The objectives of this proposal are to complete some very straightforward studies which should greatly facilitate our understanding of both the molecular and chemical basis of cystic fibrosis (CF). The proposed studies will focus on the CFTR protein (cystic fibrosis transmembrane conductance regulator), predicted from cDNA sequence analysis to be 1480 amino acids In length. The predicted protein Includes both a large membrane spanning region and a large cytoplasmic domain, the latter consisting of two putative ATP binding regions (ATP-1, and ATP-11) and a putative regulatory region (R) thought to be a protein kinase target. Significantly, almost 70% of CF patients lack a phenylalanine in the center of the ATP-1 region. Consequently, it has been suggested that the wild type CFTR may be an 'ion motive' ATPase which couples ATP hydrolysis to Cl- transport, and that the phenylalanine deletion mutation may alter the binding or hydrolysis of ATP and, therefore, Cl- transport. With these thoughts in mind, the Specific Aims of this proposal are sixfold: 1.Prepare In large amounts, using both chemical and molecular biological approaches, the two cytoplasmic regions, ATP-1 and ATP-11, of the 'wild type"""""""" CFTR protein. 2.Characterize the resultant peptides physically, and assess their capacity to bind and hydrolyze ATP In the presence and absence of Cl-. 3.Prepare as in '1' the ATP-1 region containing the single phenylalanine deletion found in most cystic fibrosis patients. 4.Characterize physically the ATP-1 phenylalanine mutant peptide and assess its capacity to bind and hydrolyze ATP in the presence and absence of Cl-. 5.Prepare, as in """"""""l"""""""", a """"""""wild type' and a phenylalanine mutant peptide containing both the regulatory region 'R' and the ATP-1 region, and compare these peptides' capacity to bind and hydrolyze ATP before and after treatment with protein kinases A or C. 6.Assess the effect of antibodies to the ATP-1, ATP-11, and R + ATP-1 regions on the ATPase activity of epithelial cell membranes. The proposed studies are fundamental for understanding the molecular and chemical basis of cystic fibrosis, and may encourage future experiments directed at replacing only a small portion of the """"""""wild type"""""""" CFTR gene in CF patients.
