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The MRP gene associated with a non-P-glycoprotein multidrug resistance encodes a 190-kDa membrane bound glycoprotein
1Division of Biology, Kansas State University, Manhattan 66506.
Abstract:
HL60 cells isolated for resistance to Adriamycin (HL60/ADR) overexpress a 190-kDa ATP binding protein which has a minor sequence homology with P-glycoprotein. It has also been observed that HL60/ADR overexpress the MRP gene which was first identified as a component of a non-P-glycoprotein mediated multidrug resistance of H69/ADR cells [Cole et al., Science (Washington DC), 258: 1650, 1992]. A complementary DNA of MRP has been cloned and based on the deduced sequence encodes a member of the superfamily of proteins which bind ATP and function in various transport processes [Cole et al., Science (Washington DC), 258: 1650, 1992]. In view of this it was of interest to identify the protein encoded by MRP and determine if it may be related to p190. In the present study we have prepared antisera against three synthetic peptides which correspond to the deduced sequence of the MRP protein. Proteins reactive with the antisera have been examined in HL60/ADR cells using Western blot analysis. All antisera react with a 190 kDa protein contained in membranes of resistant but not sensitive cells. One antiserum used for further studies is not reactive with P-glycoprotein contained in membranes of HL60 cells isolated for resistance to vincristine. Analysis of subcellular fractions demonstrates that p190 is present primarily in the endoplasmic reticulum with lower levels also present in plasma membranes. Treatment of HL60/ADR cells with tunicamycin results in the appearance of a 165-kDa resistance associated protein which reacts with the antipeptide serum. The results of this study therefore demonstrate that the MRP gene encodes a 190-kDa membrane bound glycoprotein.
Insights
The multidrug resistance-associated protein (MRP) gene encodes a 190-kDa membrane glycoprotein found in Adriamycin-resistant HL60 cells. This protein is distinct from P-glycoprotein and is primarily located in the endoplasmic reticulum and plasma membranes.
Area of Science:
- Molecular biology
- Cell biology
- Biochemistry
Background:
- HL60/ADR cells exhibit multidrug resistance and overexpress a 190-kDa ATP-binding protein with partial homology to P-glycoprotein.
- The multidrug resistance-associated protein (MRP) gene, identified in H69/ADR cells, encodes a protein belonging to the ATP-binding cassette superfamily involved in transport processes.
Purpose of the Study:
- To identify the protein encoded by the MRP gene.
- To determine if the MRP-encoded protein is related to the p190 protein observed in HL60/ADR cells.
Main Methods:
- Antisera were generated against synthetic peptides of the MRP protein sequence.
- Western blot analysis was performed on HL60/ADR cell membranes using the generated antisera.
- Subcellular fractions were analyzed to determine the localization of the p190 protein.
- Tunicamycin treatment was used to assess the glycosylation state of the resistance-associated protein.
Main Results:
- All antisera recognized a 190-kDa protein in membranes of resistant HL60/ADR cells, but not in sensitive cells.
- The identified 190-kDa protein was distinct from P-glycoprotein found in vincristine-resistant HL60 cells.
- The p190 protein was localized predominantly in the endoplasmic reticulum and also in plasma membranes.
- Tunicamycin treatment yielded a 165-kDa protein, indicating the 190-kDa protein is a glycoprotein.
Conclusions:
- The MRP gene encodes a 190-kDa membrane-bound glycoprotein.
- This MRP-encoded glycoprotein is responsible for a component of Adriamycin resistance in HL60 cells.
- The identified protein is distinct from P-glycoprotein, highlighting a different mechanism of multidrug resistance.