Abstract Body (Do not enter title and authors here): Background: Chronic thromboembolic pulmonary hypertension (CTEPH) is a lung blood vessel disease characterized by abnormal pulmonary arterial endothelial cell (PAEC) function. Adrenomedullin (AM), a potent vasodilator, positively affects pulmonary hypertension (PH) by activating the Calcitonin-Like Receptor (CLR), prevalent on PAECs. CLR's ligand specificity and activity rely on the co-expression of receptor activity-modifying proteins (RAMPs) 1-3. We found RAMP1 & 3 were upregulated, while RAMP2 was downregulated in CTEPH ECs, potentially altering AM signaling.
Objective: Our hypothesis suggests that changes in RAMP1/2/3 expression alter AM signaling via CLR in different subcellular locations, leading to distinct physiological effects. This study aims to determine how RAMP expression changes in ECs modulate location-biased AM signaling via CLR that influence distinct physiological effects, including EC phenotype in CTEPH.
Methods: HEK293T cells were transfected with plasmid constructs using the PEI protocol. To study the relationship between AM-induced CLR-RAMP1/2/3 activation and β-arrestin1/2 directed location-biased signaling, we performed a BRET assay for β-arrestin1/2 recruitment. Receptor internalization used WT CLR tagged with RLuc8 and WT RAMP1/2/3, plus a Fyve-location marker tagged mVenus to evaluate proximity to the early endosome. Global cAMP levels were measured with an EPAC-based BRET biosensor. We examined the impact of location bias on CLR-RAMP1/2/3 signaling by assessing ERK1/2 activity in different subcellular locations using BRET-based ERK biosensors, EKAR (ERK activity reporter).
Results: Upon AM stimulation, recruitment of β-arrestin-1/2 to CLR was highest with RAMP1, then RAMP2 and RAMP3. CLR-RLuc8 internalization was also highest for RAMP1, followed by RAMP2 and RAMP3. Global cAMP levels were elevated in RAMP1 and RAMP2-transfected HEK293 cells compared to RAMP3. ERK1/2 activity was highest for RAMP1, then RAMP2, but no activity was noted with RAMP3 coexpression. Phospho-ERK1/2 for RAMP1 and RAMP2 at various time points, while RAMP3 levels remained unchanged.
Conclusions: The overall findings demonstrate evidence of location-biased AM signaling through differential association of RAMPs with CLR, with differences in β-arrestin recruitment, G protein signaling, and ERK activity. Defining the mechanisms that regulate AM signaling in CTEPH could aid in the development of new CTEPH medical therapies.