Zeta (ζ), the opioid growth factor receptor: identification and characterization of binding subunits

The zeta (ζ) opioid receptor mediates the activity of the opioid growth factor, [Met⁵]-enkephalin, a peptide that regulates developmental events in a variety of normal and tumorigenic tissues and cells, including the nervous system. To identify the binding subunit(s) of the ζ receptor, protein blots of rat cerebellar proteins from 6-day-old animals were separated by sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto nitrocellulose. Ligand blotting of these blots with 1.5 nM[¹²⁵I]-[Met⁵]-enkephalin revealed four binding polypeptides of 32, 30, 17, and 16 kDa. Binding occurred at concentrations relevant to the K_d of the receptor, was blocked by cold ligand and opioid antagonists, and exhibited a stereospecific response. No binding was recorded in the adult rat cerebellum. Subcellular fractionation studies using ligand blotting and receptor-binding analysis indicated that these binding subunits were associated with the nucleus. Two-dimensional protein analysis using non-equilibrium pH gradient electrophoresis (NEPHGE) SDS-PAGE of 6-day-old cerebellum and ligand blotting showed that the 32-, 30-, 17-, and 16-kDa subunits have basic isoelectric points. Two-dimensional chymotryptic peptide mapping showed a strong homology between the 32- and 30-kDa subunits, but not with the 17- and 16-kDa polypeptides. The 17- and 16-kDa subunits had only a partial homology to each other. These results are consistent with the biology, biochemistry, and pharmacology of the ζ receptor, and are the first to identify and characterize the binding polypeptides of an opioid receptor that has important growth-related properties.