Photoaffinity labeling of the mammalian dopamine transporter.
A high affinity (1-2 nM) radioiodinated, photoaffinity probe for the dopamine transporter, 1-(2-[bis-(4-fluorophenyl)-methoxylethyl)-4-(2-[4-azido-3- [125I]iodophenyl]ethyl)piperazine ([125I]FAPP) has been synthesized. Upon photolysis, [125I]FAPP incorporates into a striatal polypeptide of apparent Mr 62,000 as visualized by autoradiography following sodium dodecyl sulfate-PAGE. Photoincorporation of [125I]FAPP into the Mr 62,000 polypeptide was stereoselectively inhibited by various dopamine uptake agents with a potency order typical of the dopamine transporter. The glycoprotein nature of the apparent Mr 62,000 polypeptide was assessed following specific exo- and endoglycosidase treatment. The dopamine transporter appears to be associated with complex-type oligosaccharides as indexed by its susceptibility to neuraminidase but not alpha-mannosidase digestion. Complete N-linked deglycosylation of the neuronal dopamine transporter with the endoglycosidase, glycopeptidase-F, increased the electrophoretic mobility of the 62 kDa polypeptide to apparent Mr 48,000. [125I]FAPP should prove to be a useful probe for the molecular characterization of the dopamine uptake site in various tissues and under certain pathophysiological states.
Sallee
F R
FR
Department of Psychiatry, Medical University of South Carolina, Charleston 29425.
Fogel
E L
EL
Schwartz
E
E
Choi
S M
SM
Curran
D P
DP
Niznik
H B
HB
D000345:Affinity Labels; D000818:Animals; D001345:Autoradiography; D001692:Biological Transport; D002352:Carrier Proteins / Q000032:analysis*; D003342:Corpus Striatum / Q000378:metabolism*; D004285:Dogs; D050483:Dopamine Plasma Membrane Transport Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D015394:Molecular Structure; D009419:Nerve Tissue Proteins; D010777:Photochemistry; D010879:Piperazines
D000345:Affinity Labels; D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010879:Piperazines; C043425:vanoxerine; D006026:Glycoside Hydrolases