Defining the Role of of Noncanonical GPCR Signalling in Pulmonary Hypertension

Duke University, Durham NC

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Abstract

ABSTRACT Pulmonary hypertension (PH) is characterized by endothelial dysfunction, irregular vascular remodeling and consistent vasoconstriction leading to eventual fatal right heart failure despite current medical therapies. The most common drug targets in PH are G protein coupled receptors (GPCRs), which are a target for almost a third of all FDA-approved drugs. Although these receptors have been studied intensely for over 40 years, several aspects of GPCR signaling remain poorly understood. Canonically, it has been well established that these receptors are able to signal through both heterotrimeric G proteins and ?-arrestins (?arrs). These events were thought to be largely separable given that G proteins primarily initiate downstream signaling while ?arrs can signal and regulate receptor desensitization and trafficking. Recent studies have suggested evidence for a combined role of G protein and ?arr in GPCRs signaling through the formation of signaling ?megaplexes? and the impairment of ?arr-based signaling in the absence of functional G proteins. However, there remains a significant knowledge gap surrounding the significance of G protein and ?arr coordinated signaling. Our long term aim is to understand the signaling mechanisms of GPCRs to provide better insight for the development of novel therapeutics for PH. In our recent studies, we have directly assessed whether G proteins and ?arrs can interact across a panel receptors and were surprised to find that all receptors tested could form a complex between the inhibitory G protein (G?i) and ?arr, including the type 1 angiotensin II receptor (AT1R) and atypical chemokine receptor 3 (ACKR3, also known as CXCR7), which are both potential drug targets in PH. We further found that these complexes could interact with secondary effectors, most notably extracellular signal-regulated kinase (ERK). These results suggested a conserved, non-canonical role for G?i:?arr signaling across GPCRs. Our overarching goal is to define the mechanism in which G?i:?arr form complexes and understand their impact on physiology. We hypothesize that G?i:?arr complex formation require a discrete set of motifs present in G?i, ?arrs and GPCRs and that these complexes regulate endothelial function in PH. To test this hypothesis, first I will determine the specific sequence motifs in G?i, ?arr and the receptor that are required to form G?i:?arr complex. Second, I will determine the signalling pathways that are regulated by G?i:?arr interaction using APEX proximity labeling and novel ?complex BRET? assays. Third, I will determine the impact of Gai:?arr within PH patient endothelial cells by targeting G?i and ?arr signaling and testing their effects on endothelial function. This study strives to understand an emerging paradigm in GPCR signalling in which G?i and ?arr work together to orchestrate unique downstream signalling. Completion of these aims will provide novel insights for cell signalling, development of new pharmacological tools targeting G?i:?arr coupling, and lay the groundwork for therapeutics for cardiovascular-related diseases. These studies will also provide an excellent opportunity for my training to develop as an independent scientist.