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
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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.
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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