Site-directed Mutagenesis Identifies Residues Involved in Ligand Recognition in the Human A2a Adenosine Receptor (*)

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.

[1]  M. Williams,et al.  Adenosine receptors: pharmacology, structure-activity relationships, and therapeutic potential. , 1992, Journal of medicinal chemistry.

[2]  G. Stiles,et al.  Molecular cloning and characterization of an adenosine receptor: the A3 adenosine receptor. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[3]  H. Okayama,et al.  A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. , 1983, Molecular and cellular biology.

[4]  C. Strader,et al.  Identification of two serine residues involved in agonist activation of the beta-adrenergic receptor. , 1989, The Journal of biological chemistry.

[5]  K. Jacobson,et al.  Characterization of human striatal A2-adenosine receptors using radioligand binding and photoaffinity labeling. , 1992, Journal of receptor research.

[6]  R. Henderson,et al.  Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. , 1990, Journal of molecular biology.

[7]  S Schiffmann,et al.  RDC8 codes for an adenosine A2 receptor with physiological constitutive activity. , 1990, Biochemical and biophysical research communications.

[8]  B. Cullen.  Use of eukaryotic expression technology in the functional analysis of cloned genes. , 1987, Methods in enzymology.

[9]  M. Jarvis,et al.  Adenosine agonists reduce conditioned avoidance responding in the rat , 1993, Pharmacology Biochemistry and Behavior.

[10]  J. Wess,et al.  Functional role in ligand binding and receptor activation of an asparagine residue present in the sixth transmembrane domain of all muscarinic acetylcholine receptors. , 1994, The Journal of biological chemistry.

[11]  U. Ungerstedt,et al.  Antagonistic interaction between adenosine A2A receptors and dopamine D2 receptors in the ventral striopallidal system. Implications for the treatment of schizophrenia , 1994, Neuroscience.

[12]  H. Normile,et al.  Role of adenosine A2a receptors in the nucleus accumbens , 1994, Progress in Neuro-Psychopharmacology and Biological Psychiatry.

[13]  J. Ballesteros,et al.  [19] Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors , 1995 .

[14]  M. Williams,et al.  [3H]CGS 21680, a selective A2 adenosine receptor agonist directly labels A2 receptors in rat brain. , 1989, The Journal of pharmacology and experimental therapeutics.

[15]  K. Jacobson,et al.  Cloning, expression, and characterization of the unique bovine A1 adenosine receptor. Studies on the ligand binding site by site-directed mutagenesis. , 1992, The Journal of biological chemistry.

[16]  J. Bockaert,et al.  D2-dopamine receptor-mediated inhibition of cyclic AMP formation in striatal neurons in primary culture. , 1985, Molecular pharmacology.

[17]  J. Baldwin.  The probable arrangement of the helices in G protein‐coupled receptors. , 1993, The EMBO journal.

[18]  K. Jacobson,et al.  A2A adenosine receptors from rat striatum and rat pheochromocytoma PC12 cells: characterization with radioligand binding and by activation of adenylate cyclase. , 1992, Molecular pharmacology.

[19]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[20]  A. Tucker,et al.  A1 adenosine receptors. Two amino acids are responsible for species differences in ligand recognition. , 1994, The Journal of biological chemistry.

[21]  X. Ji,et al.  Structure-activity relationships of N6-benzyladenosine-5'-uronamides as A3-selective adenosine agonists. , 1994, Journal of medicinal chemistry.

[22]  K. Jacobson,et al.  Molecular modeling of adenosine receptors. The ligand binding site on the rat adenosine A2A receptor. , 1994, European journal of pharmacology.

[23]  J. Wess,et al.  Site‐directed mutagenesis of the m3 muscarinic receptor: identification of a series of threonine and tyrosine residues involved in agonist but not antagonist binding. , 1991, The EMBO journal.

[24]  J. Bockaert,et al.  Pharmacological characterization of the D2 dopamine receptor negatively coupled with adenylate cyclase in rat anterior pituitary. , 1983, Molecular pharmacology.

[25]  K. Jacobson,et al.  Identification of the A2 adenosine receptor binding subunit by photoaffinity crosslinking. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[26]  K. Jacobson,et al.  Chemical modification and irreversible inhibition of striatal A2a adenosine receptors. , 1992, Molecular pharmacology.

[27]  C. Strader,et al.  The role of histidine 265 in antagonist binding to the neurokinin-1 receptor. , 1994, The Journal of biological chemistry.

[28]  R. Higuchi.  Using PCR to Engineer DNA , 1989 .

[29]  J Hoflack,et al.  Three-dimensional models of neurotransmitter G-binding protein-coupled receptors. , 1991, Molecular pharmacology.

[30]  C. Gerfen,et al.  Cloning and expression of an A1 adenosine receptor from rat brain. , 1991, Molecular pharmacology.

[31]  P. J. Richardson,et al.  Role of Histidine Residues in the Adenosine A2a Receptor Ligand Binding Site , 1994, Journal of neurochemistry.

[32]  S. Shiozaki,et al.  KF17837: a novel selective adenosine A2A receptor antagonist with anticataleptic activity. , 1994, European journal of pharmacology.

[33]  K. Jacobson,et al.  A binding site model and structure-activity relationships for the rat A3 adenosine receptor. , 1994, Molecular pharmacology.

[34]  Gebhard F. X. Schertler,et al.  Projection structure of rhodopsin , 1993, Nature.

[35]  P. Schofield,et al.  A threonine residue in the seventh transmembrane domain of the human A1 adenosine receptor mediates specific agonist binding. , 1994, The Journal of biological chemistry.

[36]  K. Jacobson,et al.  Identification of an adenosine receptor domain specifically involved in binding of 5'-substituted adenosine agonists. , 1994, The Journal of biological chemistry.

[37]  K. Jacobson,et al.  Acute treatment of mice with high doses of caffeine: An animal model for choreiform movement , 1993 .