Surface Mapping of the Ligand-Filled C-Terminal Half of the Porcine Estradiol Receptor by Restricted Proteolysis

Both the overexpression of P-glycoprotein and the broad range of substrates of this ATP-binding cassette (ABC) transporter induce the phenomenon of multidrug resistance, one major cause of the failure of cancer chemotherapy in humans. This study reports that [ 125 I]iodipine, a structural analogue of the 1,4-dihydropyridine azidopine, shares a common binding site with iodomycin, a Bolton±Hunter derivative of the anthracycline daunomycin. This binding site is different from that described for iodoarylazidoprazosin, which is presumed to share a common binding site with azidopine. Edman sequencing revealed that [ 125 I]iodipine had photolabelled the same peptide as iodomycin and spans the primary sequence of hamster isoform pgp1 from amino acid 230 to amino acid 312.Keywords: ABC transporter; P-glycoprotein; photoaffinity labeling.The overexpression of P-glycoprotein is thought to be one reason for the failure of human cancer chemotherapy. P-glycoprotein confers resistance to a broad spectrum of structurally and chemically unrelated compounds by reducing the intracellular concentration of drugs [1]. This phenomenon of multidrug resistance (MDR) can be overcome by so-called modulators such as verapamil, cyclosporins and dihydropyridine calcium channel blockers, which resensitize the tumour cells by blocking the drug exclusion function of P-glycoprotein [2]. P-glycoprotein belongs to the protein superfamily of ATP-binding cassette (ABC) transporters. Predicted by hydropathy plot analysis, the 170-kDa large plasma membrane protein is made up of 12 transmembrane domains and two cytosolic nucleotide-binding folds [3]. A central issue of P-glycoprotein research is the question of how this protein is able to recognize and transport so many unrelated compounds. [14]. Azidopine photolabels two sites on P-glycoprotein, one in the N-terminal half between amino acid residues 198 and 440 and the other in the C-terminal half [10]. The binding site for azidopine was not analysed further (Fig. 5), but it is presumed that azidopine and [ 125 I]iodoarylazidoprazosin share a common binding site on P-glycoprotein [15].Another photoreactive substrate of P-glycoprotein is iodomycin, a photoreactive Bolton±Hunter derivative of the anthracycline daunomycin [16]. Recently we were able to localize by direct amino acid sequence analysis the [ 125 I]iodomycin-binding site of hamster isoform pgp1 [13] N-terminal in a segment of the primary sequence between amino acid 230 (TM4) and amino acid 312 (TM5).The 1,4-dihydropyridine derivative iodipine, known to be a modulator of MDR [17], was used at an early stage to characterize L-type Ca 2+ channels [18]. The photobinding of the inherently photoreactive iodipine to P-glycoprotein from Chinese hamster ovary (CHO) B30 plasma-membrane vesicles was demonstrated by . Iodipine shares the Bolton±Hunter group with iodomycin but otherwise has a structure very similar to that of azidopine (Fig. 1), for which reason the latter has been extensively employed as a reference compound in studies of drug binding ...

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“…Sequence analysis was carried out with the Applied Biosystems model 477A apparatus as described by Thole et al . [25].…”

Section: Methodsmentioning

confidence: 99%

Iodomycin and iodipine, a structural analogue of azidopine, bind to a common domain in hamster P‐glycoprotein

Demmer

Andreae

Thole

et al. 1999

European Journal of Biochemistry

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“…For sequencing of the cleavage products, recombinant Maxp22 was incubated with caspase-5 or caspase-7 at 30 mC for 15 h. The cleavage products were processed and sequenced on an Applied Biosystems 477A protein sequencer with a 120A online HPLC system [30] or fractionated by reverse-phase HPLC and analysed as described previously [31].…”

Section: Caspase Cleavage Assays In Vitro and Peptide Sequencingmentioning

confidence: 99%

Targeting of the transcription factor Max during apoptosis: phosphorylation-regulated cleavage by caspase-5 at an unusual glutamic acid residue in position P1

Krippner‐Heidenreich

Talanian

Sekul³

et al. 2001

Biochem. J.

