Prostaglandin receptor EP4 induces transient membrane targeting of aquaporin‐2 through a novel intracellular signaling pathway

Abstract

In our previous studies, the prostaglandin receptor EP2 agonist butaprost increased aquaporin‐2 (AQP2) membrane targeting (MT) and phosphorylation at ser‐269 (pS269), but EP4 agonist CAY10580 (CA) increased only MT. Potential EP4 signaling pathways alongside EP2 vs EP4 mediated AQP2 trafficking events were examined in these studies. In AQP2‐MDCK cells natively expressing EP2 and EP4, the EP2 antagonist AH6809 (10μM) abolished prostaglandin E2 (1–10nM) induced cAMP and pS269, but not MT. Stimulation with 1μM CA (2 to 80 min) increased MT from 5–80 min, but did not increase pS269‐AQP2/biotin‐AQP2 at any time point. Pretreatment with the Gi inhibitor, pertussis toxin (18h, 100ng/ml), neither increased pS269 nor affected AQP2 MT during CA stimulation. 40 min agonist washout abolished CA stimulated AQP2 MT whereas butaprost (1μM) or forskolin (10μM) induced MT was upheld for at least 80 min of washout, although pS269 was strongly decreased after 20 min. In conclusion: 1) EP4 increases AQP2 MT without increasing cAMP and pS269. This is neither due to agonist functional selectivity, concomitant Gi protein signaling nor time dependent receptor desensitization 2) EP4 mediated membrane targeted AQP2 is rapidly internalized after washout, whereas the effect of forskolin or EP2 on AQP2 MT are upheld 3) the latter is not due to continued pS269 after washout. Funded by Aarhus University and The Lundbeck Foundation.