Photoaffinity labeling of the dopamine reuptake carrier protein with 3-azido[3H]GBR-12935.
A high affinity tritiated azido-diphenylpiperazine derivative, 3-azido[3H]GBR-12935, was synthesized as a potential photoaffinity probe of the dopamine transporter. Initially, the reversible binding of 3-azido[3H]GBR-12935 to crude synaptosomal membranes from the rat striatum was characterized. Specific binding was sodium dependent and inhibited by a variety of drugs that are known to potently inhibit dopamine uptake. Other neurotransmitter uptake inhibitors, as well as cis-flupenthixol, a potent inhibitor of [3H]GBR-12935 binding to piperazine binding sites, failed to inhibit specific binding at concentrations of less than or equal to 10 microM. A good correlation was observed between the relative potencies of these drugs in inhibiting dopamine uptake into synaptosomes and in inhibiting specific 3-azido[3H]GBR-12935 binding to rat striatal membranes (r = 0.95, p less than 0.01). These data suggest that 3-azido[3H]GBR-12935, like other diphenylpiperazines such as [3H]GBR-12935 and [3H]GBR-12909, binds primarily to the dopamine transporter under defined assay conditions. After UV photolysis of crude synaptosomal membranes preincubated with 3-azido[3H]GBR-12935 (1-2 nM), a single radiolabeled polypeptide with an apparent molecular mass of 80 kDa was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Photoincorporation of 3-azido[3H]GBR-12935 into this polypeptide was inhibited selectively by compounds that inhibit the uptake of dopamine (but not other biogenic amines) and was completely dependent on the presence of Na+. No photolabeled proteins were observed when cerebellar membranes were substituted for striatal membranes. Essentially complete adsorption of the radiolabeled 80-kDa polypeptide to wheat germ agglutinin and elution with N-acetyl-D-glucosamine strongly suggest that the dopamine transporter polypeptide photolabeled by 3-azido[3H]GBR-12935 is glycosylated.
Berger
S P
SP
Section on Molecular Pharmacology, National Institute of Mental Health, Bethesda, Maryland 20892.
Martenson
R E
RE
Laing
P
P
Thurkauf
A
A
Decosta
B
B
Rice
K C
KC
Paul
S M
SM
D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D003342:Corpus Striatum / Q000378:metabolism / Q000648:ultrastructure; D050483:Dopamine Plasma Membrane Transport Proteins; D006023:Glycoproteins / Q000378:metabolism; D007700:Kinetics; D008027:Light; D008297:Male; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008566:Membranes / Q000378:metabolism; D008970:Molecular Weight; D009419:Nerve Tissue Proteins; D010879:Piperazines / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D014316:Tritium
D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010879:Piperazines; D014316:Tritium; C068380:1-(2-(bis(phenyl)methoxy)ethyl)-4-(3-(3'-azidophenyl)propyl)piperazine