Dual Incorporation of Photoaffinity Ligands on Dopamine Transporters Implicates Proximity of Labeled Domains
- 1Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota (R.A.V., J.D.G.); 2Department of Environmental Health Sciences, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland (J.R.L.); 3Department of Biomedical and Therapeutic Science, University of Illinois, Peoria, Illinois (M.E.A.R.); and4Department of Pharmaceutical Sciences, Wayne State University, Detroit, Michigan (A.K.D.)
Abstract
We have recently developed novel high-affinity blockers for the dopamine transporter (DAT) by carrying out structure-activity studies of GBR 12909 molecule piperidine analogs. To investigate the molecular basis of binding of these compounds in comparison to known sites of action of GBR 12909, cocaine, and benztropine analogs, we developed a piperidine-based photoaffinity label [125I]4-[2-(diphenylmethoxy)ethyl]-1-[(4-azido- 3-iodophenyl)methyl]-piperidine [125I]AD-96-129), and used proteolysis and epitope-specific immunoprecipitation to identify the protein domains that interact with the ligand. [125I]AD-96-129 became incorporated into two different regions of the DAT primary sequence, an N-terminal site containing transmembrane domains (TMs) 1 to 2, and a second site containing TMs 4 to 6. Both of these regions have been identified previously as sites involved in the binding of other DAT photoaffinity labels. However, in contrast to the previously characterized ligands that showed nearly complete specificity in their binding site incorporation, [125I]AD-96-129 became incorporated into both sites at comparable levels. These results suggest that the two domains may be in close three-dimensional proximity and contribute to binding of multiple uptake blockers. We also found that DATs labeled with [125I]AD-96-129 or other photoaffinity labels displayed distinctive sensitivities to proteolysis of a site in the second extracellular loop, with protease resistance related to the extent of ligand incorporation in the TM4 to 6 region. These differences in protease sensitivity may indicate the relative proximity of the ligands to the protease site or reflect antagonist-induced conformational changes in the loop related to transport inhibition.
Footnotes
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Send reprint requests to: Dr. Roxanne Vaughan, Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, 501 N. Columbia Rd., Grand Forks, ND 58203-9037. E-mail: rvaughan{at}medicine.nodak.edu
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This work was supported by ND EPSCoR and National Institutes of Health Grants DA13147 (R.A.V.), DA08647 (A.K.D.), and DA08870 (J.R.L.). We thank Vickie Swift, University of North Dakota Graphics Department, for providing artwork.
- Abbreviations:
- DAT
- dopamine transporter
- TM
- transmembrane domain
- GBR
- GBR 12909 ([2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine)
- [125I]DEEP
- 1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-azido-3-iodophenyl)ethyl]piperazine
- [125I]GA II 34
- 4-(4′-azido-3′-iodophenyl)-n-butyl 3α-[bis(4′-fluorophenyl)methoxy]tropane
- [125I]RTI 82
- 3β-(p-chlorophenyl)tropane-2β-carboxylic acid, 4′-azido-3′-iodophenylethyl ester
- SERT
- serotonin transporter
- VMAT
- vesicle monoamine transporter
- [125I]AD-96-129
- 4-[2-(diphenylmethoxy)ethyl]-1-[(4-azido-3-iodophenyl)methyl]-piperidine
- PAGE
- polyacrylamide gel electrophoresis
- NET
- norepinephrine transporter
- EL
- extracellular loop
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- Received September 26, 2000.
- Accepted February 1, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
