Photoaffinity labeling and binding studies reveal the existence of two types of phencyclidine receptors in the NCB-20 cell line

The mouse neuroblastoma-Chinese hamster brain hybrid cell line NCB-20 is the only clonal cell line in which binding studies indicate the presence of phencyclidine (PCP) receptor-like sites. We report here that polypeptide components of NCB-20 cell PCP sites were identified with the photolabile PCP derivative [³H]N-[1-(3-azidophenyl)cyclohexyl]piperidine ([³H]AZ-PCP). The pharmacological selectivity of [³H]AZ-PCP binding (under reversible conditions) was similar to that observed for [³H]N-[1-(2-thienyl)cyclohexyl]-piperidine ([³H]TCP) binding to NCB-20 cell membranes. Inhibition of [³H]TCP binding by AZ-PCP, dexoxadrol or MK-801 was biphasic, suggesting the presence of two types of PCP sites on NCB-20 cells. Photolysis of NCB-20 cell membranes pre-equilibrated with [³H]AZ-PCP, followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), revealed the presence of 5 major labeled bands (M_r 90,000, 68,000, 49,000, 40,000 and 33,000), a pattern similar to that observed for rat brain membranes. MK-801 and d-2-amino-5-phosphonovaleric acid (d-(-)-AP5) selectively inhibited the labeling of M_r 68,000 and 90,000 polypeptides. These results indicate that the labeled bands represent constituents of at least two different PCP binding proteins. The M_r 68,000 and 90,000 components appear to correspond to a high-affinity site, which comprises approximately 20% of total [³H]TCP sites in these cells, and exhibits the pharmacology expected for the PCP receptor of the N-methyl-d-aspartate (NMDA)-gated channel.