Cohen, Fiona R.; (1995) Allosteric regulation of adenosine receptors. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The adenosine A1 receptor is one of the large superfamily of G protein coupled receptors. These receptors are generally considered to have 2 binding sites, one which binds the hormone or neurotransmitter, and one which binds the G protein. However it has recently been reported that an additional binding site exists on the A1 receptor. The binding of certain benzoylthiophenes (e.g. 2-amino-4, 5- dimethylthien-3-yl [3-(trifluoro methyl) phenyl] - methanone, PD81) to this site, allosterically modulate the binding and activity of agonists (and antagonists), thereby providing a novel means of modulating the actions of endogenous adenosine. Initially, the binding and functional properties of the cloned human adenosine A1 receptor, expressed in Chinese hamster ovary cells, were characterised. A novel saponin pretreatment simplified the assay system and enabled for the first time a detailed characterisation of the binding properties of adenosine itself for the A1 receptor. The core of the study was a quantitative investigation of the effects of allosteric agents on binding and function of the adenosine A1 receptor. The experimental evidence is quantitatively compatible with PD81 binding to a specific allosteric site on the A1 receptor. Functional assays demonstrate that the cloned human A2A receptor does not appear to have the same allosteric site. Replacement of regions of the A1 receptor by complementary regions of the A2a receptor was used in an attempt to elucidate the region where the allosteric ligand interacts with the receptor. Seven A1/A2A chimeric receptors were constructed using the PCR technique of splicing by overlap extension. The expression and function of the chimeric receptors, as well as the effects of the alterations on the competitive and allosteric ligand binding sites was assessed using electrophysiological and binding techniques. The results suggest that the seventh transmembrane domain and/or the intracellular COOH-terminus domain of the A1 adenosine receptor may be critical for the positive allosteric interaction between the agonist binding site and the allosteric site.

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