Cocaine and GBR photoaffinity labels as probes of dopamine transporter structure.
Several aspects of DAT structure and function have been elucidated using a combination of photoaffinity labeling, proteolysis, enzymatic deglycosylation, and epitope-specific immunoprecipitation. The two photolabels are incorporated in different regions of the protein, suggesting that the binding sites for the ligands are distinct or partially nonoverlapping, consistent with results produced by site-directed mutagenesis and analysis of chimeras. These studies have also verified several aspects of DAT structure previously hypothesized based only on theoretical considerations, including the presence of at least one transmembrane helix or other membrane-anchoring structure in two different regions of the protein, identification of the glycosylated domain, and some topological properties. It should be possible to extend and adapt these techniques to further delineate DAT structural properties and to identify other functional domains such as phosphorylation sites or active sulfhydryl moieties.
Vaughan
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Molecular Neurobiology Branch, National Institute on Drug Abuse, Intramural Research Program, Baltimore, Maryland 21224, USA.
D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000737:chemistry* / Q000493:pharmacokinetics; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002241:Carbohydrates / Q000032:analysis; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D003042:Cocaine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000493:pharmacokinetics; D050483:Dopamine Plasma Membrane Transport Proteins; D006031:Glycosylation; D006801:Humans; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D010879:Piperazines / Q000737:chemistry* / Q000493:pharmacokinetics; D011869:Radioligand Assay / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D000345:Affinity Labels; D001386:Azides; D002241:Carbohydrates; D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010879:Piperazines; D011994:Recombinant Proteins; C493214:SLC6A3 protein, human; C060753:1-(2-(diphenylmethoxy)ethyl)-4-(2-(4-azido-3-iodophenyl)ethyl)piperazine; C076379:RTI 82; D003042:Cocaine