Abstract
The prostaglandin F2alpha (PGF2alpha) receptor (FP) is a key regulator of parturition and a target for pharmacological management of preterm labor. However, an incomplete understanding of signaling pathways regulating myometrial contraction hinders the development of improved therapeutics. Here we used a peptidomimetic inhibitor of parturition in mice, PDC113.824, whose structure was based on the NH(2)-terminal region of the second extracellular loop of FP receptor, to gain mechanistic insight underlying FP receptor-mediated cell responses in the context of parturition. We show that PDC113.824 not only delayed normal parturition in mice but also that it inhibited both PGF2alpha- and lipopolysaccharide-induced preterm labor. PDC113.824 inhibited PGF2alpha-mediated, G(alpha)(12)-dependent activation of the Rho/ROCK signaling pathways, actin remodeling, and contraction of human myometrial cells likely by acting as a non-competitive, allosteric modulator of PGF2alpha binding. In contrast to its negative allosteric modulating effects on Rho/ROCK signaling, PDC113.824 acted as a positive allosteric modulator on PGF2alpha-mediated protein kinase C and ERK1/2 signaling. This bias in receptor-dependent signaling was explained by an increase in FP receptor coupling to G(alpha)(q), at the expense of coupling to G(alpha)(12). Our findings regarding the allosteric and biased nature of PDC113.824 offer new mechanistic insights into FP receptor signaling relevant to parturition and suggest novel therapeutic opportunities for the development of new tocolytic drugs.
Highlights
Premature birth due to preterm delivery is the most important cause of neonatal mortality and morbidity in industrialized countries [1, 2]
Our results suggest that this compound acts as a functional allosteric modulator for F2␣ (PGF2␣) receptor (FP) receptor, because it exerts its effects at a site distinct from the orthosteric binding site, and further biases PGF2␣-bound receptors toward increased G␣q-protein kinase C (PKC)-MAPK signaling, while blocking cell contraction and cytoskeleton reorganization through inhibition of the G␣12-Rho-ROCK signaling pathway
Taken together our results suggested that PDC113.824 acts as an allosteric modulator of FP receptormediated myometrial contraction and cytoskeletal reorganization through inhibition of the Rho/ ROCK signaling pathway
Summary
Reagents—[3H]PGF2␣, [35S]GTP␥S, and ECL were from PerkinElmer. [125I]Angiotensin was labeled as described in a previous study [17]. Ligand Binding Experiments—For binding experiments, 100 g of HEK293 cells stably expressing HA-FP receptor was incubated with 105 cpm of [3H]PGF2␣ (150 –240 Ci/mmol) in the presence of either vehicle (EtOH, 0.01% v/v), 1 M cold PGF2␣, 10 M AL-8810, 1 or 10 M PDC113.824 for 1 h at room temperature in 0.5 ml of binding buffer (described previously [16]). PKCI-GFP Translocation—FP receptor cells co-transfected with PKCI-GFP were serum-starved for 30 min and pretreated with vehicle (water) or PDC113.824 (2 M) for 30 min followed by treatment with increasing concentrations of PGF2␣ (from 10Ϫ11 to 10Ϫ7 M) for 10 min each. 48 h later, cells were serum-starved for 30 min, pre-treated or not with PDC113.824 (1 M) for 30 min or C3 exoenzyme (1 g/ml) for 3– 4 h, followed by stimulation with 1 M PGF2␣ for 25 min. Sample size (n) and p values are given in the figure legends
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