Mapping the Interface of a GPCR Dimer: A Structural Model of the A(2A) Adenosine and D(2) Dopamine Receptor Heteromer

The A(2A) adenosine (A(2A)R) and D(2) dopamine (D(2)R) receptors form oligomers in the cell membrane and allosteric interactions across the A(2A)R–D(2)R heteromer represent a target for development of drugs against central nervous system disorders. However, understanding of the molecular determinants of A(2A)R–D(2)R heteromerization and the allosteric antagonistic interactions between the receptor protomers is still limited. In this work, a structural model of the A(2A)R–D(2)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(2A)R–D(2)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(2A)R blocked heterodimer interactions and disrupted the allosteric effect of A(2A)R activation on D(2)R agonist binding. Protein–protein docking was used to construct a model of the A(2A)R–D(2)R heterodimer with a TM-IV/V interface, which was refined using molecular dynamics simulations. Mutations in the predicted interface reduced A(2A)R–D(2)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(2A)R–D(2)R interface can be extended to study the many other G protein-coupled receptors that engage in heteroreceptor complexes.