Membrane Topology and Glycosylation of the Human Multidrug Resistance-associated Protein

doi:10.1074/jbc.271.21.12322

Membrane Topology and Glycosylation of the Human Multidrug Resistance-associated Protein (*)

From the (1) Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1113 Budapest, Hungary, the
(2) National Institute of Haematology and Immunology, H-1113 Budapest, Hungary, and the
(3) Netherlands Cancer Institute and Department of Pathology, Free University Hospital, 1080 HV Amsterdam, The Netherlands

^§ Recipient of a Howard Hughes International Fellowship. To whom correspondence should be addressed:
National Institute of Haematology and Immunology, 1113 Budapest, Daróczi u. 24, Hungary
. Tel.:/Fax: 36-1-185-2234.

Abstract

The membrane topology of the human multidrug resistance-associated protein (MRP) was examined by flow cytometry phenotyping, immunoblotting, and limited proteolysis in drug-resistant human and baculovirus-infected insect cells, expressing either the glycosylated or the underglycosylated forms of this protein. Inhibition of N-linked glycosylation in human cells by tunicamycin did not inhibit the transport function or the antibody recognition of MRP, although its apparent molecular mass was reduced from 180 kDa to 150 kDa. Extracellular addition of trypsin or chymotrypsin had no effect either on the function or on the molecular mass of MRP, while in isolated membranes limited proteolysis produced three large membrane-bound fragments. These experiments and the alignment of the MRP sequence with the human cystic fibrosis transmembrane conductance regulator (CFTR) suggest that human MRP, similarly to CFTR, contains a tandem repeat of six transmembrane helices, each followed by a nucleotide binding domain, and that the C-terminal membrane-bound region is glycosylated. However, the N-terminal region of MRP contains an additional membrane-bound, glycosylated area with four or five transmembrane helices, which seems to be a characteristic feature of MRP-like ATP-binding cassette transporters.

Footnotes

↵* This work was supported by research grants from COST, PECO, OMFB, OTKA Grants F13178, T17602, T6348, and ETT Hungary. 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.
↵() The abbreviations used are:

MDR1

multidrug-resistance protein

ABC transporters

ATP-binding cassette transporters

ADR

adriamycin (doxorubicin)

AM

acetoxymethylester

cMOAT

canalicular multispecific organic anion transporter

CFTR

cystic fibrosis transmembrane conductance regulator

ECL

enhanced chemiluminescence

mAb

monoclonal antibody

MRP

multidrug resistance-associated protein

Sf9 cells

Spodoptera frugiperda ovarian cells

PBS

phosphate-buffered saline

MOPS

4-morpholinepropanesulfonic acid.
- Received February 12, 1996.
- Revision received March 28, 1996.