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Title: Identifying novel G protein-coupled receptor targets in pulmonary hypertension : uncovering the role of GPR75
Author: Iyinikkel, Jean Rose
ISNI:       0000 0004 7961 2535
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 2019
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Pulmonary arterial hypertension (PAH) is a progressive and debilitating disease characterised by increased pulmonary vascular resistance (PVR) and proliferation of pulmonary artery smooth muscle cells (PASMC). Remodelling of the pulmonary artery leads to sustained elevations in pulmonary arterial pressure, right heart failure and eventual death if left untreated. Despite the number and variety of treatment options available, the survival rate and prevalence of PAH remains poor. Several risk factors such as gender, genetics and environmental stimuli contribute to the susceptibility and development of PAH. Female bias and differential response to drugs between the sexes is known in PAH. Normal low PVR is dependent on the interaction of circulating and locally produced vasomodulators, many of which act via G protein-coupled receptors (GPCRs). The accessibility of GPCRs on the plasma membrane, their distribution and role in amplifying signalling make them excellent pharmacological targets. Despite the large number of physiological responses that are regulated by GPCRs, only a small proportion are targeted therapeutically for PAH. Our lab profiled GPCR expression in PAH-PASMC in an effort to identify novel targets for the disease. Preliminary data revealed that PASMC from PAH-patients have increased expression of the orphan GPCR, GPR75; GPR75 was also upregulated in animal models of PAH. Our overall aim was to validate GPR75 as a target for PAH by uncovering its regulation, pharmacology, and functional significance. We hypothesise that GPR75 is a key regulator of PAH pathology and is therefore a novel drug target for the disease. To validate GPR75 as a target for PAH, we investigated its expression in another model of PAH, serotonin transporter overexpressing (SERT+) mice. Using real-time PCR (RTPCR) we found female SERT+ mice, which have elevated right ventricular systolic pressures, have increased GPR75 expression in their lungs. To determine the regulation To determine the regulation of GPR75 in PASMC we investigated the effect of hypoxia, gender and co-mitogens (all of which contribute to the pathophysiology of PAH). We found hypoxia, which increased PASMC proliferation, increased GPR75; GPR75 expression correlated with a proliferative phenotype. Female-PASMC had higher basal expression of GPR75 compared to malePASMC, suggesting a role of GPR75 in the female bias of PAH. Chronic treatment of PASMCs with the female sex hormone, 17β-E2, increased proliferation of PASMC, lowered cAMP and increased GPR75 expression; the response was more pronounced in female-PASMC. Interestingly, 17β-E2 synergistically enhanced the increased GPR75 seen with hypoxia alone in female-PASMC. Since we saw a female bias in GPR75 expression and its regulation by 17β-E2 we went on to investigate sex differences in PASMC-GPCRs. Using a TaqMan® GPCR array we profiled the expression of GPCRs in male- and female-PASMC; 25 GPCRs were upregulated over 2-fold in female-PASMC compared to male-PASMC, whereas 19 GPCRs were downregulated. Of interest the Gαi/Gαq/11-coupled oxytocin receptor was upregulated over 6-fold in female-PASMC, which correlated with enhanced signalling and function. In order to dissect the function and downstream signalling of GPR75, we established an overexpressing cell model. Although the chemokine RANTES and the eicosanoid 20HETE had been proposed as possible ligands for GPR75, using [35S]GTPγS binding assays we did not see such interactions. We found GPR75 to be constitutively activity, which increased proliferation and reduced cAMP accumulation suggesting Gαi- coupling; N-terminal specific GPR75 antibodies blocked its function. Using flow cytometry and immunofluorescence we found internalisation of GPR75 to be β-arrestin independent; GPR75 recycled back to the cell surface. Using site-directed mutagenesis we examined the functional role of GPR75 single nucleotide polymorphisms (SNPs). Overexpression of SNP- GPR75-Q234X completely abolished GPR75 mediated signalling. And prevented localisation to the plasma membrane. GPR75-Q234X could be pharmacologically relevant as it blunts function. Since we established a role for GPR75 in PAH, we used the CRISPR/Cas9 technology to help dissect its physiological role. We successfully used CRISPR/Cas9 to knockdown GPR75 protein and mRNA expression both in-vitro and in-vivo. In-vitro, reduced GPR75 expression blunted GPR75-dependent activity. Interestingly the CRISPR-GPR75 KO mouse is viable and shows no obvious phenotype. We hypothesise that exposing these CRISPR-GPR75 KO mice to hypoxia will protect them from developing PAH. Taken together our data show the utility of profiling GPCRs in disease and highlight GPCR expression and function could contribute to the sex bias in PAH. We have generated a number of tools that will aide in fully uncovering the pharmacological and physiological role of GPR75. Overall we have validated GPR75 as a novel therapeutic target in PAH; inhibitors of GPR75 would be expected to blunt or reverse remodelling of the pulmonary artery associated with the disease.
Supervisor: Murray, Fiona Sponsor: Institute of Medical Sciences ; University of Aberdeen
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Pulmonary hypertension ; G proteins