Importance of the extracellular domain for prostaglandin EP2 receptor function

The ligand binding pocket of biogenic amine G protein-coupled receptors is embedded in the membrane-spanning regions of these receptors, whereas the e xtracellular domains of the peptidergic receptors play a key role in the st ructure and function of this class of receptors, To examine the role of the extracellular sequences in prostaglandin receptor-ligand interaction, chim eras were constructed with the two G(s)-coupled E-prostanoid (EP) receptors , replacing each of the extracellular sequences of the human EP2 receptor w ith the corresponding human EP, receptor residues. Replacement of the third extracellular loop (ECIII) yielded a receptor that binds [H-3]prostaglandi n E-2 (PGE(2); K-d = 6.3 nM) with similar affinity as the EP2 wild-type rec eptor (K-d = 12.9 nM). Similarly, replacement of the nonconserved carboxyl- terminal portion of ECII resulted in a receptor that maintains [H-3]PGE(2) 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 [H-3]PGE(2) binding nor receptor-evoked cAMP generation. Immuno precipitation demonstrated that each chimera is expressed at levels near th at of wild-type receptors; however, enzyme-linked immunosorbent assay revea led that inactive chimeras have reduced cell surface expression. Similarly, chimeras that exchange the multiple extracellular loop sequences N/ECl, EC II/ECIII, or all four sequences lacked detectable binding and signal transd uction, and although expressed, were not detected on the cell surface. Thes e data suggest that the extracellular sequences of the EP2 receptor are cri tical determinants of receptor structure and/or function, unlike other G pr otein-coupled receptors that bind small molecules.