Characterization and epitope mapping of several new anti-P-glycoprotein monoclonal antibodies

Shapiro, Adam B.; Duthie, Monika; Childs, Sarah J.; Okubo, Toshiteru; Ling, Victor

doi:10.1002/(sici)1097-0215(19960717)67:2<256::aid-ijc17>3.0.co;2-9

Cited by 9 publications

(9 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1). Previous studies of MRP and other ABC transport proteins indicate that these regions are cytoplasmic (18,19,27,28,36 ). Predicted Topologies of MRP-Analyses of the MRP sequence using various algorithms for predicting the locations of membrane spanning helices suggested several topological models of MRP (29 -31).…”

Section: Resultsmentioning

confidence: 93%

Membrane Topology of the Multidrug Resistance Protein (MRP)

Hipfner¹,

Almquist²,

Leslie³

et al. 1997

Journal of Biological Chemistry

192

150

View full text Add to dashboard Cite

Multidrug resistance is frequently characterized by an ATPdependent reduction in cellular drug accumulation. This phenotype can occur in mammalian cells by overexpression of either the multidrug resistance protein (MRP) 1 or P-glycoprotein (MDR1) (1-5). MRP and P-glycoprotein belong to the ATPbinding cassette (ABC) superfamily of transport proteins but share only 15% amino acid identity (1). Nevertheless, both proteins confer resistance to a broad range of cytotoxic xenobiotics including doxorubicin, vincristine, and VP-16 (etoposide), drugs that are widely used in the treatment of many human cancers. However, there is growing evidence that the mechanisms by which MRP and P-glycoprotein reduce cellular drug accumulation are not the same, suggesting that there are major differences in the drug-protein interactions of these two molecules (6 -8).Like most eukaryotic ABC proteins, MRP and P-glycoprotein contain hydrophobic membrane spanning domains (MSDs) and cytoplasmic nucleotide binding domains (NBDs) (9). To understand how drugs interact with P-glycoprotein, there has been considerable interest in determining the precise topology of this integral membrane protein. Investigations in most experimental systems support a model in which P-glycoprotein is organized as a symmetrically arranged, tandemly duplicated molecule with each half consisting of six transmembrane segments followed by a NBD (10), but alternate models have also been proposed (11,12).At present, little is known about the membrane topology of MRP. The topological model we proposed when MRP was cloned in 1992 was based on computer-assisted hydropathy analyses of its deduced amino acid sequence and alignment with the predicted structure of ltpgpA (1). LtpgpA is an ABC protein cloned from Leishmania tarentolae which was the most closely related protein to MRP known at that time (13). In the original model, we suggested that MRP consisted of eight transmembrane segments and an NBD in its NH 2 -proximal half and only four transmembrane segments and an NBD in its COOH-proximal half (1). More recently, alignment of the hydropathy profiles of human MRP with those of its murine ortholog (14) and several members of the ABC superfamily (including the related sulfonylurea receptor, SUR (15), and the yeast cadmium resistance factor, YCF1 (16), as well as Pglycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR)) suggested to us a different topology for MRP. In this later model, we predicted that MRP contains two MSDs of six transmembrane helices in a "6 ϩ 6" configuration typical of several eukaryotic ABC transporters (9), plus an extremely hydrophobic NH 2 -terminal MSD of approximately 220 amino acids (4,14,17,18). This additional hydrophobic domain is predicted to contain four to six transmembrane segments and is not present in ABC proteins such as P-glycoprotein and CFTR. Thus it is a characteristic feature of members of the MRP branch of the ABC transporter superfamily (4,14).

show abstract

Section: Resultsmentioning

confidence: 93%

Membrane Topology of the Multidrug Resistance Protein (MRP)

Hipfner¹,

Almquist²,

Leslie³

et al. 1997

Journal of Biological Chemistry

192

150

View full text Add to dashboard Cite

show abstract

“…The C219 antibody is directed to internal epitopes of Pgp near the ATP-binding cassette in the C- and N-terminal halves of the protein, 20 whereas C494 is directed to distinct internal epitope proximal to the ATP-binding site near the N-terminal half of Pgp. 21 The membrane was washed five times (5 minutes each) in TBS with 0.5% Tween 20. The nitrocellulose membrane was then incubated with anti-mouse Ig secondary antibody (Fab) (1:1250) for 90 minutes at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Ontogeny of P-Glycoprotein in Mouse Intestine, Liver, and Kidney

Mahmood

Daood²,

Hart³

et al. 2001

Journal of Investigative Medicine

View full text Add to dashboard Cite

show abstract

“…The mAb MM4.17 was a gift from Dr. Maurizio Cianfriglia, and its epitope has been mapped to loop 7 of Pgp (Cianfriglia et al, 1994). Monoclonal antibodies MD-1 and MD-7 were generated against purified hamster Pgp, and their epitopes have been mapped to 22 and 11 amino acid residues in the linker region and loop 8, respectively (Shapiro et al, 1996). These two mAbs have been shown to react with human Pgp.…”

Section: Methodsmentioning

confidence: 99%

Topological Folding and Proteolysis Profile of P-glycoprotein in Membranes of Multidrug-Resistant Cells: Implications for the Drug-Transport Mechanism

Zhang¹,

Wang²,

Shapiro³

et al. 1996

Biochemistry

View full text Add to dashboard Cite

P-glycoprotein (Pgp)1 is a polytopic membrane protein and functions as an energy-dependent drug efflux pump. It is responsible for multidrug resistance (MDR) in cancer cell lines. Recently, the topological structure of Pgp has been investigated. However, the results are in dispute. A major question concerning the Pgp topology is the membrane orientation of the loop linking TM4 and TM5 (loop 4) and the loop linking TM8 and TM9 (loop 8). In this study, we generated polyclonal antibodies specific to these two loops. In combination with a panel of other well-characterized site-specific polyclonal- and monoclonal antibodies of Pgp, we tested the membrane orientation of these two loops of Pgp in multidrug-resistant cells using immunocytochemistry and proteolysis/membrane protection assay. Our results showed that (1) both loops 4 and 8 are located extracellularly whereas other domains, such as the ATP-binding sites, are in the cytoplasm and (2) proteolysis of Pgp is not a random event and the trypsin-sensitive sites are cleaved in orders. Since the Pgp was not genetically manipulated in this study, in contrast to previous studies, we believe that naturally expressed Pgp molecules have an unconventional topology. We speculate that this alternate topology of Pgp may represent a different functional state of the protein. Further detailed analysis of Pgp topology will help to understand the fundamental mechanism of drug transport by Pgp.

show abstract

Characterization and epitope mapping of several new anti-P-glycoprotein monoclonal antibodies

Cited by 9 publications

References 36 publications

Membrane Topology of the Multidrug Resistance Protein (MRP)

Membrane Topology of the Multidrug Resistance Protein (MRP)

Ontogeny of P-Glycoprotein in Mouse Intestine, Liver, and Kidney

Topological Folding and Proteolysis Profile of P-glycoprotein in Membranes of Multidrug-Resistant Cells: Implications for the Drug-Transport Mechanism

Contact Info

Product

Resources

About