WORK RELEVANT TO CYSTIC FIBROSIS Although clearly a key to understanding why loss of CFTR expression leads to the severe pathology seen in cystic fibrosis, we still have an incomplete understanding of CFTR biosynthesis, trafficking, regulation and function, especially in native epithelial cells. Therefore we took an unbiased proteomic-based approach to identify proteins that associate with CFTR. Using a combination of approaches including multidimensional liquid chromatography coupled with MALDI-MS and tandem MS/MS, we have identified three novel CFTR-interacting proteins: protein phosphatase 2A (PP2A), filamin A (FLN-A) and sorting nexin 27 (SNX27). Our work indicates that PP2A binding (via its unique Bεregulatory subunit) regulates CFTR open probability at the cell surface (Thelin et al., 2005). In contrast, the association with FLN-A and SNX27 modulate aspects of CFTR trafficking in the endosomal system (Thelin et al., 2007;McDermot et al., in preparation). We further showed that a disease-causing mutation in the highly conserved amino terminus of CFTR (S13F CFTR) completely abrogated FLN binding in epithelial cells resulting in a mutant protein with a significantly shortened half-life in cells (Thelin et al, 2007). We are now carefully dissecting the role of each interacting protein in CFTR function and are examining more broadly their function in a variety of epithelial cells and tissues. Currently little is known about the mechanisms underlying CFTR internalization and recycling but our identification of SNX27, as a CFTR-interacting protein, may increase of understanding of how this process is regulated. We found that SNX27 localized within early and recycling endosomes through a mechanism requiring a functional PX domain, where it co-localized with internalized CFTR. Although SNX27 does not appear to effect the internalization of CFTR, interference of SNX27 function, via the expression of dominant-negative proteins or siRNA, caused a dramatic reduction of internalized CFTR recycling back to the cell surface. Our data provide further insight into the role of C-terminus of CFTR of in trafficking of the protein back to the cell surface and identify SNX27 as a novel regulator of this process. To further define the function of SNX27 - in regulating CFYR trafficking and in modulating other cellular functions, we used proteomic approaches to more fully define SNX27-interacting proteins. We found a large number of proteins that modulate the function of Rho and Rac GTPases, including a number of GTPAse-activating proteins and their regulators. We are carefully comparing the localization of SNX27 and these interacting proteins in a variety of epithelial cell model systems and are developing functional assays to fully explore the role of SNX27. WORK RELEVANT TO POLYCYSTIC KIDNEY DISEASE Autosomal Dominant Polycystic kidney disease (ADPKD) affects approximately 1 in 1000 individuals. The disease is characterized by the formation of multiple cysts on both kidneys leading to a decline in renal function and renal replacement therapy in 50% of individuals age 60 or more. Mutations in either of two transmembranous receptors Polycystin 1 and 2 are responsible for all patients manifesting the polycystic phenotype. Though a Polycystin 1-2 complex functions as an ion channel, Polycystin activation, signaling and cellular trafficking are poorly understood. We used a peptide-pulldown approach previously used in the lab to identify CFTR-interacting proteins, to identify novel PC-2-associated proteins. The most potent binding proteins to PC2-2 were found to be tubulin, FANCD2, Tel2 and GCN-1. We have since confirmed an interaction in vitro between tubulin and PC2-2, which is diminished when tubulin is incubated with a mutant PC-2 peptide (PC2-2 A190T). This mutation has been identified in a patient with ADPKD. We have also shown that the carboxy-tail of tubulin is responsible for the interaction with Polycystin-2. FANCD2 and TEL2 are proteins with known involvement in cell proliferation. A particular characteristic of Polycystic Kidney Disease is an increase in proliferation of tubular cells. How defects in Polycystin function lead to increased cell proliferation are not completely understood. Hence, identification of FANCD2 and TEL2 as potential binding partners of Polycystin-2 is of great interest. We have confirmed an interaction between endogenous FANCD2 and expressed Polycystin-2 in cells. Interaction between the two proteins is absent in cells where a truncated form of Polycystin-2 lacking the PC-2-2 sequence is expressed. Interaction with Polycystin-2 and Tel-2 remains to be confirmed. Over the coming months, interactions between Polycystin-2 and the four targets described above will be tested further recombinant proteins and in the appropriate cell types both endogenously where possible, and by expression of exogenous proteins. Analysis of the potential interaction with GCN-1 is in collaboration with Dr. Beatriz Castilho, University of Sao Paolo. Polycystin-2 has known localizations and functions in the cell cilium, plasma membrane, endoplasmic reticulum and mitotic spindles. We will address the localization of each potential Polycysin-2 interactor in a panel of kidney epithelial cells.
|Seabold, Gail K; Wang, Philip Y; Petralia, Ronald S et al. (2012) Dileucine and PDZ-binding motifs mediate synaptic adhesion-like molecule 1 (SALM1) trafficking in hippocampal neurons. J Biol Chem 287:4470-84|
|Gee, Stephen T; Milgram, Sharon L; Kramer, Kenneth L et al. (2011) Yes-associated protein 65 (YAP) expands neural progenitors and regulates Pax3 expression in the neural plate border zone. PLoS One 6:e20309|
|Garcia-Caballero, Agustin; Ishmael, Susan S; Dang, Yan et al. (2011) Activation of the epithelial sodium channel by the metalloprotease meprin * subunit. Channels (Austin) 5:14-22|
|Valdes, Julie L; Tang, Jingrong; McDermott, Mark I et al. (2011) Sorting nexin 27 protein regulates trafficking of a p21-activated kinase (PAK) interacting exchange factor (?-Pix)-G protein-coupled receptor kinase interacting protein (GIT) complex via a PDZ domain interaction. J Biol Chem 286:39403-16|
|Playford, Martin P; Nurminen, Elisa; Pentikäinen, Olli T et al. (2010) Cystic fibrosis transmembrane conductance regulator interacts with multiple immunoglobulin domains of filamin A. J Biol Chem 285:17156-65|
|Kesner, Barry A; Milgram, Sharon L; Temple, Brenda R S et al. (2010) Isoform divergence of the filamin family of proteins. Mol Biol Evol 27:283-95|
|Zachos, Nicholas C; Hodson, Caleb; Kovbasnjuk, Olga et al. (2008) Elevated intracellular calcium stimulates NHE3 activity by an IKEPP (NHERF4) dependent mechanism. Cell Physiol Biochem 22:693-704|