IDENTIFICATION OF DYNORPHINS AS ENDOGENOUS LIGANDS FOR AN OPIOID RECEPTOR-LIKE ORPHAN RECEPTOR

To identify the endogenous ligands for a cloned orphan receptor that shares high degrees of sequence homology with opioid receptors, this orphan receptor was expressed in Xenopus oocytes and in mammalian cell lines CHO-K1 and HEH-293. The coupling of the receptor to a G protein-activated K+ channel was used as a functional assay in oocytes. Endogenous opioid peptide dynorphins were found to activate the K+ channel by stimulating the orphan receptor. This activation was dose-dependent, with EC(50) values at 45 and 37 nM for dynorphin A and dynorphin A-(1-13), respectively, The dynorphin effect was antagonized by the non-selective opioid antagonist naloxone but at rather high concentrations in the micromolar range. Naloxone also caused a rightward shift of the dose-response curve for dynorphin A, suggesting a competitive antagonism mechanism, In transiently transfected cells, 5 mu M dynorphin A-(1-13) inhibited the forskolin-stimulated cyclic AMP increase by 51 and 35% in CHO-K1 and HEH-293 cells, respectively, Other classes of endogenous opioids, i.e. enkephalins and endorphins, caused very little activation of this receptor. These results suggest that this orphan receptor is a member of the opioid receptor family and that dynorphins are endogenous ligands for this receptor.