Photoaffinity-labeled ligand binding domains on dopamine transporters identified by peptide mapping

Binding domains on rat dopamine transporters for cocaine and 1-(2-diphenylmethoxy)ethyl-4-(3-phenylpropyl)piperazine compounds were identified using controlled proteolysis of photoaffinity-labeled protein and epitope-specific immunoprecipitation of the labeled fragments. Rat dopamine transporters were photoaffinity labeled with 1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-azido- 3-[125I]iodophenyl)ethyl]piperazine ([125I]DEEP) [a 1-(2-di- phenylmethoxy)ethyl-4-(3-phenylpropyl)piperazine analog] or 3 beta-(p-chlorophenyl)tropane-2 beta-carboxylic acid, 4'-azido-3'- [125I]iodophenylethyl ester ([125I]RTI 82) (a cocaine analog) and were gel purified to remove contaminating radioactivity. The resulting samples were treated with V8 protease or trypsin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The peptide maps generated with each enzyme were different for each of the ligands, suggesting that the ligands were incorporated into different regions of the protein. Identical peptide maps were generated from striatum- and nucleus accumbens-derived transporters, indicating that these polypeptides are highly similar in primary sequence. The proteolytic fragments generated by V8 protease were localized to specific domains of the protein using antipeptide antibodies corresponding to five different regions of the transporter. Fragments of 10 and 7 kDa from [125I]DEEP-labeled transporters were specifically immunoprecipitated with an antibody generated against amino acids 42-59 (near the first putative trans-membrane domain), whereas a 34-kDa fragment from [125I]RTI 82-labeled transporters was precipitated with three different sera corresponding to regions in the carboxyl-terminal two thirds of the protein. None of the V8 fragments smaller than 45 kDa, containing either photolabel, was altered in molecular mass by N-deglycosylation. The results indicate that photoincorporation of [125I]DEEP occurs in the amino half of the dopamine transporter, near the first two transmembrane helices, whereas [125I]RTI 82 labels the carboxyl-terminal region of the protein, between transmembrane domains 4 and 12.