MOLECULAR-CLONING AND EXPRESSION OF A RAT KAPPA-OPIOID RECEPTOR

At least three types of opioid receptors have been identified in the n ervous system. In this paper we report molecular cloning and expressio n of a rat kappa opioid receptor. PCR was performed on double-stranded cDNA derived from poly(A)+ RNA of the rat striatum with primers simil ar to those of Libert and co-workers [Libert, Parmentier, Lefort, Dins art, Van Sande, Maenhaut, Simons, Dumont and Vassart (1989) Science 24 4, 569-572]. One of the PCR products, which had 65 % sequence similari ty to the mouse delta opioid receptor, was used to screen a rat striat um cDNA library. Two positive clones were isolated and found to be ide ntical. The clone had a 2.1-kb insert, which was termed RKOR-1. RKOR-1 has an open reading frame of 1140 bp and encodes a 380-amino-acid pro tein. Hydropathy analysis indicates that RKOR-1 has seven putative tra nsmembrane domains with short intra- and extra-cellular loops. Membran es of Cos-7 cells transfected with RKOR-1 exhibited high specific bind ing for [H-3]diprenorphine ([H-3]DIP), a non-selective opioid ligand. Naloxone inhibited [H-3]DIP binding with stereospecificity. [H-3]DIP b inding was potently inhibited by selective kappa opioid ligands, with K(i) values in the nanomolar or subnanomolar range, but much less pote ntly inhibited by drugs selective for mu or delta receptors. Thus, RKO R-I represents an opioid receptor with kappa characteristics.