Abstracts Biological Sciences

Studies of P-glycoprotein intracellular domains by cysteine scanning mutagenesis

by Marc-Etienne. Rousseau

Interactions between the various intracellular loops and nucleotide binding domains (NBDs) of P-glycoprotein (P-gp), and the extent to which they contribute to protein structure and transport mechanism are widely unknown. Analogy to bacterial members of the ABC transporter family suggests that the nucleotide binding domains interact with a specific site on an intracellular loop and with each other, as a cooperative dimer, in order to energize the transport functions. To investigate this hypothesis, we have used a cysteine scanning mutagenesis strategy on four potentially interacting regions of P-gp. We have analyzed the biological activity of the different mouse P-gp cysteine mutants in a yeast heterologous system. We established that the biological activity of the human MDR1 and MDR1-cysteine-less proteins can also be monitored by the yeast system. We also engineered and tested a MDR1 cysteine-less protein containing a factor Xa protease recognition site located in the third extracellular loop as a tool for future studies. Finally, analysis of the biological activity of the substitution mutants reveals key residues in regions that may be involved in drug binding and/or intramolecular domain interactions.