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

The zeta (zeta) 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 zeta 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 Kd 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 zeta receptor, and are the first to identify and characterize the binding polypeptides of an opioid receptor that has important growth-related properties.