Phosphorylation and regulation of murine mdr1b P -glycoprotein
Glavy, Joseph Stephen
MetadataShow full item record
The acquisition of the multidrug resistance (MDR) phenotype in tumor cell lines is associated with the overexpression of a 170,000 dalton plasma membrane glycoprotein, P-glycoprotein. It has been shown that P-glycoprotein functions as ATP-driven drug efflux pump with a broad specificity for hydrophobic compounds, thereby reducing intracellular drug levels in resistant cells. Although P-glycoprotein is phosphorylated in vivo, a physiological role for phosphorylation in transporter function has been difficult to document. All of the in vivo phosphorylation sites in murine mdr1b P-glycoprotein are contained within the highly charged linker region (amino acids 629--686) which connects the two homologous half modules of P-glycoprotein. The linker region can be divided into two major subdomains, an the acidic region (amino acids 631--658), and a basic region (amino acids 659--686). Through the use of tryptic phosphopeptide mapping, we have demonstrated exclusive acidic-directed phosphorylation in mdr1b P-glycoprotein isolated from a colchicine-selected J.774.2 cell line. These colchicine-selected cell demonstrated greater cross-resistance to non-selective agents than a vinblastine-selected J.774.2 cell previously shown to be predominantly phosphorylated in the basic subdomain of the linker region. Furthermore, a HeLa stably transfected with mdr 1b P-glycoprotein isolated by low level vinblastine selection, exhibited extensive phosphate incorporation into both the acidic and basic subdomain. These cells exhibited approximately cross-resistance to colchicine, vinblastine, Taxol, and adriamycin. To determine whether phosphorylation within the acidic subdomain of the linker region, alone or in combination with phosphorylation within the basic subdomain, was important for transporter function, P-glycoprotein mutants were constructed in which all potential phosphorylation in the acidic subdomain (S/T7A) or in the complete linker region (S/T11A) were eliminated. Immunocytochemistry showed that this mutant transporter was located at plasma membrane. Photoaffinty labeling studies using azidopine and 7' -BzDC-Taxol demonstrated that the S/T11A mutant mdr1b glycoprotein was able to bind both drugs. The mutant transporter exhibited cyclosporin A-inhibitable rhodamine 123 efflux properties. However, we were unable to create a HeLa cell line stably expressing the S/T11A mutated P-glycoprotein using RcCMV expression vector and sequential G-418 and vinblastine selections. Given that stable expression of both the wild type and basic-directed kinase site deficient P-glycoproteins was possible under the same selection conditions, this would suggest that elimination of all potential phosphorylation sites in the linker region caused subtle alterations to the functioning and/or specificity of the transporter.
- Theses and Dissertations