jerusalem, Israel (N.K., M.Y.N.); DFG-Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany (U.R., E.P.); and Institute of Pharmacology, Medical School Hannover, Germany (M.T.R., R.S.)
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Ute Renner
Cellular Neurophysiology, Medical School Hannover, Germany (N.G., A.W., A.Z., E.P.); Institute of Biochemistry, Food Science and Nutrition, the Robert H. Smith Faculty of Agriculture, Food and Environment, and the Fritz Haber Center for Molecular Dynamics, the Hebrew University of Jerusalem, Israel (N.K., M.Y.N.); DFG-Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany (U.R., E.P.); and Institute of Pharmacology, Medical School Hannover, Germany (M.T.R., R.S.)
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Alexander Wirth
Cellular Neurophysiology, Medical School Hannover, Germany (N.G., A.W., A.Z., E.P.); Institute of Biochemistry, Food Science and Nutrition, the Robert H. Smith Faculty of Agriculture, Food and Environment, and the Fritz Haber Center for Molecular Dynamics, the Hebrew University of Jerusalem, Israel (N.K., M.Y.N.); DFG-Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany (U.R., E.P.); and Institute of Pharmacology, Medical School Hannover, Germany (M.T.R., R.S.)
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Michael T. Reinartz
Cellular Neurophysiology, Medical School Hannover, Germany (N.G., A.W., A.Z., E.P.); Institute of Biochemistry, Food Science and Nutrition, the Robert H. Smith Faculty of Agriculture, Food and Environment, and the Fritz Haber Center for Molecular Dynamics, the Hebrew University of Jerusalem, Israel (N.K., M.Y.N.); DFG-Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany (U.R., E.P.); and Institute of Pharmacology, Medical School Hannover, Germany (M.T.R., R.S.)
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Roland Seifert
Cellular Neurophysiology, Medical School Hannover, Germany (N.G., A.W., A.Z., E.P.); Institute of Biochemistry, Food Science and Nutrition, the Robert H. Smith Faculty of Agriculture, Food and Environment, and the Fritz Haber Center for Molecular Dynamics, the Hebrew University of Jerusalem, Israel (N.K., M.Y.N.); DFG-Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany (U.R., E.P.); and Institute of Pharmacology, Medical School Hannover, Germany (M.T.R., R.S.)
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Andre Zeug
Cellular Neurophysiology, Medical School Hannover, Germany (N.G., A.W., A.Z., E.P.); Institute of Biochemistry, Food Science and Nutrition, the Robert H. Smith Faculty of Agriculture, Food and Environment, and the Fritz Haber Center for Molecular Dynamics, the Hebrew University of Jerusalem, Israel (N.K., M.Y.N.); DFG-Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany (U.R., E.P.); and Institute of Pharmacology, Medical School Hannover, Germany (M.T.R., R.S.)
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Evgeni Ponimaskin
Cellular Neurophysiology, Medical School Hannover, Germany (N.G., A.W., A.Z., E.P.); Institute of Biochemistry, Food Science and Nutrition, the Robert H. Smith Faculty of Agriculture, Food and Environment, and the Fritz Haber Center for Molecular Dynamics, the Hebrew University of Jerusalem, Israel (N.K., M.Y.N.); DFG-Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany (U.R., E.P.); and Institute of Pharmacology, Medical School Hannover, Germany (M.T.R., R.S.)
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Masha Y. Niv
Cellular Neurophysiology, Medical School Hannover, Germany (N.G., A.W., A.Z., E.P.); Institute of Biochemistry, Food Science and Nutrition, the Robert H. Smith Faculty of Agriculture, Food and Environment, and the Fritz Haber Center for Molecular Dynamics, the Hebrew University of Jerusalem, Israel (N.K., M.Y.N.); DFG-Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany (U.R., E.P.); and Institute of Pharmacology, Medical School Hannover, Germany (M.T.R., R.S.)
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Abstract

Experimental evidence suggests that most members of class A G-protein coupled receptors (GPCRs) can form homomers and heteromers in addition to functioning as single monomers. In particular, serotonin (5-HT) receptors were shown to homodimerize and heterodimerize with other GPCRs, although the details and the physiological role of the oligomerization has not yet been fully elucidated. Here we used computational modeling of the 5-HT1A receptor monomer and dimer to predict residues important for dimerization. Based on these results, we carried out rationally designed site-directed mutagenesis. The ability of the mutants to dimerize was evaluated using different FRET-based approaches. The reduced levels of acceptor photobleaching-Förster resonance energy transfer (FRET) and the lower number of monomers participating in oligomers, as assessed by lux-FRET, confirmed the decreased ability of the mutants to dimerize and the involvement of the predicted contacts (Trp1754.64, Tyr1985.41, Arg1514.40, and Arg1524.41) at the interface. This information was reintroduced as constraints for computational protein-protein docking to obtain a high-quality dimer model. Analysis of the refined model as well as molecular dynamics simulations of wild-type (WT) and mutant dimers revealed compensating interactions in dimers composed of WT and W175A mutant. This provides an explanation for the requirement of mutations of Trp1754.64 in both homomers for disrupting dimerization. Our iterative computational-experimental study demonstrates that transmembrane domains TM4/TM5 can form an interaction interface in 5-HT1A receptor dimers and indicates that specific amino acid interactions maintain this interface. The mutants and the optimized model of the dimer structure may be used in functional studies of serotonin dimers.

Footnotes

  • ↵Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • This work was funded by the Niedersachsen-Israel research fund [Grant ZN2448] and Deutsche Forschungsgemeinschaft [Grant PO732].

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    http://dx.doi.org/10.1124/mol.112.079137.

  • ABBREVIATIONS:

    5-HT
    5-hydroxytryptamine (serotonin)
    GPCR
    G-protein-coupled receptor
    FRET
    Förster resonance energy transfer
    TM
    transmembrane
    3D
    three-dimensional
    PDB
    Protein Data Bank
    MD
    molecular dynamics
    GFP
    green fluorescence protein
    8-OH-DPAT
    8-hydroxy-2-dipropylaminotetralin
    PAGE
    polyacrylamide gel electrophoresis
    YFP
    yellow fluorescent protein
    WT
    wild type
    CFP
    cyan fluorescent protein
    IL
    intracellular loop
    vdW
    van der Waals
    RMSD
    root-mean-square deviation
    D3
    dopamine D3 receptor
    OR
    opioid receptor
    PNGase
    glycopeptidase
    ER
    endoplasmic reticulum
    lux-FRET
    linear unmixing FRET.

  • Received April 4, 2012.
  • Accepted June 5, 2012.
  • Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 82 (3)
Molecular Pharmacology
Vol. 82, Issue 3
1 Sep 2012
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Research ArticleArticle

5-HT1A Dimeric Interface

Nataliya Gorinski, Noga Kowalsman, Ute Renner, Alexander Wirth, Michael T. Reinartz, Roland Seifert, Andre Zeug, Evgeni Ponimaskin and Masha Y. Niv
Molecular Pharmacology September 1, 2012, 82 (3) 448-463; DOI: https://doi.org/10.1124/mol.112.079137

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Research ArticleArticle

5-HT1A Dimeric Interface

Nataliya Gorinski, Noga Kowalsman, Ute Renner, Alexander Wirth, Michael T. Reinartz, Roland Seifert, Andre Zeug, Evgeni Ponimaskin and Masha Y. Niv
Molecular Pharmacology September 1, 2012, 82 (3) 448-463; DOI: https://doi.org/10.1124/mol.112.079137
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