The long-term goal of this project is to define how protein phosphorylation contributes to the regulation of intestinal ion transport and the pathogenesis of secretory diarrhea. This project will focus on CFTR, the protein product of the gene responsible for cystic fibrosis (CF), because this protein may play a key role in the cAMP-mediated control of intestinal C1-secretion. These studies are based on the hypothesis that CFTR may function in the enterocyte as the C1-channel that is activated in diarrheal diseases associated with protein kinase A (PKA) activation (e.g., cholera) and with protein phosphatase inhibition (e.g., diarrhetic shellfish poisoning).
Four aims will be addressed:
Aim #1 will study the pathophysiologic significance of CFTR in intestinal tissues by defining the localization of CFTR in enterocytes using anti-peptide antibodies developed in this laboratory. The T84 colonocyte cell line will be used to validate Western blot and cell staining procedures for the detection of CFTR. These studies will particularly focus on cells known to contain large amounts of CFTR mRNA (e.g., Brunner glands) or to exhibit prominent cAMP-dependent C1- secretion (e.g., colonic crypt cells) Aim #2 will identify physiologically important phosphorylation sites in CFTR. CFTR will be immunoprecipitated from 32P(i)-labeled T84 cells and analyzed by phosphopeptide mapping. Phosphopeptide maps will be analyzed using bacterially synthesized CFTR peptides that have been phosphorylated at known sites by PKA or PKC. These experiments will determine which of these CFTR sites are phosphorylated by activated protein kinases in intact T84 cells.
Aim #3 will study how phosphorylation affects CFTR function in intact T84 cells. Whole-cell C1- conductance responses will be measured in cells perfused with CFTR antibodies and with bacterially expressed CFTR peptides that contain phosphoserine at sites corresponding to the serine residue labeled by PKA and by PKC in intact cells. C1- conductance responses of these perfused cells will be compared with responses in cells perfused with protein phosphatase and protein kinase inhibitors.
Aim #4 will determine whether the most common mutation in CFTR (deltaF508) affects CFTR phosphorylation in intact cells. This will be studied by comparing the effect of forskolin on phosphopeptide maps of CFTR in (32)P(i)-labeled airway epithelial cell lines that endogenously express deltaF508- vs. wt-CFTR. If these cell lines express insufficient CFTR for phosphopeptide mapping, then transfected cell lines expressing deltaF508-vs. wt-CFTR will be tested. In summary, these studies will provide information about the regulation and function of CFTR in the intestinal epithelial cell, and may also explain how CFTR mutations result in the ion transport defect characteristic of CF. This information has the potential to lead to new approaches to treating CF, secretory diarrhea, and other diseases in which CFTR function may be altered.
