| Summary: | The adenosine A} receptor (AR) is an important G protein-coupled receptor (GPCR) distributed widely throughout the human body and influences many bodily functions. The work presented here investigates in detail the complex heterogeneous nature of agonist binding to the human AR in order to explore receptor-receptor interactions that might be present in oligomeric receptor complexes. The equilibrium and kinetic binding properties of two cell lines expressing the AR at different densities were characterised in detail. This characterisation was compared with that of a series of 47 cell lines expressing different levels of either the AjR-GFP or AR-GFP-Gaj fusion proteins. Equilibrium radioligand saturation and competition experiments provided evidence for a re lationship between the fraction of high affinity agonist binding sites and the level of receptor expression. Cell lines expressing lower levels of the AR showed a greater relative ability to bind agonists with high affinity, and to promote formation of the activated agonist-receptor-G protein ternary complex. The association of Hjagonist to the AjR was biphasic and determined by two different molec ular processes. The association rate constant of the fast component was entirely dependent on the concentration of Hjagonist, whereas dependence of the slow component on concentration was inconclusive. The dissociation of a Hjinverse agonist from the AR was rapid, mono-exponential, complete, and insensitive to GTP. In contrast, the kinetics of H agonist dissociation were complex. Disso ciation of H agonist from the AR-G protein complex was biphasic and dependent on the nature of ligand used to prevent H agonist rebinding. Greater H agonist dissociation was observed in the presence of competing agonist than competing inverse agonist, a novel finding called "agonist- induced agonist dissociation," and was dependent on agonist efficacy. The mechanism behind this is unknown, but appears to involve interactions between high affinity receptor-G protein complexes, possibly in the form of receptor oligomerisation. These interactions are absent at low expression levels and progressively increase with level of expression. Agonist-induced agonist dissociation was observed even in the presence of a high concentration of GTP. Separation of cell membrane fractions by their buoyant density clearly showed the AR-GFP and AR-GFP-Gaj fusion proteins were not found in lower density caveolin-enriched "raft" frac tions. These observations have implications for the nature of the immediate receptor environment and whether the AR and other components of the receptor signalling complex are actively con centrated in regions of the cell membrane. The work presented here describes novel properties of agonist and antagonist binding at the human adenosine Aj receptor and to AjR-GFP and AR-GFP-Go fusion proteins. The ligand dependence of the kinetics of agonist dissociation provides direct evidence for receptor-receptor interactions, such as receptor oligomerisation.
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