Species- and brain region-specific dopamine transporters: immunological and glycosylation characteristics.
Dopamine transporters (DATs) from the caudate nucleus of four species (rat, mouse, dog, and human) and four regions of rat brain (striatum, nucleus accumbens, prefrontal cortex, and midbrain) were photoaffinity labeled and analyzed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis for cross-reactivity to four epitope-specific rat antipeptide antibodies. Each of these antibodies varied in its efficiency at recognizing DAT. The DATs from the rat brain regions exhibited the same degree of recognition by each of the four sera, a result compatible with these proteins being the product of a single gene. The DATs from the different species were recognized by all four sera but with different efficiencies, possibly relating to amino acid sequence differences within the immunizing epitope. All of the photolabeled, immunoprecipitated DATs migrated with a molecular mass of approximately 80 kDa, and no lower molecular mass forms were found. The DATs from all species and brain regions tested were shown by enzymatic deglycosylation to contain N-linked carbohydrates and sialic acids in amounts comparable with rat striatal DATs. The finding that no photolabeled DAT forms < 80 kDa were isolated from membranes indicates that partially or incompletely glycosylated forms are not present, even in the midbrain cell bodies where immature forms might be expected to be found. These findings verify the utility of these anti-rat antibodies as biochemical tools for studying DATs from other species and extend our knowledge of biochemical characteristics of DATs from these species and brain regions.
Vaughan
R A
RA
Neuroscience Branch, NIDA Intramural Research Program, Baltimore, Maryland 21224, USA.
Brown
V L
VL
McCoy
M T
MT
Kuhar
M J
MJ
D000345:Affinity Labels; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002236:Carbohydrate Conformation; D002352:Carrier Proteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D003429:Cross Reactions; D004285:Dogs; D050483:Dopamine Plasma Membrane Transport Proteins; D006031:Glycosylation; D006801:Humans; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011233:Precipitin Tests; D051381:Rats; D013045:Species Specificity; D014018:Tissue Distribution
D000345:Affinity Labels; D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493214:SLC6A3 protein, human; C493215:Slc6a3 protein, mouse; C493216:Slc6a3 protein, rat