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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.

SUBMITTER: Naranjo AN

PROVIDER: S-EPMC4565196 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Conserved disulfide bond is not essential for the adenosine A2A receptor: Extracellular cysteines influence receptor distribution within the cell and ligand-binding recognition.

Naranjo Andrea N AN Chevalier Amy A Cousins Gregory D GD Ayettey Esther E McCusker Emily C EC Wenk Carola C Robinson Anne S AS

Biochimica et biophysica acta 20141116 2

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 cystein ...[more]

PMID: 25445670

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Project description:The A2a adenosine receptor is a member of the G-protein coupled receptor family, and its activation stimulates cyclic AMP production. To determine the residues which are involved in ligand binding, several residues in transmembrane domains 5-7 were individually replaced with alanine and other amino acids. The binding properties of the resultant mutant receptors were determined in transfected COS-7 cells. To study the expression levels in COS-7 cells, mutant receptors were tagged at their amino terminus with a hemagglutinin epitope, which allowed their immunological detection in the plasma membrane by the monoclonal antibody 12CA5. The functional properties of mutant receptors were determined by measuring stimulation of adenylate cyclase. Specific binding of [3H]CGS 21680 (15 nM) and [3H]XAC (4 nM), an A2a agonist and antagonist, respectively, was absent in the following Ala mutants: F182A, H250A, N253A, I274A, H278A, and S281A, although they were well expressed in the plasma membrane. The hydroxy group of Ser-277 is required for high affinity binding of agonists, but not antagonists. An N181S mutant lost affinity for adenosine agonists substituted at N6 or C-2, but not at C-5'. The mutant receptors I274A, S277A, and H278A showed full stimulation of adenylate cyclase at high concentrations of CGS 21680. The functional agonist potencies at mutant receptors that lacked radioligand binding were > 30-fold less than those at the wild type receptor. His-250 appears to be a required component of a hydrophobic pocket, and H-bonding to this residue is not essential. On the other hand, replacement of His-278 with other aromatic residues was not tolerated in ligand binding. Thus, some of the residues targeted in this study may be involved in the direct interaction with ligands in the human A2a adenosine receptor. A molecular model based on the structure of rhodopsin, in which the 5'-NH in NECA is hydrogen bonded to Ser-277 and His-278, was developed in order to visualize the environment of the ligand binding site.

Project description:The A2a adenosine receptor, a member of the G protein-coupled receptor family, is important in the regulation of dopaminergic pathways of the brain and in platelet and cardiovascular functions. In this study, the role of extracellular loops in ligand binding to the human A2a receptor was explored through site-directed mutagenesis. Four glutamate/aspartate residues (Glu151, Glu161, Glu169, and Asp170) in the second extracellular loop (E2) and a cysteine residue (Cys262) in the third extracellular loop (E3) were individually replaced with alanine and other amino acids. A proline residue (Pro173) in E2 was mutated to arginine, the homologous amino acid in A3 receptors. The binding properties of the resultant mutant receptors were determined in transfected COS-7 cells. The mutant receptors were tagged at their amino terminus with a hemagglutinin epitope, thus allowing their detection in the plasma membrane with immunological techniques. High affinity specific binding of [3H]2-[4-[(2-carboxyethyl)phenyl]ethyl-amino]-5'-N-ethylcarboxamidoad eno sine (15 nM) and [3H]8-[4-[[[[2-aminoethyl)-amino]carbonyl]methyl]oxy]phenyl]-1,3- dipropylxanthine (4nM), an A2a agonist and antagonist, respectively, was not observed with four of the mutant receptors, E151A, E151Q, E151D, and E169A, although they were well expressed at the cell surface. The E151A and E169A mutant receptors showed nearly full stimulation of adenylyl cyclase at approximately 10(3)-fold higher concentrations of 2-[4-[(2-carboxyethyl)phenyl]ethyl-amino]-5'-N-ethylcarboxamidoadenosine . The E161A mutant receptor showed as increase in affinity for the nonxanthine adenosine antagonist 9-chloro-2-(furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine(6 fold) but not for other ligands. An E169Q mutant gained affinity (5-22 fold) for adenosine derivatives (agonists) substituted at N6 but not at C2 or C5' positions. Mutant receptors D170K and P173R were similar to wild-type receptors in binding of both agonist and antagonist radioligands. A C262G mutant also resembled the wild-type receptor in radioligand binding, indicating that a potential disulfide bridge with another cysteine residue in proximity is not required for the structural integrity of the receptor. Our data suggest that certain amino acids in the second extracellular loop may be directly or indirectly involved in ligand binding.

Dataset Information

Conserved disulfide bond is not essential for the adenosine A2A receptor: Extracellular cysteines influence receptor distribution within the cell and ligand-binding recognition.

Publications

Conserved disulfide bond is not essential for the adenosine A2A receptor: Extracellular cysteines influence receptor distribution within the cell and ligand-binding recognition.

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