Site-directed mutagenesis of the human adenosine A2A receptor. Critical involvement of Glu13 in agonist recognition.
A glutamic acid residue in the first transmembrane domain of the human adenosine A2A receptor was mutated to glutamine. Radioligand binding studies on COS-7 cell membranes expressing either the wild-type or the mutant receptor revealed that the affinity of the prototypic agonist CGS21680 (2-[4-[(2-carboxyethyl)phenyl]ethylamino]-5'-N-ethylcarboxamidoadenosine ) for the mutant receptor was 15-fold lower than for the wild-type receptor. This was confirmed in functional studies with intact cells. The EC50 values of CGS21680 for the stimulation of cAMP production differed in a similar way. Antagonists of various chemical structure were equally effective on both mutant and wild-type receptors, thus the mutation selectively diminishes agonist affinity. We propose an indirect perturbation of the binding site, perhaps through a proton transfer mechanism as suggested by molecular modelling.
IJzerman
A P
AP
Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Netherlands.
Von Frijtag Drabbe Künzel
J K
JK
Kim
J
J
Jiang
Q
Q
Jacobson
K A
KA
D000242:Cyclic AMP / Q000096:biosynthesis; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D058906:Purinergic P1 Receptor Agonists; D011869:Radioligand Assay; D018047:Receptors, Purinergic P1 / Q000235:genetics* / Q000378:metabolism
D058906:Purinergic P1 Receptor Agonists; D018047:Receptors, Purinergic P1; D018698:Glutamic Acid; D000242:Cyclic AMP