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

Zhang, Mei; Wang, Guichun; Shapiro, Adam B.; Zhang, Jianting

doi:10.1021/bi960400s

Cited by 42 publications

(62 citation statements)

References 29 publications

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“…This led to the previous conclusion that TM8 (along with TM7 C-terminal flanking residues) was oriented in the ER lumen, a result consistent with the lack of PK accessibility of the short cytosolic peptide segment connecting TM7b and TM8. Given the topological specificities of TM7a, TM7b, and TM8 demonstrated here, our previous data (as well as those of Zhang et al, 1996) are entirely consistent with the topology reported by Beja and Bibi (1995). However, it should be noted that the cooperative topogenic behavior of TM7b and TM8 is such that only a subset of chains, slightly less than half, exhibit this unconventional topology.…”

Section: Molecular Biology Of the Cell 2688supporting

confidence: 92%

“…Although the orientation of TM7b and TM8 in doubly spanning chains in Figure 4 is precisely the orientation reported by Beja and Bibi (1995) for murine Pgp expressed in E. coli, it differs from the topology previously proposed by us and Zhang et al (1996), in which it was assumed that TM7 spanned the membrane as a single TM segment (Zhang and Ling, 1991;. This led to the previous conclusion that TM8 (along with TM7 C-terminal flanking residues) was oriented in the ER lumen, a result consistent with the lack of PK accessibility of the short cytosolic peptide segment connecting TM7b and TM8.…”

Section: Molecular Biology Of the Cell 2688supporting

confidence: 53%

“…The topology of these chains therefore indicates that translocation termination by TM7b results in translocation reinitiation by the type II signal anchor activity of TM8. The inefficient ST activity of TM7b, coupled together with the signal anchor activity of TM8, therefore appears to be sufficient to generate topological heterogeneity previously observed for MDR1-Pgp.Although the orientation of TM7b and TM8 in doubly spanning chains in Figure 4 is precisely the orientation reported by Beja and Bibi (1995) for murine Pgp expressed in E. coli, it differs from the topology previously proposed by us and Zhang et al (1996), in which it was assumed that TM7 spanned the membrane as a single TM segment (Zhang and Ling, 1991;. This led to the previous conclusion that TM8 (along with TM7 C-terminal flanking residues) was oriented in the ER lumen, a result consistent with the lack of PK accessibility of the short cytosolic peptide segment connecting TM7b and TM8.…”

supporting

confidence: 53%

“…In this latter case, antibody mapping, cysteine labeling, and epitope insertion studies of functional Pgp at the plasma membrane of mammalian cells (Yoshimura et al, 1989;Georges et al, 1993;Schinkel et al, 1993;Loo and Clarke, 1995;Kast et al, 1996) have supported a conventional 12-spanning topology predicted by initial hydropathy analyses (Chen et al, 1986;Gros et al, 1986). In contrast, other studies of full-length, truncated, and chimeric Pgps expressed in mammalian, cell-free, Xenopus oocyte (XO) and prokaryotic expression systems (Zhang and Ling, 1991;Zhang et al, 1996;Beja and Bibi, 1995;Poloni et al, 1995) have found that a predicted cytosolic peptide loop between TM segments 8 and 9 appears to reside in an extracytosolic location. Moreover, at the ER membrane, this TM8 -9 loop appears to be extracytosolic in only a subpopulation (40 -50%) of chains, suggesting that in certain cellular compartments Pgp might be capable of achieving both conventional as well as unconventional topologies .…”

Section: Introductionmentioning

confidence: 94%

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Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

Moss

Helm

et al. 1998

MBoC

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Topogenic determinants that direct protein topology at the endoplasmic reticulum membrane usually function with high fidelity to establish a uniform topological orientation for any given polypeptide. Here we show, however, that through the coupling of sequential translocation events, native topogenic determinants are capable of generating two alternate transmembrane structures at the endoplasmic reticulum membrane. Using defined chimeric and epitope-tagged full-length proteins, we found that topogenic activities of two C-trans (type II) signal anchor sequences, encoded within the seventh and eighth transmembrane (TM) segments of human P-glycoprotein were directly coupled by an inefficient stop transfer (ST) sequence (TM7b) contained within the C-terminus half of TM7. Remarkably, these activities enabled TM7 to achieve both a single-and a doublespanning TM topology with nearly equal efficiency. In addition, ST and C-trans signal anchor activities encoded by TM8 were tightly linked to the weak ST activity, and hence topological fate, of TM7b. This interaction enabled TM8 to span the membrane in either a type I or a type II orientation. Pleiotropic structural features contributing to this unusual topogenic behavior included 1) a short, flexible peptide loop connecting TM7a and TM7b, 2) hydrophobic residues within TM7b, and 3) hydrophilic residues between TM7b and TM8. INTRODUCTIONTransmembrane (TM) 1 topology of most eukaryotic integral membrane proteins is established at the endoplasmic reticulum (ER) membrane as specific topogenic determinants (e.g., signal, stop transfer [ST], and signal anchor sequences) emerge from the ribosome and engage their cognate cytosolic and/or membrane bound receptors (Blobel, 1980). This process of topogenesis involves at least four distinct steps: 1) ER membrane targeting, 2) translocation initiation, 3) translocation termination, and 4) membrane integration (reviewed in Rapoport et al., 1996;Johnson, 1997). Topogenic determinants in naturally occurring proteins usually direct these events efficiently and completely, thus ensuring a single uniform topology for any given cohort of nascent polypeptide chains. Failure to complete one or more of these steps, however, may result in proteins with heterogeneous topological outcomes. For example, mutagenesis studies of ST (Kuroiwa et al., 1991) and signal anchor (Parks et al., 1989;Parks and Lamb, 1991) sequences have shown that under certain conditions, topogenic determinants may generate mixed populations of nascent chains exhibiting secretory, N-trans (type I) and/or C-trans (type II) TM topology. Specialized topogenic determinants in naturally occurring proteins may also direct alternate topologies. In the case of murine plasminogen activator inhibitor 2, cytosolic and secretory iso-* Corresponding author. Present address: Division of Molecular Medicine, Oregon Health Sciences University, Portland, OR 97201-3098. 1 Abbreviations used: ER, endoplasmic reticulum; MDR1-Pgp, human P-glycoprotein; Pgp, P-glycoprotein; PK, proteinase K; ST, s...

