title: Role of extracellular cysteine residues in the adenosine A2A receptor.
abstract: The G protein-coupled A2A adenosine receptor represents an important drug target. Crystal structures and modeling studies indicated that three disulfide bonds are formed between ECL1 and ECL2 (I, Cys71(2.69)-Cys159(45.43); II, Cys74(3.22)-Cys146(45.30), and III, Cys77(3.25)-Cys166(45.50)). However, the A2BAR subtype appears to require only disulfide bond III for proper function. In this study, each of the three disulfide bonds in the A2AAR was disrupted by mutation of one of the cysteine residues to serine. The mutant receptors were stably expressed in Chinese hamster ovary cells and analyzed in cyclic adenosine monophosphate (cAMP) accumulation and radioligand binding studies using structurally diverse agonists: adenosine, NECA, CGS21680, and PSB-15826. Results were rationalized by molecular modeling. The observed effects were dependent on the investigated agonist. Loss of disulfide bond I led to a widening of the orthosteric binding pocket resulting in a strong reduction in the potency of adenosine, but not of NECA or 2-substituted nucleosides. Disruption of disulfide bond II led to a significant reduction in the agonists' efficacy indicating its importance for receptor activation. Disulfide bond III disruption reduced potency and affinity of the small adenosine agonists and NECA, but not of the larger 2-substituted agonists. While all the three disulfide bonds were essential for high potency or efficacy of adenosine, structural modification of the nucleoside could rescue affinity or efficacy at the mutant receptors. At present, it cannot be excluded that formation of the extracellular disulfide bonds in the A2AAR is dynamic. This might add another level of G protein-coupled receptor (GPCR) modulation, in particular for the cysteine-rich A2A and A2BARs.
authors.affiliation: Pharma Center Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: De Filippo
authors.affiliation: Pharma Center Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany.
authors.forename: Vigneshwaran
authors.initials: V
authors.lastname: Namasivayam
authors.affiliation: Pharma Center Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany.
authors.forename: Lukas
authors.initials: L
authors.lastname: Zappe
authors.affiliation: Pharma Center Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany.
authors.forename: Ali
authors.initials: A
authors.lastname: El-Tayeb
authors.affiliation: Pharma Center Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany.
authors.forename: Anke C
authors.initials: AC
authors.lastname: Schiedel
authors.affiliation: Pharma Center Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany. christa.mueller@uni-bonn.de.
authors.forename: Christa E
authors.initials: CE
authors.lastname: Müller
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D005110:Extracellular Space / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D043705:Receptor, Adenosine A2A / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D043705:Receptor, Adenosine A2A; D003545:Cysteine
keywords: A2A adenosine receptor; A2B adenosine receptor; Cysteine; Disulfide bonds; GPCR; Mutagenesis