Cloning of G Protein-Coupled Opioid Receptors Using Degenerate PCR and Low-Stringency Homology Screening

In summary we have described methods that we have used to clone the classical μ, δ, and κ opioid receptors and a new receptor, LC132, that based on structural arguments we predict is also a member of the opioid receptor gene family. The combination of low-stringency homology screening and degenerate PCR provides a powerful strategy for the cloning of members of this G protein-coupled receptor family. When these cloning techniques are combined with transient and/or stable expression and classical pharmacological analyses it is often possible to determine which receptor subtype is encoded by the cloned cDNA. Given the sensitivity of the molecular approaches, in particular PCR, and what has been observed for other G protein-coupled receptors that bind the same, or similar ligands, we expect that more receptor genes will be identified than were orginally predicted from earlier pharmacological data. The cloning of LC132 is a perfect example of the situation that molecular neuropharmacologists now face; that is the ability to clone and express a new receptor type whose sequence allows its inclusion within an established gene family yet whose endogenous ligand is unknown. We are confident, however, that in time the endogenous ligand for orphan receptors such as LC132 will be determined by combining expression in tissue culture with pharmacological and physiological analyses.