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G-protein coupled receptors (GPCRs) are a predominant class of cell-surface receptors in eukaryotic life. They are responsible for the perception of a broad range of ligands and involved in a multitude of physiological functions. GPCRs are therefore of crucial interest for biological and pharmaceutical research. Molecular analysis and functional characterisation of GPCRs is frequently hampered by challenges in efficient large-scale production, non-destructive purification and long-term stability. Cell-free protein synthesis (CFPS) provides new production platforms for GPCRs by extracting the protein synthesis machinery of the cell in an open system that allows target-oriented modulations of the synthesis process and direct access to the nascent polypeptide chain. CFPS is fast, reliable and highly adaptable. Unfortunately, highly productive cell-free synthesis of GPCRs is often opposed by low product quality. This thesis was aimed to adapt and improve some of the new possibilities for the cell-free production of GPCRs in high yield and quality for structural and pharmaceutical analysis. An E. coli based CFPS system was applied to synthesise various turkey and human Beta-adrenergic receptor (Beta1AR) derivatives as well as human Endothelin receptors type A and B (ETA and ETB) constructs. Both receptor families are important drug targets and pharmacologically addressed in the treatment of several cardiovascular diseases. CF-synthesis was mainly performed in presence of nanodiscs (ND), which are reconstituted high density lipoprotein particles forming discoidal bilayer patches with a diameter varyring from 6 to approx. 15 nm. The supplementation of ND in the CF-synthesis reaction caused the co-translational solubilisation of the freshly synthesised GPCRs. The fraction of the solubilised GPCR that was correctly folded was analysed by the competence to bind its ligand alprenolol or Endothelin-1, respectively. Both the solubilisation efficiency and the ability to fold in a ligand binding competent state was strongly affected by the lipid composition of the supplied ND. Best results were generally achieved with lipids having phosphoglycerol headgroups and unsaturated fatty acid chains with 18 carbon atoms. Furthermore, thermostabilisation by introduction of point mutations had a large positive impact on the folding efficiency of both Beta1AR and ETB receptor. Formation of a conserved disulphide bridge in the extracellular region was additionally found to be crucial for the function of the ETB receptor. Disulphide bridge formation could be enhanced by applying a glutathione-based redox system in the CFPS. Further improvements in the quality of ETB receptor could be made by the enrichment of heat-shock chaperones in the CF-reaction. Depending on the receptor type and DNA-template, roughly 10 – 30 nmol (350 – 1500 µg) of protein could be synthesised in 1 ml of CF-reaction mixture. After the applied optimisation steps, the fractions of correctly folded receptor could be improved by several orders of magnitude and were finally in between 35% for the thermostabilised turkey Beta1AR, 9% for the thermostabilised ETB receptor, 6.5% for the non-stabilised ETB receptor, 1 - 5% for non-stabilised turkey Beta1AR and for human Beta1AR isoforms and 0.1% for ETA receptor. Therefore, between 2 and 120 µg of GPCR could be synthesised in a ligand binding competent form, depending on the receptor and its modifications. Correctly folded turkey Beta1AR and ETB receptors were thermostable at 30°C and could be stored at 4°C for several weeks after purification. Yields of the thermostabilised turkey Beta1AR were sufficient to purify the receptor in a two-step process by ligand-binding chromatography to obtain pure and correctly folded receptor in the lipid bilayer of a ND. Furthermore, a lipid dependent ligand screen could be demonstrated with the turkey Beta1AR and significant alterations in binding affinities to currently in-use pharmaceuticals were found. The established protocols are therefore suitable and highly competetive for a variety of applications such as screening of GPCR ligands, analysis of lipid effects on GPCR function or for the systematical biochemical characterisation of GPCRs. Most promising for future approaches appears to address the suspected bottlenecks of intial insertion of the GPCR-polypeptide chain in the ND bilayer and the thermal stability of the receptors. Nevertheless, the estabilised protocols for the analysed targets in this thesis are already highly competitive to previously published production protocols either in cell-based or cell-free systems with regard to yield of functional protein, speediness and costs. Moreover, the direct accessibility and other general characteristics of cell-free synthesis open a large variety of possible applications and this work can therefore contribute to the molecular characterisation of this important receptor type and to the development of new pharmaceuticals.
Neuropathic pain, a form of chronic pain, is a steadily rising health problem due to health costs and increasing numbers of patients. Neuropathic pain conditions arise upon metabolic disorders, infections, chemotherapeutic treatment, trauma or nerve injury. Especially nerve injury induced neuropathic pain is characterized by spontaneous or ongoing pain due to neuroimmune interactions. Thereby, inflammatory mediators, released by the injured nerve, recruit to and activate immune cells at the site of injury. Those mediators further activate transient receptor potential vanilloid 1 (TRPV1), a known channel involved in pain perception, or bind to G-protein coupled receptors (GPCR) in peripheral nerve endings. The following activated second messenger signaling pathways lead to sensitization of TRPV1. One of those GPCRs is G2A.
The overall aim of this thesis was to investigate the role of G2A in nerve-injury induced neuropathic pain. For this, the common mouse model of nerve-injury induced neuropathic pain, the spared-nerve injury, was used. As measurements with dynamic plantar aesthesiometer showed, G2A-deficiency leads to reduced mechanical hypersensitivity. Upon analysis with FACS, ELISA and Luminex a reduced number of macrophages and neutrophils at the injured nerve, as well as less inflammatory mediators (TNFα, IL-6, VEGF) in G2A-deficient animals was observed. In dorsal root ganglia (DRGs) there was only a reduced number of macrophages and less IL-12 observed in G2A-deficient animals. Additionally, in wild-type mice, G2A agonist 9-HODE was elevated at the injured nerve, as a LC-MS/MS analysis showed.
To investigate the underlying pathways of G2A-9-HODE signaling, a proteom screen was performed. This screen revealed upregulation of multiple proteins involved in migration in wild-type macrophages. Additionally, Ca-Imaging and transwell migration assays showed that the G2A antagonist G2A11, had desensitizing effects on DRG neurons and inhibited macrophage migration.
Overall, the results suggest that loss of G2A has dual effects. On the one hand loss of G2A is antinociceptive. On the other hand, G2A-deficiency leads to reduced inflammation, suggesting G2A as promising target in treatment of neuropathic pain. Here, an antagonist had inhibitory effects on the migration and the sensitization.