Multi-drug resistance is a situation encountered in cancer patients in which the tumor becomes resistant to a variety of cytotoxic anti-cancer chemotheraputic agents. It often involves overexpression of P-glycoprotein (Pgp), a plasma membrane protein of 1280 amino acids, composed of two related halves, each of which contains six predicted transmembrane helices and one nucleotide binding site. There is firm evidence that Pgp acts in an ATP-dependent manner to exclude drugs and a wide range of other hydrophobic compounds from cells. The investigator's lab and others have established that Pgp displays substantial drug-stimulated ATPase activity, and the most widely considered current model is that Pgp is an ATP-driven drug efflux pump. The investigator has generated a Chinese hamster ovary cell line that over-expresses Pgp. Two defined systems for biochemical investigation of Pgp have been developed from these cells: 1) isolated plasma membanes which are considerably enriched in Pgp (up to 32 percent w/w) and 2) purified, reconstituted Pgp. A further system will be developed to allow combined mutagenic and biochemical analyses.
The aim of this proposal is to characterize the structure and function of the Pgp catalytic sites. Approaches include covalent labeling, specific insertion of tryptophans as fluorescent probes, mutational analyses, tests of the proposed catalytic cycle and direct structural analysis by scanning force microscopy. Basic knowledge of this kind will be invaluable in devising ways to disable P-glycoprotein in cells and overcome multidrig resistanmce in patients

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM050156-04
Application #
2022783
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1994-01-01
Project End
2000-12-31
Budget Start
1997-01-01
Budget End
1997-12-31
Support Year
4
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Rochester
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
208469486
City
Rochester
State
NY
Country
United States
Zip Code
14627
Lee, Jyh-Yeuan; Urbatsch, Ina L; Senior, Alan E et al. (2008) Nucleotide-induced structural changes in P-glycoprotein observed by electron microscopy. J Biol Chem 283:5769-79
Tombline, Gregory; Donnelly, David J; Holt, Jason J et al. (2006) Stimulation of P-glycoprotein ATPase by analogues of tetramethylrosamine: coupling of drug binding at the ""R"" site to the ATP hydrolysis transition state. Biochemistry 45:8034-47
Tombline, Gregory; Urbatsch, Ina L; Virk, Navneet et al. (2006) Expression, purification, and characterization of cysteine-free mouse P-glycoprotein. Arch Biochem Biophys 445:124-8
Tombline, Gregory; Senior, Alan E (2005) The occluded nucleotide conformation of p-glycoprotein. J Bioenerg Biomembr 37:497-500
Delannoy, Sabine; Urbatsch, Ina L; Tombline, Gregory et al. (2005) Nucleotide binding to the multidrug resistance P-glycoprotein as studied by ESR spectroscopy. Biochemistry 44:14010-9
Tombline, Gregory; Muharemagic, Alma; White, Lori Bartholomew et al. (2005) Involvement of the ""occluded nucleotide conformation"" of P-glycoprotein in the catalytic pathway. Biochemistry 44:12879-86
Tombline, Gregory; Bartholomew, Lori A; Urbatsch, Ina L et al. (2004) Combined mutation of catalytic glutamate residues in the two nucleotide binding domains of P-glycoprotein generates a conformation that binds ATP and ADP tightly. J Biol Chem 279:31212-20
Tombline, Gregory; Bartholomew, Lori A; Tyndall, Grace A et al. (2004) Properties of P-glycoprotein with mutations in the ""catalytic carboxylate"" glutamate residues. J Biol Chem 279:46518-26
Tombline, Gregory; Bartholomew, Lori; Gimi, Khursheed et al. (2004) Synergy between conserved ABC signature Ser residues in P-glycoprotein catalysis. J Biol Chem 279:5363-73
Urbatsch, Ina L; Tyndall, Grace A; Tombline, Gregory et al. (2003) P-glycoprotein catalytic mechanism: studies of the ADP-vanadate inhibited state. J Biol Chem 278:23171-9

Showing the most recent 10 out of 31 publications