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.

In neuroendocrine cells, immature secretory granules (ISGs) bud from the trans Golgi network (TGN), and then undergo a maturation process that includes ISG-ISG homotypic fusion, removal of excess membranes via clathrin-coated vesicles (CCVs) and acidification of the granules. These mature secretory granules (MSGs) can then fuse with the plasma membrane upon receiving an external signal, and finally release their content in the extracellular space. In this thesis, I will present evidence that Synaptotagmin IV (Syt IV) is one of the components involved in the regulation of granule maturation in neuroendocrine cells. Syt IV is a neuron and neuroendocrine cell-specific isoform, which belongs to the synaptotagmin family of membrane trafficking proteins. After confirming that Syt IV is localised on ISGs and absent from MSGs in PCI2 cells, I investigated whether Syt IV is involved in ISG homotypic fusion by adding the purified Syt IV cytoplasmic domain into an in vitro assay, where it functions as a dominant negative. Addition of this domain, but not a similar domain from Syt I, resulted in a dose-dependent inhibition of ISG-ISG fusion. Furthermore, I found that Syt IV binds to the ISG-SNARE Syntaxin 6 (Stx6), suggesting that the two proteins might be part of the same machinery that regulates ISG maturation. In addition, I used an in vivo approach based on the processing of secretogranin II (Sgll) into its degradation product pi8, which occurs during ISG maturation, to assay Syt IV function. I show that the Syt IV cytoplasmic domain, as well as siRNA-mediated knockdown of Syt IV inhibits Sgll processing by prohormone convertase 2 (PC2). Interestingly, PC2 is found mostly in the pro-form, suggesting that activation of PC2 is also inhibited. We conclude that Syt IV is an essential component for the process of secretory granule maturation. Lastly, I found that Syt IV binds preferentially to membrane-bound clathrin adaptor protein-1 (AP-1). This suggests that Syt IV might be sorted from maturing SGs in a complex, possibly via an ISG-SNARE protein that interacts directly with AP-1.

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