Affinity labeling the dopamine transporter ligand binding site

Photoaffinity labeling is a positive function approach that has been used in an effort to identify the cocaine-binding site on the dopamine transporter (DAT). Radioactive and non-radioactive analogs of cocaine and other dopamine uptake blockers are used to irreversibly label the DAT ligand-binding site and the protein is subjected to chemical or enzymatic treatments that cleave at specific amino acid residues. Analysis of cleavage products from radioactively photolabeled DAT using epitope-specific immunoprecipitation, gel electrophoresis, and autoradiography has identified the site of origin in the primary sequence of labeled fragments as small as 4 kDa. More precise localization of the site of labeling is done by subjecting photolabeled DAT to parallel or serial digestion with multiple cleavage methods, followed by analysis of radiolabeled peptides by reverse-phase HPLC. Fragment retention times are compared to calculated retention times of predicted digest peptides and to chemically or photochemically labeled synthetic peptides. The presence of authentic DAT sequence in HPLC fractions of digests from DAT labeled with non-radioactive ligands is further supported by MALDI and nanoelectrospray mass spectrometry. Using these methods we have identified two distinct regions of DAT that interact with multiple structurally related and diverse irreversible ligands, suggesting that these regions may be involved in the formation of ligand binding sites.