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

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