Pharmacological and molecular properties of opioid binding sites synthesized in a cell-free translation system

Cell-free translation of mRNA, extracted from NG108-15 cells, was used to examine some properties of the opioid binding sites synthesized in vitro. A monoclonal antiidiotype antibody directed against the delta opioid receptor immunoprecipitated a major band of Mr 51,000. Translational immunoassays of poly[A]+RNA, size fractionated by methylmercury agarose gel electrophoresis, demonstrated that the 51,000 Mr protein specifically immunoprecipitated by the anti-opioid receptor antiidiotype antibodies was coded by a transcript which length was in the 6 to 8 kb range. Displacement binding studies of tritiated ligands (either bremazocine or delta or mu selective peptides) with type selective opioid ligands showed that only one type of opioid binding site was synthesized in vitro. Although the pharmacological profiles of ligands binding to NG108-15 cells were characteristic of the delta receptor type, the de novo synthesized opioid binding site had lost its delta selectivity and showed equal affinity for both the mu and delta but not for the kappa ligands. Similar to our finding using the immunoprecipitation system, size fractionation of the NG108-15 poly[A]+RNA demonstrated that the transcript coding for the "mu-delta" binding site had a length of 6,500 to 7,500 nucleotides.