Antipeptide antibodies confirm the topology of the human norepinephrine transporter.
We have raised polyclonal antibodies (N6-28, L211-226, L371-384, and C590-607) against peptides corresponding to hydrophilic sequences of the human norepinephrine transporter (hNET). The antisera immunoprecipitated the [35S]Met-labeled hNET. Antiserum L211-226, directed against a sequence of the putative second (large) extracellular loop of hNET, also immunoprecipitated the human dopamine transporter. Antisera N6-28 and C590-607, raised against a hNET peptide region of the N and the C termini, respectively, recognized a 58-kDa protein from transfected COS-7 cells expressing the hNET. This 58-kDa species represents a functional, glycosylated form of the hNET and not a degradation product. Tunicamycin treatment of transfected COS-7 cells as well as peptide-N-glycosidase F digestion of the transporter converted the 58-kDa species to a 50-kDa form, indicating that the latter represents the hNET core protein. In indirect immunofluorescence studies, our antisera confirmed the originally proposed topology of hNET. Antisera N6-28 and C590-607 detected hNET only in permeabilized cells. In contrast, antisera L211-226 and L371-384 directed against peptide sequences of the second and fourth putative extracellular loop displayed fluorescence signals with the intact cells.
Brüss
M
M
Institut für Pharmakologie und Toxikologie, Universität Bonn, Federal Republic of Germany.
Hammermann
R
R
Brimijoin
S
S
Bönisch
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002352:Carrier Proteins / Q000737:chemistry* / Q000276:immunology; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D006801:Humans; D008969:Molecular Sequence Data; D009638:Norepinephrine / Q000378:metabolism*; D050484:Norepinephrine Plasma Membrane Transport Proteins; D011817:Rabbits; D027981:Symporters
D000906:Antibodies; D002352:Carrier Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; C493227:SLC6A2 protein, human; D027981:Symporters; D009638:Norepinephrine