Structural and functional characterisation of neuronal Gq protein coupled receptors
Both G protein coupled receptor 10 (GPR10) and the muscarinic IVh receptor are members of family A of the G protein coupled receptor superfamily. GPR10 is the human orthologue of a former orphan receptor for which prolactin releasing peptides (PrRP) have been identified as the cognate ligands. In contrast, the N/h receptor represents a well-established receptor for which up until the discovery of AC-42 (4-(4-butyl-1-piperidinyl)-1-(2-methylphenyl)-1-butanone) it has been difficult to identify a selective agonist. Little is known of the pharmacology of PrRPs and AC-42 at GPR10 and the human Mi (hMi) receptor, respectively. Similarly, the molecular nature of their respective binding sites is as yet unknown. In the studies in this thesis, the interaction of PrRPs with GPR10 and of AC-42 (and other ectopic agonists) with the hMi receptor have been extensively characterised using a range of pharmacological techniques in both recombinant cell lines stably and transiently expressing the receptor(s) of interest and native tissue preparations. Furthermore, the molecular interactions between ligand and receptor have been probed using homology modelling, site-directed mutagenesis (SDM) and pharmacological evaluation. The results generated reveal that PrRPs are high affinity, potent agonists at GPR10 that cause the receptor to activate effector systems that are known to couple to Gq/n proteins. Radioligand binding studies suggest both high and low affinity sites for binding. In addition, homology modelling and SDM have been used to reveal key interactions of the C-terminal region of PrRP with transmembrane domain (TM) 6 (D302) and TM7 (Q317) and extracellular loop (ECL) 2 (E213). Selective activation of the Mi receptor can be achieved using AC-42 and other novel ligands. Using functional calcium mobilisation assays, inositol phosphate assays and radioligand binding studies it has been possible to demonstrate that this class of compounds interacts with the receptor in an allosteric manner. SDM studies also suggest that residues distinct from the orthosteric binding site form part of the binding site for AC-42 and related compounds. These studies provide the first extensive pharmacological analysis characterising the interaction of PrRP and GPR10 and identify the signal transduction cascade activated by this former orphan receptor. Furthermore, SDM studies have partly elucidated the molecular nature of the PrRP binding site. Both pharmacological and SDM based examination of the muscarinic Mi receptor have revealed a unique allosteric method of activation by AC-42 and a novel class of allosteric agonists.