title: Conserved disulfide bond is not essential for the adenosine A2A receptor: Extracellular cysteines influence receptor distribution within the cell and ligand-binding recognition.
abstract: G protein-coupled receptors (GPCRs) are integral membrane proteins involved in cellular signaling and constitute major drug targets. Despite their importance, the relationship between structure and function of these receptors is not well understood. In this study, the role of extracellular disulfide bonds on the trafficking and ligand-binding activity of the human A2A adenosine receptor was examined. To this end, cysteine-to-alanine mutations were conducted to replace individual and both cysteines in three disulfide bonds present in the first two extracellular loops. Although none of the disulfide bonds were essential for the formation of plasma membrane-localized active GPCR, loss of the disulfide bonds led to changes in the distribution of the receptor within the cell and changes in the ligand-binding affinity. These results indicate that in contrast to many class A GPCRs, the extracellular disulfide bonds of the A2A receptor are not essential, but can modulate the ligand-binding activity, by either changing the conformation of the extracellular loops or perturbing the interactions of the transmembrane domains.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy Street, Newark, DE 19716, USA. Electronic address: anaranjo@udel.edu.
authors.forename: Andrea N
authors.initials: AN
authors.lastname: Naranjo
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy Street, Newark, DE 19716, USA. Electronic address: amychev@seas.upenn.edu.
authors.forename: Amy
authors.initials: A
authors.lastname: Chevalier
authors.affiliation: Department of Computer Science, Tulane University, 6823 St. Charles Ave, New Orleans, LA 70118, USA.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Cousins
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy Street, Newark, DE 19716, USA.
authors.forename: Esther
authors.initials: E
authors.lastname: Ayettey
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy Street, Newark, DE 19716, USA. Electronic address: emily.mccusker@tevapharm.com.
authors.forename: Emily C
authors.initials: EC
authors.lastname: McCusker
authors.affiliation: Department of Computer Science, Tulane University, 6823 St. Charles Ave, New Orleans, LA 70118, USA. Electronic address: cwenk@tulane.edu.
authors.forename: Carola
authors.initials: C
authors.lastname: Wenk
authors.affiliation: Department of Chemical and Biomolecular Engineering, Tulane University, 300 Lindy Boggs Laboratory, 6823 St. Charles Ave, New Orleans, LA 70118, USA; Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy Street, Newark, DE 19716, USA. Electronic address: asr@tulane.edu.
authors.forename: Anne S
authors.initials: AS
authors.lastname: Robinson
mesh_terms: D000409:Alanine / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry*; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D021381:Protein Transport; D043705:Receptor, Adenosine A2A / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
chemical_list: D004220:Disulfides; D008024:Ligands; D043705:Receptor, Adenosine A2A; D011994:Recombinant Proteins; D003545:Cysteine; D000409:Alanine
keywords: Agonist; Conformational stability; Extracellular loop; G protein-coupled receptor; Hausdorff ratio