Uncovering the molecular pharmacology of the G protein-coupled receptor GPR75: role in age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of blindness in the industrialised world affecting over 50 million people worldwide; 5% of over 70’s suffer from AMD. AMD is characterised by progressive degeneration of the retinal pigment epithelial cells (RPE) in the macula leading to destruction of the underlying photoreceptors in the eye.

There is currently no effective treatment for the most common atrophic form of the disease. Since the prevalence of AMD is predicted to increase with the expansion of the ageing population understanding the pathogenesis and discovering novel drug targets is a priority.

G protein-coupled receptors are a family of cell-surface receptors, which respond to various extracellular mediators and regulate a vast array of physiological processes. GPCRs interact with G proteins, which in turn trigger the production or inhibition of second messengers and thus a wide number of signalling pathways. Circulating mediators regulate RPE proliferation and apoptosis, therefore GPCRs are key in maintaining RPE integrity, which in turn is vital for the survival of photoreceptors. We have shown that the microenvironment present as AMD develops is capable of inducing apoptosis of RPE1.

The accessibility of GPCRs on the plasma membrane and their tissue-selective distribution make them excellent pharmacological targets (>30% of prescription drugs target GPCRs)2. However, the endogenous ligands of a large number of GPCRs remain unidentified, leaving their physiological role unknown. Studying these GPCRs, namely orphan GPCRs, could advance the understanding of disease and highlight their physiological relevance. Functional or neutralising antibodies provide a useful research tool to target orphan GPCRs and a route for target validation and even offer potential therapeutics.

GPR75 (also named retinal GPCR), which is an orphan GPCR and a focus of our lab, has been shown by ourselves and others to be highly expressed in human RPEs3, which we routinely isolate and culture. Six potential pathogenic variants of GPR75 have been identified in AMD patients, however their functional significance has yet to be determined3. Although no agonists/antagonists are known for GPR75 our lab has begun to elucidate its function by creating GPR75 over-expressing HEK-293 cells and using siRNA in-vitro and CRISPR/Cas technology in-vivo to knock down GPR75.

Using cutting edge technologies such as molecular biology, signalling and G-protein binding assays, CRISPR/Cas technology, antibody generation and a mechanistically-based animal model reflecting the early pathology of AMD, the specific aims of the studentship are to:
1.Uncover the pharmacology of GPR75, in part through the generation of functional or neutralising GPR75 antibodies. G protein coupling, and the intracellular pathways regulated by GPR75 in vitro in HEK293-GPR75 and RPEs will be determined and its influence on RPE survival.
2.Investigate the significance of GPR75 variants that are associated with AMD on receptor function by site-directed mutagenesis of wild-type GPR75.
3.Determine the physiological relevance of GPR75 in RPEs in a model of AMD, both using CRISPR/Cas GPR75-knockout mice and GPR75 functional antibodies.

This proposed PhD project brings together areas of expertise in pharmacology, immunology and the blood–ocular barrier from the University of Aberdeen with those from local industry. Expertise and training in the generation of an antibody targeting GPR75 will be given by Elasmogen, a biologics drug discovery company that specialises in antibody-like proteins called soloMERs. soloMERs are sophisticated antibodies with high affinity binding, single-domains; that are smaller than traditional antibodies (9% of a traditional antibody). The small size of soloMERs allows for both site-specific delivery and penetration, which is ideal to cross the ocular barrier in-vivo.

Completion of the specific aims will reveal the basic pharmacology of GPR75 and functional effects of receptor variants. Furthermore, understanding the role of GPR75 in RPE cells could uncover the biological mechanisms of the ageing process in the eye that predispose patients to AMD, thereby highlight the this orphan receptor as a new target for the disease, thus providing translational impact.