Functional Selectivity of Natural and Synthetic Prostaglandin EP₄ Receptor Ligands

Abstract

Classically, the prostaglandin E₂ (PGE₂) receptor EP₄ has been classified as coupling to the Gα_s subunit, leading to intracellular cAMP increases. However, EP₄ signaling has been revealed to be more complex and also involves coupling to pertussis toxin-sensitive Gα_i proteins and β-arrestin-mediated effects. There are now many examples of selective activation of independent pathways by G protein-coupled receptor (GPCR) ligands, a concept referred to as functional selectivity. Because most EP₄ ligands had thus far only been functionally characterized by their ability to stimulate cAMP production, we systematically determined the potencies and efficacies of a panel of EP₄ ligands for activation of Gα_s, Gα_i, and β-arrestin relative to the endogenous ligand PGE₂. For this purpose, we adapted three bioluminescence resonance energy transfer (BRET) assays to evaluate the respective pathways in living cells. Our results suggest considerable functional selectivity among the tested, structurally related agonists. PGE₂ was the most selective in activating Gα_s, whereas PGF_2α and PGE₁ alcohol were the most biased for activating Gα_i1 and β-arrestin, respectively. We observed reversal in order of potencies between β-arrestin 2 and Gα_i1 functional assays comparing PGE₁ alcohol and either PGF_2α, PGD₂, or 7-[(1R,2R)-2-[(E,3R)-3-hydroxy-4-(phenoxy)but-1-enyl]-5-oxocyclopentyl]heptanoic acid (M&B28767). Most ligands were full agonists for the three pathways tested. Our results have implications for the use of PGE₂ analogs in experimental and possibly clinical settings, because their activity spectra on EP₄ differ from that of the native agonist. The BRET-based methodology used for this first systematic assessment of a set of EP₄ agonists should be applicable for the study of other GPCRs.