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 G(s)-coupled E-prostanoid (EP) receptors, replacing each of the extracellular sequences of the human EP(2) receptor with the corresponding human EP(4) receptor residues. Replacement of the third extracellular loop (ECIII) yielded a receptor that binds [(3)H]prostaglandin E(2) (PGE(2); K(d) = 6.3 nM) with similar affinity as the EP(2) wild-type receptor (K(d) = 12.9 nM). Similarly, replacement of the nonconserved carboxyl-terminal portion of ECII resulted in a receptor that maintains [(3)H]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 [(3)H]PGE(2) 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 EP(2) receptor are critical determinants of receptor structure and/or function, unlike other G protein-coupled receptors that bind small molecules.