TheE. coli EnvY gene encodes a high affinity opioid binding site

In an attempt to isolate a cDNA encoding an opioid receptor, a cDNA library was cionstructed in the ?ZAP vector using NG108-15 mRNA as template. Using an in vitro transcription-translation assay and a sib selection strategy, a single phage was isolated. An RNA transcribed from this cDNA was able to direct in vitro translation of opioid binding sites. The insert was sequenced and comparison with data banks showed a 100% homology with theE. coli envY gene. We assume that the presence of the envY sequence in the NG108-15 cDNA libary was due to a contamination of the ?ZAP vector withE. coli DNA. A search for opioid binding sites, onE. coli strains showed that envY⁺ strains, but not envY^- mutants were able to bind opiates. On envY⁺ cells, the sites are stereospecific, saturable and of high affinity for the opiate ligands. These sites bind opiate agonists and antagonists but neither µ nor d opioid peptides. In contrast, rabbit reticulocyte lysate primed with RNA transcribed in vitro from, the envY sequence elicited the synthesis of an opioid binding site with mixed µ and d properties. In addition, transfection of the envY sequence into mammalian cells resulted in the expression of opioid binding sites. Depending on the type of cells transfected, these sites were selective for either the µ or d ligands.