BIOGENESIS AND FUNCTIONAL RECONSTITUTION OF HUMAN MDR1

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.