Identification of the binding subunit of the D1-dopamine receptor by photoaffinity crosslinking

The D1-dopamine receptor from rat striatum has been successfully identified by photoaffinity crosslinking using a newly synthesized radioiodinated derivative of the selective D1-antagonist SCH-23390. This compound, (R,S)-5-(3'-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl-[1H]-3- benzazepin-7-ol(SCH-38548), has been radioiodinated by a chloramine T procedure yielding three radioiodinated products. One of these separated congeners (with Rf = 0.35 on thin layer chromatography; CH2Cl2/MeOH/triethylamine; 82.5:17.5:0.01) binds reversibly to rat striatal membranes with high affinity (KD approximately equal to 200 pM), appropriate stereoselectivity, and D1-dopaminergic specificity. [125I]SCH-38548 can be covalently incorporated into a peptide of Mr approximately equal to 72,000 using the heterobifunctional crosslinking reagent N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. Covalent incorporation of [125I]SCH-38548 into the Mr approximately equal to 72,000 peptide can be blocked by dopaminergic agents with D1-dopaminergic specificity (for agonists: SKF-38393 greater than apomorphine greater than dopamine; for antagonists: SCH-23390 much much greater than, SCH-23388 and cis-flupentixol much much greater than trans-flupentixol). The D1-dopaminergic selectivity and specificity of the labeling were further demonstrated by the fact that other antagonists such as domperidone, ketanserin, phentolamine, and alprenolol did not compete for the covalent labeling of the Mr approximately equal to 72,000 peptide. These results indicate that the ligand-binding subunit of the D1-dopamine receptor resides on peptide distinct from that of the D2-dopamine receptor (Mr = 94,000). This new radioligand should be useful in the molecular characterization of the D1-dopaminergic receptor from various sources.