Fiona Murray

Background and purpose Orphan G protein-coupled receptors (GPCRs) represent a significant challenge as, due to the the absence of a known endogenous ligand, their pharmacology remain elusive. GPR75 is an orphan GPCR that has been implicated in metabolic syndrome. Loss-of-function variants of GPR75 exhibit lower body mass index and reduced risk of hepatic steatosis. CRISPR-GPR75 mice are protected against diet-induced weight gain and metabolic disease. However, little is known about the regulation and signalling of GPR75. We aimed to elucidate the pharmacology and characterise the signalling of GPR75 to provide insight into its physiological relevance and to validate this receptor as a drug target. Experimental approach Using N-terminal signal sequence FLAG-tagged GPR75 expressed in HEK and CHO cells we investigated constitutive and ligand-induced activity by cAMP accumulation (cAMP ELISA), [35S] GTPγS binding, intracellular Ca2+ release (Fluo-4AM), receptor internalisation (by flow cytometry and immunofluorescence microscopy) and G protein- and β-arrestin association by bioluminescence resonance energy transfer (BRET, GPR75- Rluc8 and venus-mini-Gαsi or β-arrestin-2-YFP). The role of G-protein receptor kinases (GRK), β-arrestins, and adaptor protein2 (AP2) were investigated CRISPR-Cas9 edited cells, knockdown, and inhibition. Key results GPR75 demonstrated ligand-independent recruitment of mGαsi and β-arrestin-2 (p<0.001) that was reduced by cholesterol depletion (p<0.05). Forskolin-induced cAMP accumulation, which was pertussis toxin-sensitive, was decreased by ~22% (p<0.05) in HEK293-GPR75 cells compared to control. Our data failed to validate RANTES or 20-HETE as GPR75-ligands. Constitutive GPR75 internalisation, which was GRK2/3 dependent, was attenuated by Dyngo-4a, but not by β-arrestin1/2 or AP2 knockdown. Conclusions and implications Our data show that GPR75 is a constitutively active Gαi-coupled GPCR, whose internalisation is β-arrestin-independent and GRK2/3- and dynamin-dependent. In conclusion, we believe GPR75 remains an orphan GPCR, whose constitutive activity could represent a pharmacologic “handle” that provides insight into its physiological role and therapeutic potential.

Background: The association between systemic hypertension and cerebrovascular disease is well documented. However, the impact of pulmonary hypertension (PH) on acute ischemic stroke outcomes is unknown despite PH being recognized as a risk factor for acute ischemic stroke. We aimed to determine the association between PH and adverse in‐hospital outcomes after acute ischemic stroke, as well as whether there are sex differences in this association. Methods and Results: Acute ischemic stroke admissions from the US National Inpatient Sample between October 2015 and December 2017 were included. The relationship between PH and outcomes (mortality, prolonged hospitalization >4 days, and routine home discharge) was analyzed using logistic regressions adjusting for demographics, comorbidities, and revascularization therapies. Interaction terms between PH and sex and age groups were also included. A total of 221 249 records representative of 1 106 045 admissions were included; 2.9% of patients had co‐morbid PH, and 35.34% of those were male. PH was not associated with in‐hospital mortality (odds ratio [OR], 0.96; 95% CI, 0.86–1.09) but was associated with increased odds of prolonged hospitalization (OR, 1.15; 95% CI, 1.09–1.22) and decreased odds of routine discharge (OR, 0.87; 95% CI, 0.81–0.94) for both sexes. Older patients with PH were significantly less likely to be discharged routinely ( P =0.028) than their younger counterparts. Compared with female patients with PH, men were 31% more likely to die in hospital ( P =0.024). Conclusions: PH was not significantly associated with in‐hospital mortality but was associated with prolonged hospitalization and adverse discharge status. Male patients with PH were more likely to die in hospital than female patients.

Significance: Growth factors and heterotrimeric G proteins are two of the most widely studied signaling pathways in eukaryotes; their cross-talk shapes some of the most fundamental cellular responses in both health and disease. Although mechanisms by which G protein pathways transactivate growth factor receptor tyrosine kinases (RTKs) have been well-defined, how the reverse may happen is less understood. This study defines the key steps and cellular consequences of a fundamental mechanism of signal cross-talk that enables RTKs to transactivate heterotrimeric G protein, Gαi. Mutations found in tumors shed light on how derailing this mechanism impacts tumor cell behavior. Thus, findings not only show how cells integrate extracellular signals via pathway cross-talk, but also demonstrate the relevance of this pathway in cancers.

