RT Journal Article
SR Electronic
T1 Dual Incorporation of Photoaffinity Ligands on Dopamine Transporters Implicates Proximity of Labeled Domains
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 1157
OP 1164
DO 10.1124/mol.59.5.1157
VO 59
IS 5
A1 Roxanne A. Vaughan
A1 Jon D. Gaffaney
A1 John R. Lever
A1 Maarten E. A. Reith
A1 Aloke K. Dutta
YR 2001
UL http://molpharm.aspetjournals.org/content/59/5/1157.abstract
AB 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.