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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 [ 3 H]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 H]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 [ 3 H]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. |
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