RT Journal Article SR Electronic T1 Importance of the Extracellular Domain for Prostaglandin EP2 Receptor Function JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 545 OP 551 DO 10.1124/mol.56.3.545 VO 56 IS 3 A1 Brett A. Stillman A1 Matthew D. Breyer A1 Richard M. Breyer YR 1999 UL http://molpharm.aspetjournals.org/content/56/3/545.abstract AB The ligand binding pocket of biogenic amine G protein-coupled receptors is embedded in the membrane-spanning regions of these receptors, whereas the extracellular domains of the peptidergic receptors play a key role in the structure and function of this class of receptors. To examine the role of the extracellular sequences in prostaglandin receptor-ligand interaction, chimeras were constructed with the two Gs-coupled E-prostanoid (EP) receptors, replacing each of the extracellular sequences of the human EP2receptor with the corresponding human EP4 receptor residues. Replacement of the third extracellular loop (ECIII) yielded a receptor that binds [3H]prostaglandin E2(PGE2; K d = 6.3 nM) with similar affinity as the EP2 wild-type receptor (K d = 12.9 nM). Similarly, replacement of the nonconserved carboxyl-terminal portion of ECII resulted in a receptor that maintains [3H]PGE2 binding (K d = 8.8 nM). In contrast, replacement of the amino terminus, ECI, the entire ECII region, or the residues within the highly conserved motif of the amino-terminal half of ECII yielded chimeras that displayed neither detectable [3H]PGE2 binding nor receptor-evoked cAMP generation. Immunoprecipitation demonstrated that each chimera is expressed at levels near that of wild-type receptors; however, enzyme-linked immunosorbent assay revealed that inactive chimeras have reduced cell surface expression. Similarly, chimeras that exchange the multiple extracellular loop sequences N/ECI, ECII/ECIII, or all four sequences lacked detectable binding and signal transduction, and although expressed, were not detected on the cell surface. These data suggest that the extracellular sequences of the EP2receptor are critical determinants of receptor structure and/or function, unlike other G protein-coupled receptors that bind small molecules.