Journal
FEBS JOURNAL
Volume 276, Issue 3, Pages 760-775Publisher
WILEY
DOI: 10.1111/j.1742-4658.2008.06822.x
Keywords
Arg-rich motif; dopamine D-1 receptor; dopamine D-2 receptor; FRET; GPCR oligomerization
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Funding
- Ministry of Science [2P05A 071 29, 2P04A 070 29]
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We investigated the influence of an epitope from the third intracellular loop (ic3) of the dopamine D-2 receptor, which contains adjacent arginine residues (217RRRRKR222), and an acidic epitope from the C-terminus of the dopamine D-1 receptor (404EE405) on the receptors' localization and their interaction. We studied receptor dimer formation using fluorescence resonance energy transfer. Receptor proteins were tagged with fluorescence proteins and expressed in HEK293 cells. The degree of D-1-D-2 receptor heterodimerization strongly depended on the number of Arg residues replaced by Ala in the ic3 of D2R, which may suggest that the indicated region of ic3 in D2R might be involved in interactions between two dopamine receptors. In addition, the subcellular localization of these receptors in cells expressing both receptors D-1-cyan fluorescent protein, D-2-yellow fluorescent protein, and various mutants was examined by confocal microscopy. Genetic manipulations of the Arg-rich epitope induced alterations in the localization of the resulting receptor proteins, leading to the conclusion that this epitope is responsible for the cellular localization of the receptor. The lack of energy transfer between the genetic variants of yellow fluorescent protein-tagged D2R and cyan fluorescent protein-tagged D1R may result from differing localization of these proteins in the cell rather than from the possible role of the D2R basic domain in the mechanism of D-1-D-2 receptor heterodimerization. However, we find that the acidic epitope from the C-terminus of the dopamine D-1 receptor is engaged in the heterodimerization process.
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