Molecular Basis of Ligand Dissociation from the Adenosine A_2A Receptor

Abstract

How drugs dissociate from their targets is largely unknown. We investigated the molecular basis of this process in the adenosine A 2A receptor (A 2A R), a prototypical G protein-coupled receptor (GPCR). Through kinetic radioligand binding experiments, we characterized mutant receptors selected based on molecular dynamic simulations of the antagonist ZM241385 dissociating from the A 2A R. We discovered mutations that dramatically altered the ligand's dissociation rate despite only marginally influencing its binding affinity, demonstrating that even receptor features with little contribution to affinity may prove critical to the dissociation process. Our results also suggest that ZM241385 follows a multistep dissociation pathway, consecutively interacting with distinct receptor regions, a mechanism that may also be common to many other GPCRs.

Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

“…Antagonist binding (energetically favourable conformation) to the vestibule of the binding pocket was also demonstrated in simulation of molecular dynamics (MD) of tiotropium binding6. Interaction of orthosteric ligands with the allosteric site was also illustrated at β-adrenergic and adenosine receptors suggesting that tandem two-site mechanism of orthosteric ligand biding is common among aminergic GPCRs272829.…”

“…Interaction of orthosteric ligands with extracellular domains was also described at other aminergic GPCRs, e.g. β-adrenergic, adenosine272829 suggesting common molecular mechanism of binding in this sub-class of GPCRs.…”

A covalent antagonist for the human adenosine A2A receptor

“…Hence, the K153A ECL2 mutant construct, potentially preventing the covalent bond from being formed, was made to perform a similar wash-out experiment as described above. Since ZM241385 showed a similar affinity for both the K153A ECL2 mutant (p K i = 7.83 ± 0.04) and WT receptors (p K i = 7.91 ± 0.05) [20], we assumed that the difference in radioligand binding recovery was not due to a point mutation within a receptor binding site, which has the potential of altering ligand binding properties. Moreover, in the absence of either LUF7445 or ZM241385, the apparently same binding capacity (data not shown) proved that the washing steps had little influence on the integrity of both WT hA 2A AR and mutant A2AAR-K153A ECL2 .…”

“…Site-directed receptor mutant hA 2A AR-K153A ECL2 was constructed by the same procedure reported previously [20]. The wild type pcDNA3.1-A 2A R plasmid DNA with N-terminal HA and FLAG tags and C-terminal His tag was used as a template for polymerase chain reaction (PCR) mutagenesis.…”

“…We followed the procedures reported previously [20, 21]. Briefly, human embryonic kidney (HEK) 293 cells were grown as monolayers in Dulbecco’s modified Eagle’s medium supplemented with stable glutamine, 10% newborn calf serum, 50 μg/mL streptomycin, and 50 IU/mL penicillin at 37 °C and 7% CO 2 atmosphere.…”