The key residue within the second extracellular loop of human EP3 involved in selectively turning down PGE2- and retaining PGE1-mediated signaling in live cells

Abstract

Key residues and binding mechanisms of PGE₁ and PGE₂ on prostanoid receptors are poorly understood due to the lack of X-ray structures for the receptors. We constructed a human EP3 (hEP3) model through integrative homology modeling using the X-ray structure of the β₂-adrenergic receptor transmembrane domain and NMR structures of the thromboxane A2 receptor extracellular loops. PGE₁ and PGE₂ docking into the hEP3 model showed differing configurations within the extracellular ligand recognition site. While PGE₂ could form possible binding contact with S211, PGE₁ is unable to form similar contacts. Therefore, S211 could be the critical residue for PGE₂ recognition, but is not a significant for PGE₁. This prediction was confirmed using HEK293 cells transfected with hEP3 S211L cDNA. The S211L cells lost PGE₂ binding and signaling. Interestingly, the S211L cells retained PGE₁-mediated signaling. It indicates that S211 within the second extracellular loop is a key residue involved in turning down PGE₂ signaling. Our study provided information that S211L within EP3 is the key residue to distinguish PGE₁ and PGE₂ binding to mediate diverse biological functions at the initial recognition step. The S211L mutant could be used as a model for studying the binding mechanism and signaling pathway specifically mediated by PGE₁.

Keywords: EP selectivity; G protein-coupled receptor (GPCR); Prostaglandin; Prostaglandin E1; Prostaglandin E2; Receptor regulation.