Opioid peptide receptor studies. 1. Identification of a novel δ-opioid receptor binding site in rat brain membranes

Our laboratory was among the first to propose the existence of δ receptor subtypes: a δ site thought to be associated with a μ-δ-opioid receptor complex termed the δ_cx binding site and δ site not associated with the μ-δ-opioid receptor complex, termed the δ_ncx site. In previous studies, we assayed the δ_cx site with [³H][d-Ala²,d-Leu⁵]enkephalin using rat brain membranes depleted of δ_ncx sites by pretreatment with the site-directed acylating agent, (+)-trans-SUPERFIT. In the present study, we investigated, using (+)-trans-SUPERFIT-pretreated membranes, the possibility of heterogeneity of the δ_cx binding site. Two sites were resolved: the δ_cx-1 site at which μ ligands are potent noncompetitive inhibitors and δ ligands are weak competitive inhibitors, and the δ_cx-2 site has a δ-like ligand-selectivity profile, several experiments distinguished it from the δ_ncx site. Two lines of evidence suggest that the δ_ncx site corresponds to the cloned δ receptor. One, the δ receptor was cloned from the NG108-15 cell line, and this receptor, like the δ_ncx binding site, irreversibly binds SUPERFIT and (+)-trans-SUPERFIT. Secondly, administration of δ-antisense DNA selectively decreases δ_ncx binding. Viewed collectively, the major finding of this study is the discovery of a novel SUPERFIT-insensitive and δ-antisense-insensitive δ_cx-2 binding site.