Characterization of the A2AR-D2R interface: focus on the role of the C-terminal tail and the transmembrane helices.
A single serine point mutation (S374A) in the adenosine A(2A) receptor (A(2A)R) C-terminal tail reduces A(2A)R-D(2)R heteromerization and prevents its allosteric modulation of the dopamine D(2) receptor (D(2)R). By means of site directed mutagenesis of the A(2A)R and synthetic transmembrane (TM) α-helix peptides of the D(2)R we further explored the role of electrostatic interactions and TM helix interactions of the A(2A)R-D(2)R heteromer interface. We found evidence that the TM domains IV and V of the D(2)R play a major role in the A(2A)R-D(2)R heteromer interface since the incubation with peptides corresponding to these domains significantly reduced the ability of A(2A)R and D(2)R to heteromerize. In addition, the incubation with TM-IV or TM-V blocked the allosteric modulation normally found in A(2A)R-D(2)R heteromers. The mutation of two negatively charged aspartates in the A(2A)R C-terminal tail (D401A/D402A) in combination with the S374A mutation drastically reduced the physical A(2A)R-D(2)R interaction and lost the ability of antagonistic allosteric modulation over the A(2A)R-D(2)R interface, suggesting further evidence for the existence of an electrostatic interaction between the C-terminal tail of A(2A)R and the intracellular loop 3 (IL3) of D(2)R. On the other hand, molecular dynamic model and bioinformatic analysis propose that specific AAR, AQE, and VLS protriplets as an important motive in the A(2A)R-D(2L)R heteromer interface together with D(2L)R TM segments IV/V interacting with A(2A)R TM-IV/V or TM-I/VII.
Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden. Dasiel.Borroto-Escuela@ki.se
Dasiel O
DO
Borroto-Escuela
Wilber
W
Romero-Fernandez
Alexander O
AO
Tarakanov
Maricel
M
Gómez-Soler
Fidel
F
Corrales
Daniel
D
Marcellino
Manuel
M
Narvaez
Malgorzata
M
Frankowska
Marc
M
Flajolet
Nathaniel
N
Heintz
Luigi F
LF
Agnati
Francisco
F
Ciruela
Kjell
K
Fuxe
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D002460:Cell Line; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D043705:Receptor, Adenosine A2A / Q000737:chemistry* / Q000235:genetics; D017448:Receptors, Dopamine D2 / Q000737:chemistry* / Q000235:genetics; D012694:Serine / Q000737:chemistry / Q000235:genetics
D043705:Receptor, Adenosine A2A; D017448:Receptors, Dopamine D2; C113776:dopamine D2L receptor; D001224:Aspartic Acid; D012694:Serine