Studies on the role of the receptor protein motifs possibly involved in electrostatic interactions on the dopamine D1 and D2 receptor oligomerization.
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 D(2)R, which may suggest that the indicated region of ic3 in D(2)R 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 D(2)R and cyan fluorescent protein-tagged D(1)R may result from differing localization of these proteins in the cell rather than from the possible role of the D(2)R 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.
Łukasiewicz
Sylwia
S
Department of Physical Biochemistry, Jagiellonian University, Kraków, Poland.
Faron-Górecka
Agata
A
Dobrucki
Jerzy
J
Polit
Agnieszka
A
Dziedzicka-Wasylewska
Marta
M
D020816:Amino Acid Motifs; D002460:Cell Line; D006801:Humans; D008856:Microscopy, Fluorescence; D009154:Mutation / Q000235:genetics; D055503:Protein Multimerization; D011869:Radioligand Assay; D017447:Receptors, Dopamine D1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017448:Receptors, Dopamine D2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D055672:Static Electricity
D017447:Receptors, Dopamine D1; D017448:Receptors, Dopamine D2