Data_Sheet_1_Mapping the Interface of a GPCR Dimer: A Structural Model of the A2A Adenosine and D2 Dopamine Receptor Heteromer.pdf

<p>The A<sub>2A</sub> adenosine (A<sub>2A</sub>R) and D<sub>2</sub> dopamine (D<sub>2</sub>R) receptors form oligomers in the cell membrane and allosteric interactions across the A<sub>2A</sub>R–D<sub>2</sub>R heteromer represent a target for development of drugs against central nervous system disorders. However, understanding of the molecular determinants of A<sub>2A</sub>R–D<sub>2</sub>R heteromerization and the allosteric antagonistic interactions between the receptor protomers is still limited. In this work, a structural model of the A<sub>2A</sub>R–D<sub>2</sub>R heterodimer was generated using a combined experimental and computational approach. Regions involved in the heteromer interface were modeled based on the effects of peptides derived from the transmembrane (TM) helices on A<sub>2A</sub>R–D<sub>2</sub>R receptor–receptor interactions in bioluminescence resonance energy transfer (BRET) and proximity ligation assays. Peptides corresponding to TM-IV and TM-V of the A<sub>2A</sub>R blocked heterodimer interactions and disrupted the allosteric effect of A<sub>2A</sub>R activation on D<sub>2</sub>R agonist binding. Protein–protein docking was used to construct a model of the A<sub>2A</sub>R–D<sub>2</sub>R heterodimer with a TM-IV/V interface, which was refined using molecular dynamics simulations. Mutations in the predicted interface reduced A<sub>2A</sub>R–D<sub>2</sub>R interactions in BRET experiments and altered the allosteric modulation. The heterodimer model provided insights into the structural basis of allosteric modulation and the technique developed to characterize the A<sub>2A</sub>R–D<sub>2</sub>R interface can be extended to study the many other G protein-coupled receptors that engage in heteroreceptor complexes.</p>