CLONING OF A HUMAN KAPPA-OPIOID RECEPTOR FROM THE BRAIN

By using a rat kappa. opioid receptor cDNA as a probe to screen a huma n brain cDNA library, we isolated a 4.0-kb clone (z115) which encompas ses a major portion of a human kappa opioid receptor (hkor), extending from the amino acid residue #6 to the 3'-untranslated region. The ext reme 5'-region 232-bp fragment of z115 was used as a probe to screen a human genomic DNA library. A 1.6-kb fragment (d2) of one positive clo ne was found to extend from 5'-untranslated region to beyond the exon/ intron junction at residue Arg(86). The genomic DNA fragment d2 and th e cDNA clone z115 were assembled to generate a clone (d2-z115) contain ing the entire coding sequence of hkor. Clone d2-z115 has an open read ing frame of 1140 bp, which encodes for a 380-amino acid protein. The deduced amino acid sequence has 93.9% and 93.2% identity to rat and mo use kappa receptors, respectively. It also displays similar to 60% ide ntity to both human mu and delta receptors. Northern blot analysis sho wed that in the human brain there was a single hkor mRNA transcript of 6.0 kb. Among brain regions examined, the amygdala, caudate nucleus, hypothalamus and subthalamic nucleus contained high levels of hkor mRN A. Hkor was cloned into the expression vector pBK-CMV and transiently expressed in COS-1 cells. Hkor had high affinity for [H-3] diprenorphi ne, a nonselective opioid antagonist, and displayed stereospecific bin ding to naloxone. kappa selective ligands (U50,488H and nor-BNI) had h igh affinities, whereas mu and delta selective ligands bound with much lower affinities. Dynorphin A (1-17) and alpha-neoendorphin, both end ogenous kappa peptides, bound with high affinities. These binding char acteristics confirmed that hkor is a kappa receptor, most likely kappa (1) type. Cloning of the human kappa receptor allows investigation of interactions of compounds with the human receptor, instead of rodent r eceptors, for development of better therapeutic agents.