Cystic fibrosis (CF) is the most common lethal genetic disease among caucasians. The gene which is defective in cystic fibrosis has been identified, and the function of the gene product has been shown to be associated with epithelial C1- transport. However, there are many features of the predicted CF gene product (termed cystic fibrosis transmembrane conductance regulator, CFTR) which are poorly understood. Based upon homology with known genes and their gene products, CFTR is predicted to contain two domains capable of nucleotide binding and hydrolysis, as well as phosphorylation sites for protein kinase A, and is therefore felt to be important in normal CAMP mediated activation of epithelial C1- channels.
The Specific Aims of this project are to use recombinant DNA techniques and standard chromatographic methods to isolate preparative amounts of CFTR polypeptides from recombinant and non-recombinant sources. Studies of nucleotide binding, ATP hydrolysis, phosphorylaiton, cellular processing and cellular localization, as well as the relationship of these processes to C1- transport will be investigated using these polypeptides. The same methods will be applied to mutant CFTR proteins containing many clinically relevant gene defects, and to CF protein containing mutations which have been designed to disrupt nucleotide binding, hydrolysis and other properties, as a means of characterizing these functions. The long term objective of this application is to identify and characterize biochemical features of CFTR. Our understanding of the disease will be enhanced by this work, both in terms of the ways in which defects in the CF gene product cause clinical CF, at a level of investigation proximal to the C1- transport phenotype. Development of rational approaches to therapy of the disease may also be aided by improved understanding of the biochemical basis of CFTR dysfunction.
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