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Max is the central component of the Myc/Max/Mad network of transcription factors that regulate growth, differentiation and apoptosis. Whereas the Myc and Mad genes and proteins are highly regulated, Max expression is constitutive and no post-translational regulation is known. We have found that Max is targeted during Fas-induced apoptosis. Max is first dephosphorylated and subsequently cleaved by caspases. Two specific cleavage sites for caspases in Max were identified, one at IEVE(10) decreasing S and one at SAFD(135) decreasing G near the C-terminus, which are cleaved in vitro by caspase-5 and caspase-7 respectively. Mutational analysis indicates that both sites are also used in vivo. Thus Max represents the first caspase-5 substrate. The unusual cleavage after a glutamic acid residue is observed only with full-length, DNA-binding competent Max protein but not with corresponding peptides, suggesting that structural determinants might be important for this activity. Furthermore, cleavage by caspase-5 is inhibited by the protein kinase CK2-mediated phosphorylation of Max at Ser-11, a previously mapped phosphorylation site in vivo. These findings suggest that Fas-mediated dephosphorylation of Max is required for cleavage by caspase-5. The modifications that occur on Max in response to Fas signalling affect the DNA-binding activity of Max/Max homodimers. Taken together, our findings uncover three distinct processes, namely dephosphorylation and cleavage by caspase-5 and caspase-7, that target Max during Fas-mediated apoptosis, suggesting the regulation of the Myc/Max/Mad network through its central component.

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“…Sequence analysis was carried out with the Applied Biosystems model 477A apparatus as described by Thole et al [26].…”

Section: Edman Sequence Analysismentioning

confidence: 99%

Identification and localization of three photobinding sites of iodoarylazidoprazosin in hamster P‐glycoprotein

Isenberg

Thole

Tümmler

et al. 2001

European Journal of Biochemistry

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P-glycoprotein is an ATP-dependent drug-efflux pump which can transport a diverse range of structurally and functionally unrelated substrates across the plasma membrane. Overexpression of this protein may result in multidrug resistance and is a major cause of the failure of cancer chemotherapy. The most commonly used photoreactive substrate is iodoarylazidoprazosin. Its binding domains within the P-glycoprotein have so far been inferred from indirect methods such as epitope mapping. In this study, the binding sites were refined and relocalized by direct analysis of photolabeled peptides. P-glycoproteincontaining plasma membrane vesicles of Chinese hamster ovary B30 cells were photoaffinity-labeled with iodoarylazidoprazosin. After chemical cleavage behind tryptophan residues or enzymatic cleavage behind lysine residues, the resulting 125 I-labeled peptides were separated by tricine/ PAGE and HPLC and subjected to Edman sequencing. The major photoaffinity binding sites of iodoarylazidoprazosin were localized in the amino-acid regions 248±312 [transmembrane segment (TM)4 to TM5], 758±800 (beyond TM7 to beyond TM8) and 1160±1218 (after the Walker A motif of the second nucleotide-binding domain). Therefore the binding pocket of iodoarylazidoprazosin is made up of at least three binding epitopes.Keywords: ABC transporter; multidrug resistance; P-glycoprotein; photoaffinity labeling.Chemotherapy is one of the three major options for the treatment of cancer. However, after the initial treatment with cytostatic drugs, tumor cells can become resistant to a broad range of structurally different drugs. Such resistance during chemotherapy is known as multidrug resistance (MDR). MDR is mainly based on overexpression of P-glycoprotein, an ATP-binding cassette (ABC) transporter. P-glycoprotein actively transports many amphiphilic cytostatic drugs out of cancer cells (causing resistance against these drugs) [1±4].The large 1276 amino-acid plasma membrane protein P-glycoprotein is made up of 12 transmembrane segments (TMs) and two nucleotide-binding domains (NBDs) [3]. The major issue of how P-glycoprotein can handle so many substances has been mainly approached by photoaffinity labeling and mutagenesis studies [5,6]. Active mutagenesis identified numerous motifs and positions in the transmembrane domains and both nucleotide-binding folds which are directly or indirectly involved in drug binding [7]. These data demonstrate the complexity of the 3D structure of the substrate-binding pocket of P-glycoprotein. Moreover, binding and transport of the drug were found to be coupled to ATP hydrolysis [8] Epitope mapping revealed two major photobinding sites for the prazosin derivative [ 125 I]iodoarylazidoprazosin in each half of the P-glycoprotein (TM6 and TM12) and one minor photobinding site from TM4 up to, but not including, TM6 [15]. The minor photolabeling site overlaps that found for its photoreactive P-glycoprotein substrates iodipine and iodomycin [10,11], the second cytoplasmic loop between TM4 and TM5. Drug-transport studies ...

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Surface Mapping of the Ligand-Filled C-Terminal Half of the Porcine Estradiol Receptor by Restricted Proteolysis

Cited by 16 publications

References 31 publications

Iodomycin and iodipine, a structural analogue of azidopine, bind to a common domain in hamster P‐glycoprotein

Iodomycin and iodipine, a structural analogue of azidopine, bind to a common domain in hamster P‐glycoprotein

Targeting of the transcription factor Max during apoptosis: phosphorylation-regulated cleavage by caspase-5 at an unusual glutamic acid residue in position P1

Identification and localization of three photobinding sites of iodoarylazidoprazosin in hamster P‐glycoprotein

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