ANTIPEPTIDE ANTIBODIES CONFIRM THE TOPOLOGY OF THE HUMAN NOREPINEPHRINE TRANSPORTER

We have raised polyclonal antibodies (N6-28, L211-226, L371-384, and C 590-607) against peptides corresponding to hydrophilic sequences of th e human norepinephrine transporter (hNET). The antisera immuno precipi tated the [S-35]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 N6-2 8 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 func tional, glycosylated form of the hNET and not a degradation product, T unicamycin treatment of transfected COS-7 cells as well as peptide-N-g lycosidase 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 confirm ed the originally proposed topology of hNET, Antisera N6-28 and C590-6 07 detected hNET only in permeabilized cells, In contrast, antisera L2 11-226 and L371-384 directed against peptide sequences of the second a nd fourth putative extracellular loop displayed fluorescence signals w ith the intact cells.