Extracellular Loop 2 of the Adenosine A1 Receptor Has a Key Role in Orthosteric Ligand Affinity and Agonist Efficacy.
The adenosine A1G protein-coupled receptor (A1AR) is an important therapeutic target implicated in a wide range of cardiovascular and neuronal disorders. Although it is well established that the A1AR orthosteric site is located within the receptor's transmembrane (TM) bundle, prior studies have implicated extracellular loop 2 (ECL2) as having a significant role in contributing to orthosteric ligand affinity and signaling for various G protein-coupled receptors (GPCRs). We thus performed extensive alanine scanning mutagenesis of A1AR-ECL2 to explore the role of this domain on A1AR orthosteric ligand pharmacology. Using quantitative analytical approaches and molecular modeling, we identified ECL2 residues that interact either directly or indirectly with orthosteric agonists and antagonists. Discrete mutations proximal to a conserved ECL2-TM3 disulfide bond selectively affected orthosteric ligand affinity, whereas a cluster of five residues near the TM4-ECL2 juncture influenced orthosteric agonist efficacy. A combination of ligand docking, molecular dynamics simulations, and mutagenesis results suggested that the orthosteric agonist 5'-N-ethylcarboxamidoadenosine binds transiently to an extracellular vestibule formed by ECL2 and the top of TM5 and TM7, prior to entry into the canonical TM bundle orthosteric site. Collectively, this study highlights a key role for ECL2 in A1AR orthosteric ligand binding and receptor activation.
Monash Institute of Pharmaceutical Sciences (A.T.N.N., J.-A.B., T.T., L.L.M, K.J.G, P.J.W, P.M.S, A.C., L.T.M), Monash e-Research Centre (T.D.N), and Department of Pharmacology (A.T.N.N, J.-A.B., K.J.G., P.M.S., A.C., L.T.M), Monash University, Parkville, Victoria, Australia.
Anh T N
AT
Nguyen
Monash Institute of Pharmaceutical Sciences (A.T.N.N., J.-A.B., T.T., L.L.M, K.J.G, P.J.W, P.M.S, A.C., L.T.M), Monash e-Research Centre (T.D.N), and Department of Pharmacology (A.T.N.N, J.-A.B., K.J.G., P.M.S., A.C., L.T.M), Monash University, Parkville, Victoria, Australia.
Jo-Anne
JA
Baltos
Monash Institute of Pharmaceutical Sciences (A.T.N.N., J.-A.B., T.T., L.L.M, K.J.G, P.J.W, P.M.S, A.C., L.T.M), Monash e-Research Centre (T.D.N), and Department of Pharmacology (A.T.N.N, J.-A.B., K.J.G., P.M.S., A.C., L.T.M), Monash University, Parkville, Victoria, Australia.
Trayder
T
Thomas
Monash Institute of Pharmaceutical Sciences (A.T.N.N., J.-A.B., T.T., L.L.M, K.J.G, P.J.W, P.M.S, A.C., L.T.M), Monash e-Research Centre (T.D.N), and Department of Pharmacology (A.T.N.N, J.-A.B., K.J.G., P.M.S., A.C., L.T.M), Monash University, Parkville, Victoria, Australia.
Toan D
TD
Nguyen
Monash Institute of Pharmaceutical Sciences (A.T.N.N., J.-A.B., T.T., L.L.M, K.J.G, P.J.W, P.M.S, A.C., L.T.M), Monash e-Research Centre (T.D.N), and Department of Pharmacology (A.T.N.N, J.-A.B., K.J.G., P.M.S., A.C., L.T.M), Monash University, Parkville, Victoria, Australia.
Laura López
LL
Muñoz
Monash Institute of Pharmaceutical Sciences (A.T.N.N., J.-A.B., T.T., L.L.M, K.J.G, P.J.W, P.M.S, A.C., L.T.M), Monash e-Research Centre (T.D.N), and Department of Pharmacology (A.T.N.N, J.-A.B., K.J.G., P.M.S., A.C., L.T.M), Monash University, Parkville, Victoria, Australia.
Karen J
KJ
Gregory
Monash Institute of Pharmaceutical Sciences (A.T.N.N., J.-A.B., T.T., L.L.M, K.J.G, P.J.W, P.M.S, A.C., L.T.M), Monash e-Research Centre (T.D.N), and Department of Pharmacology (A.T.N.N, J.-A.B., K.J.G., P.M.S., A.C., L.T.M), Monash University, Parkville, Victoria, Australia.
Paul J
PJ
White
Monash Institute of Pharmaceutical Sciences (A.T.N.N., J.-A.B., T.T., L.L.M, K.J.G, P.J.W, P.M.S, A.C., L.T.M), Monash e-Research Centre (T.D.N), and Department of Pharmacology (A.T.N.N, J.-A.B., K.J.G., P.M.S., A.C., L.T.M), Monash University, Parkville, Victoria, Australia.
Patrick M
PM
Sexton
Monash Institute of Pharmaceutical Sciences (A.T.N.N., J.-A.B., T.T., L.L.M, K.J.G, P.J.W, P.M.S, A.C., L.T.M), Monash e-Research Centre (T.D.N), and Department of Pharmacology (A.T.N.N, J.-A.B., K.J.G., P.M.S., A.C., L.T.M), Monash University, Parkville, Victoria, Australia arthur.christopoulos@monash.edu lauren.may@monash.edu.
Arthur
A
Christopoulos
Monash Institute of Pharmaceutical Sciences (A.T.N.N., J.-A.B., T.T., L.L.M, K.J.G, P.J.W, P.M.S, A.C., L.T.M), Monash e-Research Centre (T.D.N), and Department of Pharmacology (A.T.N.N, J.-A.B., K.J.G., P.M.S., A.C., L.T.M), Monash University, Parkville, Victoria, Australia arthur.christopoulos@monash.edu lauren.may@monash.edu.
Lauren T
LT
May
D000241:Adenosine / Q000494:pharmacology; D058907:Adenosine A1 Receptor Agonists / Q000494:pharmacology*; D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D008024:Ligands; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000378:metabolism; D017433:Protein Structure, Secondary; D043682:Receptor, Adenosine A1 / Q000737:chemistry* / Q000378:metabolism*; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D014970:Xanthines / Q000494:pharmacology
D058907:Adenosine A1 Receptor Agonists; D008024:Ligands; D050505:Mutant Proteins; D043682:Receptor, Adenosine A1; D014970:Xanthines; D000242:Cyclic AMP; D000241:Adenosine; D000409:Alanine