Photoaffinity labeling of the D2-dopamine receptor using a novel high affinity radioiodinated probe

The ligand binding subunit of the D2 subtype of the dopamine receptor has been identified by photoaffinity labeling. In order to develop a specific covalent receptor probe, an analogue of the potent D2 selective antagonist spiperone, N-(p-aminophenethyl)spiperone (NAPS) has been synthesized. The aminophenethyl substituent of NAPS can be radioiodinated to theoretical specific radioactivity (2,175 Ci/mmol) and then the arylamine group converted to an arylazide to yield a photosensitive probe [( 125I]N3-NAPS). In rat striatal membranes, the nonradiolabeled azide probe (N3-NAPS) binds to the receptor with high affinity (KD congruent to 1.6 +/- 0.05 nM) and upon photoactivation irreversibly decreases the number of available receptors in these membranes as measured by [3H]spiperone binding. More importantly, however, incubation of rat striatal membranes with [125I]N3-NAPS leads to the photodependent covalent incorporation of the probe into a peptide of Mr = 94,000 as assessed by autoradiography of gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling of this Mr = 94,000 peptide can be blocked specifically and stereoselectively by dopaminergic antagonists such as (+)- and (-)-butaclamol but not by non-dopaminergic antagonists. Moreover, dopaminergic agonists also attenuate the covalent labeling of this peptide with an order of potency which is typically D2-dopaminergic. Therefore, the specificity of [125I]N3-NAPS labeling of the Mr = 94,000 peptide suggests that this peptide represents the ligand binding subunit of the D2-dopamine receptor.