Functional Selectivity of Natural and Synthetic Prostaglandin EP4 Receptor Ligands

Classically, the prostaglandin E-2 (PGE(2)) receptor EP4 has been classified as coupling to the G alpha(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 G alpha(i) proteins and beta-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 G alpha(s), G alpha(i), and beta-arrestin relative to the endogenous ligand PGE(2). 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. PGE(2) was the most selective in activating G alpha(s), whereas PGF(2 alpha) and PGE(1) alcohol were the most biased for activating G alpha(i1) and beta-arrestin, respectively. We observed reversal in order of potencies between beta-arrestin 2 and G alpha(i1) functional assays comparing PGE(1) alcohol and either PGF(2 alpha), PGD(2), 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 PGE(2) 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.