| The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized primarily at the apical surfaces of epithelial cells lining the airway, gut, and exocrine glands. It has been argued that the active form of CFTR Cl- channel is a PDZ-dependent multimer. In the first part of this dissertation, we demonstrate that although CFTR in vitro can self-associate into multimers, which depends on PDZ-based interactions; this may not be the case in cell membrane. Using chemical cross-linking, we demonstrate that CFTR exists as a higher order complex in cell membrane. However, this higher order complex is predominantly CFTR dimers, and the PDZ-based interacting partners (NHERF1 and NHERF2) constitute ∼2% of this complex. Solubilizing membrane expressing CFTR in detergents tends to destabilize the CFTR dimers and dissociate them into monomeric form. The dimerization of CFTR is regulated by PKA-dependent phosphorylation and does not depend on the active form of the channel. In addition, the dimerization is not influenced by either the PDZ motif or its interacting partners (NHERF1 and NHERF2). We also demonstrate that other signaling-related proteins (i.e., Gbeta, syntaxin 1A) can be in this CFTR-containing complex as well. These findings from our study provide a deeper understanding of how the CFTR assembly takes place in native cell membrane.; It is well established that CFTR plays a pivotal role in cholera toxin-induced diarrhea. Lysophosphatidic acid (LPA), a naturally occurring phospholipid present in blood and foods, has been reported to play a vital role in a variety of conditions involving GI wound repair, apoptosis, inflammatory bowel disease, and diarrhea. In the second part of this dissertation, we demonstrate that type-2 LPA receptors (LPA2) were expressed at the apical surface of intestinal epithelial cells, where they formed a macromolecular complex with NHERF2 and CFTR. LPA inhibited CFTR-mediated Cl- currents in cultured cells and excised mouse intestinal epithelia. CFTR-dependent intestinal fluid secretion induced by cholera toxin in mice was significantly reduced by LPA administration, and disruption of this complex using an LPA 2-specific peptide reversed LPA2-mediated CFTR inhibition. Thus, LPA-rich foods may represent an alternative method of treating certain forms of diarrhea. |
