Browning, Christopher; (2003) The effect of receptor density on the binding and functional properties of the human adenosine A1 receptor. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The adenosine A1 receptor is a member of the 7-transmembrane G-protein coupled receptor family. It has an important signalling role in both normal physiology and disease. In addition to containing binding sites for adenosine and G-proteins, the A1 receptor contains an allosteric binding site which binds synthetic molecules such as the allosteric enhancer PD 81,723. The availability of this compound, together with high affinity antagonists, highly potent agonists with a range of efficacies, the ability to determine ligand binding to the G-protein coupled and uncoupled states of the receptor and to readily measure function in membranes, make the A1 receptor an ideal candidate for testing the predictions of mathematical models of drug receptor interactions. The effects of receptor density, guanine nucleotides, and agonist efficacy on the ligand binding properties of the human adenosine A1 receptor, recombinantly expressed at different densities, in membranes from Chinese hamster ovary cells have been examined. The proportions and ligand affinities of receptors in the G- protein coupled and uncoupled states of the A1 receptor have been measured. The results suggest that there is a significant amount of receptor-G protein precoupling in the absence of ligand but the overall results are not quantitatively compatible with all the predictions of the ternary complex model of agonism. Radioligand dissociation kinetic studies show the novel phenomenon of agonist-induced agonist dissociation when the receptors are expressed at high but not low expression levels. These results are compatible with the presence of receptor dimers, or another form of receptor cross-talk that occurs when agonists are bound to receptor G-protein complexes at high expression levels. The effects of receptor density and agonist efficacy on the functional properties of the adenosine A1 receptor were examined using a [35S]GTP S binding assay. Marked novel biphasic dose-response curves were observed for high efficacy agonists in membranes from a high expressing A1 receptor cell line. Experiments were performed to investigate the nature of this response, its reversibility, and the effects of an inverse agonist and agonists of different efficacy. A detailed study of the kinetics of association of [35S]GTP S following agonist exposure suggest that a reversible feedback mechanism is operating, with a short lag-time for its induction. The mechanism of action of the allosteric enhancer, PD 81,723, on the binding of a series of ligands with a range of intrinsic activities to the coupled and uncoupled states of the adenosine A1 receptor was investigated. The effects of PD 81,723 on the functional properties of these ligands, measured at different receptor expression levels, were also studied. The results of these studies were not compatible with the detailed predictions of the ternary complex model of agonism if PD 81,723 simply enhanced the affinity of the receptor or if it increased agonist efficacy. A quaternary complex model of allosterism and agonism was developed which is compatible with the actions of PD 81,723 in binding and function. These data can be explained by PD 81,723 activating the receptors from the allosteric site and by its ability to act as a co-agonist with agonist binding to the orthosteric site to increase affinity of the A1 receptor for its cognate G protein.

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