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 ß-arrestins (ßarrs). These events
were thought to be largely separable given that G proteins primarily initiate downstream signaling while ßarrs
can signal and regulate receptor desensitization and trafficking. Recent studies have suggested evidence
for a combined role of G protein and ßarr in GPCRs signaling through the formation of signaling
“megaplexes” and the impairment of ßarr-based signaling in the absence of functional G proteins. However,
there remains a significant knowledge gap surrounding the significance of G protein and ßarr 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 ßarrs can interact across a panel receptors and were surprised to find that all receptors tested
could form a complex between the inhibitory G protein (Gai) and ßarr, 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 Gai:ßarr signaling across GPCRs. Our overarching goal is to define the mechanism in which
Gai:ßarr form complexes and understand their impact on physiology. We hypothesize that Gai:ßarr complex
formation require a discrete set of motifs present in Gai, ßarrs and GPCRs and that these complexes regulate
endothelial function in PH. To test this hypothesis, first I will determine the specific sequence motifs in Gai, ßarr
and the receptor that are required to form Gai:ßarr complex. Second, I will determine the signalling pathways
that are regulated by Gai:ßarr interaction using APEX proximity labeling and novel “complex BRET” assays.
Third, I will determine the impact of Gai:ßarr within PH patient endothelial cells by targeting Gai and ßarr
signaling and testing their effects on endothelial function. This study strives to understand an emerging
paradigm in GPCR signalling in which Gai and ßarr work together to orchestrate unique downstream signalling.
Completion of these aims will provide novel insights for cell signalling, development of new pharmacological
tools targeting Gai:ßarr 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.