An investigation of the molecular pharmacology of G protein-coupled receptor 35

G protein-coupled receptors (GPCRs) are seven-pass integral membrane proteins that act as transducers of extracellular signals across the lipid bilayer. Their location and involvement in basic and pathological physiological processes has secured their role as key targets for pharmaceutical intervention. GPCRs are targeted by many of the best-selling drugs on the market and there are a substantial number of GPCRs that are yet to be characterised; these could offer interest for therapeutic targeting. GPR35 is one such receptor that, as a result of gene knockout and genome wide association studies, has attracted interest through its association with cardiovascular and gastrointestinal disease. Elucidation of the basic physiological function of GPR35 has, however, been difficult due a paucity of potent and selective ligands in addition to a lack of consensus on the endogenous ligand. Herein, a focussed drug discovery effort was carried out to identify agonists of GPR35. Various in vitro cellular assays were employed in conjunction with N- or C-terminally manipulated forms of the receptor to investigate GPR35’s signalling profile and to provide an assay format suitable for the characterisation of newly identified ligands. Although GPR35 associates with both Gαi/o and Gα13 families of small heterotrimeric G proteins, the G protein-independent β-arrestin-2 recruitment format was found to be the most suited to drug screening efforts. Small molecule compound screening, carried out in conjunction with the Medical Research Council Technology, identified compound 1 as the most potent ligand of human GPR35 reported at that time. However, the lower efficacy and potency of compound 1 at the rodent species orthologues of GPR35 prevented its use in in vivo studies. A subsequent effort, carried out with Novartis, focused on mast cell stabilisers as putative agonists of GPR35, revealed lodoxamide and bufrolin as highly potent agonists that activated human and rat GPR35 with equal potency. This finding offered–for the first time–the opportunity to employ the same GPR35 ligand between species at a similar concentration, an important factor to consider when translating rodent in vivo functional studies to those in man. Additionally, using molecular modelling and site directed mutagenesis studies, these newly identified compounds were used to aid characterisation of the ligand binding pockets of human and rat GPR35 to reveal the molecular basis of species selectivity at this receptor. In summary, this research effort presents GPR35 tool compounds that can now be used to dissect the basic biology of GPR35 and investigate its contribution to disease.

A biochemical analysis into the co-translational folding of a G protein-coupled receptor; GPR35

The folding and targeting of membrane proteins poses a major challenge to the cell, as they must remain insertion competent while their highly hydrophobic transmembrane (TM) domains are transferred from the ribosome, through the aqueous cytosol and into the lipid bilayer. The biogenesis of a mature membrane protein takes place through the insertion and integration into the lipid bilayer. A number of TM proteins have been shown to gain some degree of secondary structure within the ribosome tunnel and to retain this conformation throughout maturation. Although studies into the folding and targeting of a number of membrane proteins have been carried out to date, there is little information on one of the largest class of eukaryotic membrane proteins; the G-protein-coupled receptors (GPCRs). This project studies the early folding events of the human ortholog of GPR35. To analyse the structure of the 1st TM domain, intermediates were generated and assessed by the biochemical method of pegylation (PEG-MAL). A structurally-similar microbial opsin (Bacterioopsin) was also used to investigate the differences in the early protein folding within eukaryotic and prokaryotic translation systems. Results showed that neither the 1st TM domain of GPR35 nor Bacterioopsin were capable of compacting in the ribosome tunnel before their N-terminus reached the ribosome exit point. The results for this assay remained consistent whether the proteins were translated in a eukaryotic or prokaryotic translation system. To examine the communication mechanism between the ribosome, the nascent chain and the protein targeting pathway, crosslinking experiments were carried out using the homobifunctional lysine cross-linker BS3. Specifically, the data generated here show that the nascent chain of GPR35 reaches the ribosomal protein uL23 in an extended conformation and interacts with the SRP protein as it exits the ribosome tunnel. This confirms the role of SRP in the co-translational targeting of GPR35. Using these methods insights into the early folding of GPCRs has been obtained. Further experiments using site-directed mutagenesis to reduce hydrophobicity in the 1st TM domain of GPR35, highlighted the mechanisms by which GPCRs are targeted to the endoplasmic reticulum. Confirming that hydrophobicity within the signal anchor sequence is essential of SRP-dependent targeting. Following the successful interaction of the nascent GPR35 and SRP, GPR35 is successfully targeted to ER membranes, shown here as dog pancreas microsomes (DPMs). Glycosylation of the GPR35 N-terminus was used to determine nascent chain structure as it is inserted into the ER membrane. These glycosylation experiments confirm that TM1 has obtained its compacted state whilst residing in the translocon. Finally, a site-specific cross-linking approach using the homobifunctional cysteine cross-linker, BMH, was used to study the lateral integration of GPR35 into the ER. Cross-linking of GPR35 TM1 and TM2 could be detected adjacent to a protein of ~45kDa, believed to be Sec61α. The loss of this adduct, as the nascent chain extends, showed the lateral movement of GPR35 TM1 from the translocon was dependent on the subsequent synthesis of TM2.

