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Studies on the role of the receptor protein motifs possibly involved in electrostatic interactions on the dopamine D₁ and D₂ receptor oligomerization

Łukasiewicz, Sylwia, Faron-Górecka, Agata, Dobrucki, Jerzy, Polit, Agnieszka, Dziedzicka-Wasylewska, Marta
FEBS journal 2009 v.276 no.3 pp. 760-775
arginine, confocal microscopy, dopamine, dopamine receptors, electrostatic interactions, energy transfer, epitopes, fluorescence, fluorescent proteins, genetic engineering, genetic variation, mutants, protein synthesis
We investigated the influence of an epitope from the third intracellular loop (ic3) of the dopamine D₂ receptor, which contains adjacent arginine residues (217RRRRKR222), and an acidic epitope from the C-terminus of the dopamine D₁ 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 D₁-D₂ 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 D₁-cyan fluorescent protein, D₂-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 D₁-D₂ receptor heterodimerization. However, we find that the acidic epitope from the C-terminus of the dopamine D₁ receptor is engaged in the heterodimerization process.