Role of the Second Extracellular Loop of the Adenosine A1 Receptor on Allosteric Modulator Binding, Signaling, and Cooperativity.
Allosteric modulation of adenosine A1 receptors (A1ARs) offers a novel therapeutic approach for the treatment of numerous central and peripheral disorders; however, despite decades of research, there is a relative paucity of structural information regarding the A1AR allosteric site and mechanisms governing cooperativity with orthosteric ligands. We combined alanine-scanning mutagenesis of the A1AR second extracellular loop (ECL2) with radioligand binding and functional interaction assays to quantify effects on allosteric ligand affinity, cooperativity, and efficacy. Docking and molecular dynamics (MD) simulations were performed using an A1AR homology model based on an agonist-bound A2AAR structure. Substitution of E172ECL2 for alanine reduced the affinity of the allosteric modulators PD81723 and VCP171 for the unoccupied A1AR. Residues involved in cooperativity with the orthosteric agonist NECA were different in PD81723 and VCP171; positive cooperativity between PD81723 and NECA was reduced on alanine substitution of a number of ECL2 residues, including E170ECL2 and K173ECL2, whereas mutation of W146ECL2 and W156ECL2 decreased VCP171 cooperativity with NECA. Molecular modeling localized a likely allosteric pocket for both modulators to an extracellular vestibule that overlaps with a region used by orthosteric ligands as they transit into the canonical A1AR orthosteric site. MD simulations confirmed a key interaction between E172ECL2 and both modulators. Bound PD81723 is flanked by another residue, E170ECL2, which forms hydrogen bonds with adjacent K168ECL2 and K173ECL2. Collectively, our data suggest E172ECL2 is a key allosteric ligand-binding determinant, whereas hydrogen-bonding networks within the extracellular vestibule may facilitate the transmission of cooperativity between orthosteric and allosteric sites.
Monash Institute of Pharmaceutical Sciences (A.T.N.N., E.A.V., T.T., L.A., P.J.S., P.J.W., P.M.S., K.J.G., L.T.M., A.C.), Monash e-Research Centre (T.D.N.), and Department of Pharmacology (A.T.N.N., E.A.V., P.M.S., K.J.G., L.T.M., A.C), Monash University, Victoria, Australia.
Anh T N
AT
Nguyen
Monash Institute of Pharmaceutical Sciences (A.T.N.N., E.A.V., T.T., L.A., P.J.S., P.J.W., P.M.S., K.J.G., L.T.M., A.C.), Monash e-Research Centre (T.D.N.), and Department of Pharmacology (A.T.N.N., E.A.V., P.M.S., K.J.G., L.T.M., A.C), Monash University, Victoria, Australia.
Elizabeth A
EA
Vecchio
Monash Institute of Pharmaceutical Sciences (A.T.N.N., E.A.V., T.T., L.A., P.J.S., P.J.W., P.M.S., K.J.G., L.T.M., A.C.), Monash e-Research Centre (T.D.N.), and Department of Pharmacology (A.T.N.N., E.A.V., P.M.S., K.J.G., L.T.M., A.C), Monash University, Victoria, Australia.
Trayder
T
Thomas
Monash Institute of Pharmaceutical Sciences (A.T.N.N., E.A.V., T.T., L.A., P.J.S., P.J.W., P.M.S., K.J.G., L.T.M., A.C.), Monash e-Research Centre (T.D.N.), and Department of Pharmacology (A.T.N.N., E.A.V., P.M.S., K.J.G., L.T.M., A.C), Monash University, Victoria, Australia.
Toan D
TD
Nguyen
Monash Institute of Pharmaceutical Sciences (A.T.N.N., E.A.V., T.T., L.A., P.J.S., P.J.W., P.M.S., K.J.G., L.T.M., A.C.), Monash e-Research Centre (T.D.N.), and Department of Pharmacology (A.T.N.N., E.A.V., P.M.S., K.J.G., L.T.M., A.C), Monash University, Victoria, Australia.
Luigi
L
Aurelio
Monash Institute of Pharmaceutical Sciences (A.T.N.N., E.A.V., T.T., L.A., P.J.S., P.J.W., P.M.S., K.J.G., L.T.M., A.C.), Monash e-Research Centre (T.D.N.), and Department of Pharmacology (A.T.N.N., E.A.V., P.M.S., K.J.G., L.T.M., A.C), Monash University, Victoria, Australia.
Peter J
PJ
Scammells
Monash Institute of Pharmaceutical Sciences (A.T.N.N., E.A.V., T.T., L.A., P.J.S., P.J.W., P.M.S., K.J.G., L.T.M., A.C.), Monash e-Research Centre (T.D.N.), and Department of Pharmacology (A.T.N.N., E.A.V., P.M.S., K.J.G., L.T.M., A.C), Monash University, Victoria, Australia.
Paul J
PJ
White
Monash Institute of Pharmaceutical Sciences (A.T.N.N., E.A.V., T.T., L.A., P.J.S., P.J.W., P.M.S., K.J.G., L.T.M., A.C.), Monash e-Research Centre (T.D.N.), and Department of Pharmacology (A.T.N.N., E.A.V., P.M.S., K.J.G., L.T.M., A.C), Monash University, Victoria, Australia.
Patrick M
PM
Sexton
Monash Institute of Pharmaceutical Sciences (A.T.N.N., E.A.V., T.T., L.A., P.J.S., P.J.W., P.M.S., K.J.G., L.T.M., A.C.), Monash e-Research Centre (T.D.N.), and Department of Pharmacology (A.T.N.N., E.A.V., P.M.S., K.J.G., L.T.M., A.C), Monash University, Victoria, Australia.
Karen J
KJ
Gregory
Monash Institute of Pharmaceutical Sciences (A.T.N.N., E.A.V., T.T., L.A., P.J.S., P.J.W., P.M.S., K.J.G., L.T.M., A.C.), Monash e-Research Centre (T.D.N.), and Department of Pharmacology (A.T.N.N., E.A.V., P.M.S., K.J.G., L.T.M., A.C), Monash University, Victoria, Australia arthur.christopoulos@monash.edu lauren.may@monash.edu.
Lauren T
LT
May
Monash Institute of Pharmaceutical Sciences (A.T.N.N., E.A.V., T.T., L.A., P.J.S., P.J.W., P.M.S., K.J.G., L.T.M., A.C.), Monash e-Research Centre (T.D.N.), and Department of Pharmacology (A.T.N.N., E.A.V., P.M.S., K.J.G., L.T.M., A.C), Monash University, Victoria, Australia arthur.christopoulos@monash.edu lauren.may@monash.edu.
Arthur
A
Christopoulos
D000241:Adenosine / Q000494:pharmacology; D000409:Alanine / Q000235:genetics; D000494:Allosteric Regulation / Q000187:drug effects; D000495:Allosteric Site; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D008024:Ligands; D056004:Molecular Dynamics Simulation; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D043682:Receptor, Adenosine A1 / Q000737:chemistry* / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship
D008024:Ligands; D043682:Receptor, Adenosine A1; D000242:Cyclic AMP; D000241:Adenosine; D000409:Alanine