The mechanisms of biogenesis and intracellular trafficking of complex integral transmembrane proteins are diverse, representing a numberous variations on common themes. The steps in this process occur at the site and time of synthesis, as well as subsequently, in the endoplasmic reticulum and beyond. Study of the biogenesis of several integral membrane proteins to date has uncovered unexpected features, some of which have important implications for structure, topology and function. The human multi-drug resistance P-glycoprotein (MDR-1) serves as an ATP-dependent transporter of chemotherapeutic agents out of cells. It has been proposed to span the bilayer twelve times and is structurally related to a number of other biologically important proteins including the cystic fibrosis transmembrane regulator (CFTR) and the hepatocyte bile canalicular anion transporter. Yet, little is known about the biogenesis and aquisition of functional competence of any member of this family of proteins. In the proposed work, MDR1 will be studied as a proptotype of the P-glycoprotein family. MDR1 will be expressed in both cell-free and whole cell systems and its intracellular trafficking studied. Assays will be developed with which to follow its functional maturation and determine where and when during its biogenesis functional maturation occurs. Through the use of protein chimeras, the sequences responsible for MDR1 biogenesis will be dissected and contrasted to topogenic sequences from simpler proteins as well as from other members of the P-glycoprotein superfamily. From this work a detailed understanding of the steps by which MDR1 achieves functional competence will emerge. A system will have been established by which newly synthesized MDR-1 can be manipulated in hope of understanding its physiologic function and altering its pharmacologic properties. Moreover, these studies will provide insight into the relationship of MDR-1 to other members of the P-glycoprotein superfamily.
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