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Section: Molecular Biology Of the Cell 2688supporting

confidence: 92%

Section: Molecular Biology Of the Cell 2688supporting

confidence: 53%

supporting

confidence: 53%

Section: Introductionmentioning

confidence: 94%

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Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

Moss

Helm

et al. 1998

MBoC

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“…The generation and purification of fusion protein antigen and antibody AbD were performed as previously described (Zhang et al, 1996). Briefly, a cDNA fragment encoding the loop linking putative TM7 and TM8 of hamster Pgp (see Figure 1 for the sequence) was generated by PCR.…”

Section: Generation Of Polyclonal Antibody Abdmentioning

confidence: 99%

Identification of a 170-kDa protein over-expressed in lung cancers

2001

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Summary Lung cancer is the leading cause for cancer death in both male and female populations. Although many molecular markers for lung cancer have been developed and useful for early detection of lung cancer, their function remains unknown. In this paper, we report our findings that a 170-kDa protein (p170) is over-expressed in all types of human lung cancers compared with normal tissues and it is identified as a subunit of translation initiation factor eIF3 by cDNA cloning. Translation initiation factors are a family of proteins that promote the initiation step of protein synthesis and are regulators of cell growth at the translational level. Further studies showed that p170 mRNA is ubiquitously expressed with higher levels in adult proliferating tissues (e.g. bone marrow) and tissues during development (e.g. fetal tissues). This study suggests that p170 and eIF3 may be important factors for cell growth, development, and tumorigenesis.

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Human P-Glycoprotein Pseudoreceptor Modeling: 3D-QSAR Study on Thioxanthene Type Multidrug Resistance Modulators

Pajeva¹,

Wiese²

2001

Quant. Struct.-Act. Relat.

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P-glycoprotein (P-gp) is the main multidrug resistance (MDR) bioregulator responsible for the active outward transport of antitumor drugs in resistant tumor cells. The primary sequence of human P-gp is known and its lowresolution structure has recently been determined. Basing on hydropathy plots a secondary structure with 12 membrane spanning helixes is assumed. However, no direct correlation between any structural feature observed and primary structure of P-gp has yet been identified. Mutation and labeling studies suggest that P-gp interaction sites are located in the transmembrane domains (TMs) and TM6 and TM12 are actively involved in the interactions with many MDR modulators. The purpose of this study is structure-based fitting to the putative P-gp 3D receptor of TM6 and TM12 using pseudoreceptor modeling approach. It is based on the ''six plus six'' topology of P-gp with TM6 neighboring TM12. Data on 24 MDR ligands of thioxanthene and phenothiazine type and the program PrGen were used. A stepwise strategy was applied: (i) single domain models (TM6 or TM12) -to decide on the appropriate orientation of the ligands towards the single domain; (ii) combined domain models (TM6 and TM12) -to simulate the behavior of the ligands in the protein environment of interest; (iii) extended models -to validate the models. Training was done on thioxanthenes only and test -on thioxanthenes and phenothiazines. A number of pseudoreceptor models were derived. Scramble tests were performed with the extended models. The best model involved TM6 and TM12 acting together and yielding 1.6 factor of uncertainty in the binding constants of the test ligands. Among the mostly contributing AAs were those proven by mutation to influence P-gp functioning. TM12 was suggested to be involved through Gln990 in specific H-bond interactions with Phe983 and Gln990 being mostly contributing to the model. TM6 was suggested to perform hydrophobic and shape regulating functions through its bulky and hydrophobic AAs with Phe335 and Val338 mainly involved. As this is the first attempt to model the interaction between P-gp and MDR modulators, the obtained results have to be further proven on larger and structurally diverse data sets.

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

Cited by 42 publications

References 29 publications

Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

Identification of a 170-kDa protein over-expressed in lung cancers

Human P-Glycoprotein Pseudoreceptor Modeling: 3D-QSAR Study on Thioxanthene Type Multidrug Resistance Modulators

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