The long range objectives of this project are to understand the fundamental mechanisms by which a membrane glycoprotein matures and folds into a functional three dimensional structure. Significantly, alteration of these processes is central to the development of several human pathologies. The proposed studies focus on the 1480 amino acid membrane glycoprotein CFTR. (cystic fibrosis transmembrane conductance regulator). Greater than 90% of all cystic fibrosis (CF) patients lack a single phenylalanine residue at position 508 in CFTR. The deltaF5O8 mutation of CFTR leads to altered thermodynamic stability of a peptide fragment of CFTR in vitro and decreased amounts of the mutant protein in the apical membrane of CF epithelial cells. Thus, description of the folding of CFTR will not only provide information essential for understanding the molecular pathogenesis of this disease, but may offer a paradigm for understanding the development of membrane glycoprotein structure.
The specific aims of the proposal are: 1. DETERMINE THE FOLDING THERMODYNAMICS AND KINETlCS OF WILD TYPE AND MUTANT FORMS OF CFTR NBD1. Spectroscopic methods for monitoring folding of the CFTR model peptides we developed will be used to test the hypothesis that the deltaF508 mutation alters the folding pathway rather than the final native state stability of CFTR. 2. EVALUATE THE ROLE OF CHAPERONE PROTEINS IN CFTR FOLDING IN VIVO. High stringency immunoprecipitations in our laboratory show that CFTR interacts with a specific subset of proteins, some of which may be molecular chaperones. Using this method and a yeast two-hybrid screen we will identify candidate chaperones and test the hypothesis that differential interaction with chaperones is responsible for retention of the deltaF508- CFTR in the endoplasmic reticulum. 3. CHARACTERIZE THE SITES OF INTERACTION OF THESE PROTEINS WITH CFTR. Preliminary experiments presented here demonstrate that a CFTR peptide binds to Hsp70 in vitro. Using this biochemical approach and the two- hybrid method, we will identify and characterize chaperone sites in CFTR and test the hypothesis that cystic fibrosis mutations and suppressor mutations may affect chaperone binding sites. 4. ASSESS THE EFFECT OF ALTERED CHAPERONE AND LIGAND LEVELS ON CFTR FOLDING. Using these systems we will test the hypothesis that alteration of chaperone and/or ligand levels may permit transit of mutant forms of CFTR to the plasma membrane in functional form. The proposed studies are both necessary and fundamental to understanding the development of membrane protein structure, and may provide novel information relevant to several human diseases.
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