Opioids cause multiple physiological effects. Several distinct opioid receptors have been described, but their genes remain unknown. We propose to identify and clone opioid receptor genes to deduce their amino acid sequences and transfect the genes into mammalian cells. We have identified a human neuroblastoma cell line, SK-N-SH-SY5Y, which expresses abundant mu- , and also delta-, receptors. Strategies for gene isolation include 1) use of oligonucleotide probes deduced from peptide sequences of the mu receptor, 2) direct mammalian expression cloning using cDNA libraries from neuroblastoma cell lines; 3) application of the polymerase chain reaction using sequence identities among G protein coupled receptors; 4) screening cDNA of genomic libraries with probes derived from any known opioid receptor gene. The extended search will include the three main opioid receptor classes, and their putative subtypes. Knowledge of distinct opioid receptor genes, their deduced molecular structure, their tissue specific expression, and the availability of stably transfected cell lines will permit the development of unequivocal opioid receptor subtype assays, and thereby, provide a powerful tools for development of highly specific drugs.