The role of oxidative stress as an underlying mechanism in life-history trade-offs

A key aspect underpinning life-history theory is the existence of trade-offs. Trade-offs occur because resources are limited, meaning that individuals cannot invest in all traits simultaneously, leading to costs for traits such as growth and reproduction. Such costs may be the reason for the sub-maximal growth rates that are often observed in nature, though the fitness consequences of these costs would depend on the effects on lifetime reproductive success. Recently, much attention has been given to the physiological mechanism that might underlie these life-history trade-offs, with oxidative stress (OS) playing a key role. OS is characterised by a build-up of oxidative damage to tissues (e.g. protein, lipids and DNA) from attack by reactive species (RS). RS, the majority of which are by-products of metabolism, are usually neutralised by antioxidants, however OS occurs when there is an imbalance between the two. There are two main theories linking OS with growth and reproduction. The first is that traits like growth and reproduction, being metabolically demanding, lead to an increase in RS production. The second involves the diversion of resources away from self-maintenance processes (e.g. the redox system) when individuals are faced with enhanced growth or reproductive expenditure. Previous research investigating trade-offs involving growth or reproduction and self-maintenance has been equivocal. One reason for this could be that associations among redox biomarkers can vary greatly so that the biomarker selected for analysis can influence the conclusion reached about an individual’s oxidative status. Therefore the first aim of my thesis was to explore the strength and pattern of integration of five biomarkers of OS (three antioxidants, one damage and one general oxidation measure) in wild blue tit (Cyanistes caeruleus) adults and nestlings (Chapter 2). In doing so, I established that all five biomarkers should be included in future analyses, thus using this collection of biomarkers I explored my next aims; whether enhanced growth (Chapters 3 and 4) or reproductive effort (Chapter 5) can lead to increased OS levels, if these traits are traded off against self-maintenance. I accomplished these aims using both a meta-analytic and experimental approach, the latter involving manipulation of brood size in wild blue tits in order to experimentally alter growth rate of nestlings and provisioning rate (a proxy for reproductive expenditure) of adults. I also investigated the potential for redox integration to be used as an index of body condition (Chapter 2), allowing predictions about future fitness consequences of changes to oxidative state to be made. A growth – self-maintenance trade off was supported by my meta-analytic results (Chapter 4) which found OS to be a constraint on growth. However, when faced with experimentally enhanced growth, animals were typically not able to adjust this trade-off so that oxidative damage resulted. This might support the idea that energetically expensive growth causes resources to be diverted away from the redox system; however, antioxidants did not show an overall reduction in response to growth in the meta-analysis suggesting that oxidative costs of growth may result from increased RS production due to the greater metabolism needed for enhanced growth. My experimental data (Chapter 3) showed a similar pattern, with raised protein damage levels (protein carbonyls; PCs) in the fastest growing blue tit chicks in a brood, compared with their slower growing sibs. These within-brood differences in OS levels likely resulted from within-brood hierarchies and might have masked any between-brood differences, which were not observed here. Despite evidence for a growth – self-maintenance trade off, my experimental results on blue tits found no support for the hypothesis that self-maintenance is also traded off against reproduction, another energetically demanding trait. There was no link between experimentally altered reproductive expenditure and OS, nor was there a direct correlation between reproductive effort and OS (Chapter 5). However, there are various factors that likely influence whether oxidative costs are observed, including environmental conditions and whether such costs are transient. This emphasises the need for longitudinal studies following the same individuals over multiple years and across a wide range of habitats that differ in quality. This would allow investigation into how key life events interact; it might be that raised OS levels from rapid early growth have the potential to constrain reproduction or that high parental OS levels constrain offspring growth. Any oxidative costs resulting from these life-history trade-offs have the potential to impact on future fitness. Redox integration of certain biomarkers might prove to be a useful tool in making predictions about fitness, as I found in Chapter 2, as well as establishing how the redox system responds, as a whole, to changes to growth and reproduction. Finally, if the tissues measured can tolerate a given level of OS, then the level of oxidative damage might be irrelevant and not impact on future fitness at all.