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.