Photoaffinity labelling of dopamine D2 receptors by [3H]azidomethylspiperone

Abstract

We have characterized the dopamine D₂ receptor photaffinity probe, [³H]azido‐N‐methylspiperone ([³H]AMS). In the absence of light, [³H]AMS bound reversibly and with high affinity (K _d 70 pM) to sites in canine striatal membranes and was competitively inhibited by dopaminergic agonists and antagonists with an appropriate D₂ receptor specificity. Upon photolysis, [³H]AMS covalently incorporated into a peptide of M _r 92000 as assessed by fluorography following SDS‐polyacrylamide gel electrophoresis. Labelling of this peptide was specifically and stereoselectively blocked by D ₂ antagonists and agonists. Minor specifically labelled peptides of M _r 70000‐55000 were observed under some conditions and were the result of proteolytic degradation of the peptide at M _r, 92000.