Site-directed mutagenesis studies of human A2A adenosine receptors: Involvement of glu13 and his278 in ligand binding and sodium modulation

doi:10.1016/S0006-2952(00)00357-9

Biochemical Pharmacology

Volume 60, Issue 5, 1 September 2000, Pages 661-668

https://doi.org/10.1016/S0006-2952(00)00357-9 Get rights and content

Abstract

To provide insights into interactions between ligands and A_2A adenosine receptors, site-directed mutagenesis was used to test the roles of a glutamic acid residue in the first transmembrane domain (Glu13) and a histidine residue in the seventh transmembrane domain (His278). The two residues, which have been suggested to be closely linked in molecular modeling studies, were mutated to glutamine (E13Q) and tyrosine (H278Y), respectively. Saturation experiments revealed that [³H]ZM241385 (4-{2-[7-amino-2-(2-furyl)-1,2,4-triazolo[1,5-a][1,3,5]triazin-5-yl-amino]ethyl}phenol) bound wild-type and mutant receptors in membranes from COS-7 cells expressing human A_2A adenosine receptors with high affinity and low non-specific binding. It was found from the competition experiments that the affinity of the A_2A adenosine receptor agonists for the mutant receptors was 3- to 200-fold lower than for the wild-type receptor. Among antagonist competitors of binding at E13Q and H278Y mutant receptors, there was variation in the affinity depending on their different structures, although changes were relatively minor (<3-fold) except in the case of theophylline, whose affinity was decreased approximately 20 times on the H278Y mutant. The possible involvement of the two residues in sodium ion regulation was also tested. The agonist competition curves for [³H]ZM241385 were shifted to the right in both wild-type and mutant receptors in the presence of 1 M sodium ions, but the extent of shift (2- to 27-fold) in wild-type receptor was generally larger than for the mutant receptors. Sodium ions also decreased [³H]ZM241385 dissociation from both wild-type and mutant receptors, being more influential on the former than the latter. The results suggest that the two closely linked residues Glu13 and His278 in A_2A adenosine receptor are most important for agonist recognition and are partly responsible for the allosteric regulation by sodium ions.

Section snippets

Materials

Human A_2A adenosine receptor cDNA (expression vector pSVL-A_2A) was kindly provided by Dr. Marlene Jacobson (Merck Research Labs). Taq polymerase for the polymerase chain reaction (PCR) was purchased from Perkin Elmer. All enzymes used in this study were obtained from New England Biolabs. Oligonucleotides used were synthesized by Bioserve Biotechnologies. [³H]ZM241385 (17 Ci/mmol) was from Tocris Cookson Ltd. GTP was purchased from Aldrich and BSA from Sigma. Adenosine deaminase was obtained

Saturation binding of [³H]ZM241385 to human wild-type and mutant A_2A adenosine receptors and the effects of sodium ions and GTP

Saturation experiments were carried out with both wild-type and mutant receptors using [³H]ZM241385 as a radioligand. The experiments revealed that [³H]ZM241385 bound to a single class of wild-type receptors (K_d = 1.8 ± 0.20 nM; B_max = 7617 ± 858 fmol/mg protein) displaying low non-specific binding (<10%). The K_d values for [³H]ZM241385 binding at the E13Q and H278Y mutant receptors were 2–3 times higher than for the wild-type receptor. NaCl (1 M) produced a <2-fold decrease in K_d values in

Discussion

In the present study, the interactions between the human A_2A adenosine receptor and its ligands were investigated by site-directed mutagenesis. In a previous molecular modeling study [17], it had been suggested that a glutamic acid in the first transmembrane domain (Glu13) and a histidine in the seventh transmembrane domain (His278) are somehow linked and that they are involved in agonist binding to the A_2A adenosine receptor and its ligands. We followed up on this study by analyzing the

Acknowledgements

We thank Miriam de Groote, Jacobien K. von Frijtag Drabbe Künzel, and David S. Johnson for technical assistance.

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Molecular and cellular pharmaclogySite-directed mutagenesis studies of human A2A adenosine receptors: Involvement of glu13 and his278 in ligand binding and sodium modulation

Abstract

Section snippets

Materials

Saturation binding of [3H]ZM241385 to human wild-type and mutant A2A adenosine receptors and the effects of sodium ions and GTP

Discussion

Acknowledgements

Prog Neuropsychopharmacol Biol Psychiatry

Neuroscience

Pharmacol Biochem Behav

Eur J Pharmacol

J Biol Chem

Eur J Pharmacol

Eur J Pharmacol

J Mol Biol

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

Cardiovascular purinoceptors

Physiol Rev

[3H]CGS21680, a selective A2 adenosine receptor agonist directly labels A2 receptors in rat brain

J Pharmacol Exp Ther

Acute treatment of mice with high-doses of caffeineAn animal model for choreiform movement

Drug Dev Res

Molecular and cellular pharmaclogy
Site-directed mutagenesis studies of human A_2A adenosine receptors: Involvement of glu¹³ and his²⁷⁸ in ligand binding and sodium modulation

Saturation binding of [³H]ZM241385 to human wild-type and mutant A_2A adenosine receptors and the effects of sodium ions and GTP

[³H]CGS21680, a selective A₂ adenosine receptor agonist directly labels A₂ receptors in rat brain