Introduction: G protein coupled receptor dimerization is proposed to have great impact on receptor signaling. Allosteric communication between the protomers of a dimer can alter ligand binding, receptor conformation and the interactions with different effector proteins. Although the concept of intradimeric interactions is widely accepted, the molecular mechanism behind them is not clearly understood. In this study we investigated the allosteric interactions within the type I angiotensin receptor (AT1R) homodimer transiently expressed in CHO cells.
Methods: To detect the intradimeric interactions, one protomer of AT1R homodimer was selectively stimulated, while the activation process of the other protomer was followed by different bioluminescent resonance energy transfer (BRET) based assays. For the selective stimulation we used an antagonist resistant mutant AT1R. We monitored the activation of the non-stimulated protomer through its interaction with β-arrestin2 and followed the conformation alterations directly with an intramolecular receptor biosensor. Also cooperative ligand binding of the homodimer was examined by ligand dissociation experiments. To evaluate the molecular mechanism behind the allosteric interactions, different mutants of AT1R were investigated in our assays.
Results: We observed that agonist binding of one protomer is followed by the increased ligand dissociation, altered conformation and β-arrestin2 binding of the other protomer. The mutation of the conserved DRY sequence in the activated protomer abolished all the observed effects between the receptors.
Conclusion: The detected effects on the non-stimulated protomer are presumable the consequence of direct intradimeric allosteric interactions. Our results with AT1 receptor mutated in the DRY sequence suggest the crucial role of this motif in intradimeric interactions, and also highlight the molecular mechanisms behind them.
Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.
Funding: This work was supported, however funding details unavailable.