Antipeptide Antibodies Confirm the Topology of the Human Norepinephrine Transporter

doi:10.1074/jbc.270.16.9197

Antipeptide Antibodies Confirm the Topology of the Human Norepinephrine Transporter (*)

From the (1) Institut für Pharmakologie und Toxikologie, Universität Bonn, Reuterstrasse 2b, D-53113 Bonn, Federal Republic of Germany and the
(2) Department of Pharmacology, Mayo Clinic, Rochester, Minnesota 55905

^§ To whom correspondence should be addressed. Tel.: 49-228-735431; Fax: 49-228-735404.

Abstract

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 [S]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.

Footnotes

↵* This work was supported by Deutsche Forschungsgemeinschaft Grants Bo 521/8-2 and SFB 400 and by the A. v. Humboldt Foundation (fellowship award to S. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

NE

norepinephrine

NET

norepinephrine transporter

hNET

human norepinephrine transporter

bNET

bovine norepinephrine transporter

DA

dopamine

DAT

dopamine transporter

hDAT

human dopamine transporter

KLH

keyhole limpet hemocyanine

MAP49-57

multiple antigenic peptide whose identical eight branches correspond to the amino acid of the hNET indicated by the numbers

N6-28, L211-226, L371-384, C590-607

human norepinephrine transporter peptides with a sequence corresponding to the indicated numbers

PAGE

polyacrylamide gel electrophoresis

PNGase F

peptide- N-glycosidase F

GABA

-aminobutyric acid

TM

transmembrane domain

PBS

phosphate-buffered saline

FITC

fluorescein isothiocyanate

ELISA

enzyme-linked immunosorbent assay

DTAF

5-([4,6-dichlorotriazin-2-yl]amino)fluorescein

PBS

phosphate-buffered saline.
↵²M. Brüss, R. Hammermann, S. Brimijoin, and H. Bönisch, unpublished results.