IL-33 and its receptor ST2 are contributing factors to airway inflammation and asthma exacerbation. The IL-33/ST2 signaling pathway is involved in both the onset and the acute exacerbations of asthma. In this study, we address the role of endogenous IL-33 and its autoamplification of the IL-33/ST2 pathway in Ag-dependent and Ag-independent asthma-like models. Wild-type, IL-33 knockout, ST2 knockout mice were either intratracheally administrated with 500 ng of rIL-33 per day for four consecutive days or were sensitized and challenged with OVA over 21 d. In wild-type mice, IL-33 or OVA induced similar airway hyperresponsiveness and eosinophilic airway inflammation. IL-33 induced its own mRNA and ST2L mRNA expression in the lung. IL-33 autoamplified itself and ST2 protein expression in airway epithelial cells. OVA also induced IL-33 and ST2 protein expression. In IL-33 knockout mice, the IL-33– and OVA-induced airway hyperresponsiveness and eosinophilic airway inflammation were both significantly attenuated, whereas IL-33–induced ST2L mRNA expression was preserved, although no autoamplification of IL-33/ST2 pathway was observed. In ST2 knockout mice, IL-33 and OVA induced airway hyperresponsiveness and eosinophilic airway inflammation were both completely diminished, and no IL-33/ST2 autoamplification was observed. These results suggest that endogenous IL-33 and its autoamplification of IL-33/ST2 pathway play an important role in the induction of asthma-like phenotype. Thus an intact IL-33/ST2 pathway is necessary for both Ag-dependent and Ag-independent asthma-like mouse models.

Caveolin-1 (Cav-1) is an integral membrane protein that plays an important role in proliferative and terminally differentiated cells. As a structural component of Caveolae, Cav-1 interacts with signaling molecules via a caveolin scaffolding domain (CSD) regulating cell signaling. Recent reports have shown that Cav-1 is a negative regulator in tumor metastasis. Therefore, we hypothesize that Cav-1 inhibits cell migration through its CSD. HeLa cells were engineered to overexpress Cav-1 (Cav-1 OE), Cav-1 without a functional CSD (∆CSD), or enhanced green fluorescent protein (EGFP) as a control. HeLa cell migration was suppressed in Cav-1 OE cells while ∆CSD showed increased migration, which corresponded to a decrease in the tight junction protein, zonula occludens (ZO-1). The migration phenotype was confirmed in multiple cancer cell lines. Phosphorylated STAT-3 was decreased in Cav-1 OE cells compared to control and ∆CSD cells; reducing STAT-3 expression alone decreased cell migration. ∆CSD blunted HeLa proliferation by increasing the number of cells in the G2/M phase of the cell cycle. Overexpressing the CSD peptide alone suppressed HeLa cell migration and inhibited pSTAT3. These findings suggest that Cav-1 CSD may be critical in controlling the dynamic phenotype of cancer cells by facilitating the interaction of specific signal transduction pathways, regulating STAT3 and participating in a G2/M checkpoint. Modulating the CSD and targeting specific proteins may offer potential new therapies in the treatment of cancer metastasis.

G protein-coupled receptors (GPCRs), the largest family of targets for approved drugs, are rarely targeted for cancer treatment, except for certain endocrine and hormone-responsive tumors. Limited knowledge regarding GPCR expression in cancer cells likely has contributed to this lack of use of GPCR-targeted drugs as cancer therapeutics. We thus undertook GPCRomic studies to define the expression of endoGPCRs (which respond to endogenous molecules such as hormones, neurotransmitters and metabolites) in multiple types of cancer cells. Using TaqMan qPCR arrays to quantify the mRNA expression of ∼340 such GPCRs, we found that human chronic lymphocytic leukemia (CLL) cells/stromal cells associated with CLL, breast cancer cell lines, colon cancer cell lines, pancreatic ductal adenocarcinoma (PDAC) cells, cancer associated fibroblasts (CAFs), and PDAC tumors express 50 to >100 GPCRs, including many orphan GPCRs (which lack known physiologic agonists). Limited prior data exist regarding the expression or function of most of the highly expressed GPCRs in these cancer cells and tumors. Independent results from public cancer gene expression databases confirm the expression of such GPCRs. We propose that highly expressed GPCRs in cancer cells (for example, GPRC5A in PDAC and colon cancer cells and GPR68 in PDAC CAFs) may contribute to the malignant phenotype, serve as biomarkers and/or may be novel therapeutic targets for the treatment of cancer.