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

Growth hormone secretagogues and growth hormone releasing peptides act as orthosteric super-agonists but not allosteric regulators for activation of the G protein G{alpha}o1 by the ghrelin receptor

A series of growth hormone secretagogues act as agonists at the ghrelin receptor and have been described as 'ago-allosteric' ligands due to an ability to also modulate the maximum efficacy and potency of ghrelin (Holst et al., 2005). In membranes prepared from cells co-expressing the human ghrelin receptor and the G protein G{alpha}o1 each of MK-677, GHRP-6 and L-692585 functioned as direct agonists, and each displayed higher efficacy than ghrelin. The effect of multiple, fixed concentrations of each of these ligands on the function and concentration-dependence of ghrelin and the effect of multiple, fixed concentrations of ghrelin on the action of MK-677, GHRP-6 and L-692585 was analyzed globally according to a modified version of an operational model of allosterism which accounts for allosteric modulation of affinity, efficacy and allosteric agonism. Each of the data sets was best fitted by a model of simple competition between a partial and a full agonist. Both positive and negative allosteric modulators are anticipated to alter the kinetics of binding of an orthosteric agonist. However, none of the proposed ago-allosteric regulators tested had any effect on the dissociation kinetics of [His[125I]]-ghrelin and GHRP-6 and MK-677 were able to fully displace [His[125I]]-ghrelin from the receptor. At least in the system tested, each of the ligands acted in a simple competitive fashion with ghrelin as demonstrated by analysis according to a model whereby ghrelin is a partial agonist with respect to each of the synthetic agonists teste

A Bayesian approach for determining protein side-chain rotamer conformations using unassigned NOE data

Abstract. A major bottleneck in protein structure determination via nuclear magnetic resonance (NMR) is the lengthy and laborious process of assigning resonances and nuclear Overhauser effect (NOE) cross peaks. Recent studies have shown that accurate backbone folds can be determined using sparse NMR data, such as residual dipolar couplings (RDCs) or backbone chemical shifts. This opens a question of whether we can also determine the accurate protein sidechain conformations using sparse or unassigned NMR data. We attack this question by using unassigned nuclear Overhauser effect spectroscopy (NOESY) data, which record the through-space dipolar interactions between protons nearby in 3D space. We propose a Bayesian approach with a Markov random field (MRF) model to integrate the likelihood function derived from observed experimental data, with prior information (i.e., empirical molecular mechanics energies) about the protein structures. We unify the side-chain structure prediction problem with the side-chain structure determination problem using unassigned NMR data, and apply the deterministic dead-end elimination (DEE) and A * search algorithms t