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A7261 - Arrestins Differentially Regulate OGR1 Signaling in Airway Smooth Muscle Cells
Author Block: T. Pera, E. Tompkins, D. A. Deshpande, A. P. Nayak, R. B. Penn; Center for Translational Medicine, Thomas Jefferson University, Philadelphia, PA, United States.
Rationale: OGR1 (GPR68) is a proton-sensing G protein-coupled receptor (GPCR) capable of sensing small changes in extracellular pH (pH 8.0 - 6.8) and coupling to both Gq and Gs proteins (Pera et al. FASEB J 2017). We have recently demonstrated that the benzodiazepine lorazepam induces OGR1 signaling in airway smooth muscle cells. Upon ligand binding and activation GPCRs can recruit arrestins which may lead to GPCR desensitization or arrestin-mediated signaling. Our previous studies have shown that arrestin subtypes differentially regulate GPCRs; beta-arrestin 2 desensitizes the beta-2-adrenoceptor whereas beta-arrestin 1 expression supports muscarinic receptor-mediated calcium mobilization. Here we assess the role of arrestins in OGR1 signaling in ASM cells. Methods: HEK293 cells expressing recombinant HA-OGR1 were stimulated with lorazepam and/or acidic media (pH 6.8); to determine receptor internalization, cell surface expression of HA-OGR1 was determined by ELISA using HA-epitope antibodies. Human ASM cells were transfected with siRNA against beta-arrestin 1 or 2 and stimulated with lorazepam and/or acid (pH 6.8), and signaling was assessed: Gq signaling was assessed by measuring intracellular calcium concentration by Flexstation, and activation of mitogen-activated protein kinase, ERK1/2 by immunoblotting. We assessed Gs signaling by measuring VASP phosphorylation by immunoblotting. Results: In HEK293 cells lorazepam (50 µM) or pH 6.8 resulted in a ~30% internalization of recombinant HA-OGR1 after 30 min of stimulation. In ASM cells, lorazepam induced intracellular calcium mobilization (up to 10-fold basal); beta-arrestin 1 knockdown inhibited lorazepam-induced calcium mobilization by 90% whereas beta-arrestin 2 knockdown had no effect. Histamine-induced calcium mobilization (10-fold basal) was not affected by arrestin knockdown. Lorazepam- or pH 6.8-induced p-ERK1/2 (2.5- and 3.5-fold basal, respectively; 30 min stimulation) were reduced by 25% under beta-arrestin 1 knockdown whereas beta-arrestin 2 knockdown had no effect. With combined lorazepam and pH 6.8 stimulation p-ERK1/2 (4-fold basal) was inhibited by 50% by beta-arrestin 1 knockdown. pH 6.8-induced VASP phosphorylation (3-fold basal; 10 min stimulation) was inhibited by 50% under beta-arrestin 1 knockdown whereas it doubled under beta-arrestin 2 knockdown (6-fold basal). Combined lorazepam- and pH 6.8-induced VASP phosphorylation (4-fold basal) was not affected by beta-arrestin 1 knockdown; beta-arrestin 2 knockdown increased VASP phosphorylation by 50%. Conclusions: These data indicate that beta-arrestin 1 and 2 differentially regulate OGR1 signaling in human ASM cells; beta-arrestin 1 expression facilitates calcium mobilization and ERK signaling whereas beta-arrestin 2 does not. OGR1-mediated Gs signaling is promoted by beta-arrestin 1- but is abrogated by beta-arrestin 2 expression.
Funding: HL58506, HL140064