TY - JOUR
T1 - Photoaffinity labeling and binding studies reveal the existence of two types of phencyclidine receptors in the NCB-20 cell line
AU - Haring, Rachel
AU - Zukin, R. Suzanne
AU - Zukin, Stephen R.
N1 - Funding Information:
This work was supportedi n part by USPHS Grants NS20752a nd DA 00069 (R.S.Z.) and DA 03383 (S.R.Z.); by National SciencesF oundationGrant BNS 8607398t,h e Ritter Foundation,a nd The Departmento f Psychiatry,D r. H.M. van Praag, Chairman (S.R.Z.); and the Camp David Institutef or InternationalH ealth (R.H.).
PY - 1990/4/20
Y1 - 1990/4/20
N2 - 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 (Mr 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 Mr 68,000 and 90,000 polypeptides. These results indicate that the labeled bands represent constituents of at least two different PCP binding proteins. The Mr 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.
AB - 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 (Mr 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 Mr 68,000 and 90,000 polypeptides. These results indicate that the labeled bands represent constituents of at least two different PCP binding proteins. The Mr 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.
KW - Azidophencyclidine
KW - N-Methyl-d-aspartate receptor
KW - Neuroblastoma cell
KW - Phencyclidine receptor
KW - Photoaffinity labeling
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U2 - 10.1016/0304-3940(90)90328-7
DO - 10.1016/0304-3940(90)90328-7
M3 - Article
C2 - 2166931
AN - SCOPUS:0025274829
SN - 0304-3940
VL - 112
SP - 92
EP - 98
JO - Neuroscience Letters
JF - Neuroscience Letters
IS - 1
ER -