Signaling Complexes in Renal Epithelial Cells

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DESCRIPTION (provided by applicant): The hypothesis guiding these studies is that the character and specificity of cell signaling systems is determined by structural proteins that organize multiple signaling proteins into discrete complexes in the cell. Some components are common to many signaling systems, while others may be unique. These studies will focus on the interaction of the extracellular Ca-sensing receptor (CaR) and filamin (ABP-280), a cytoskeletal protein, in renal epithelial cells with emphasis on MTAL and INCO cells. The CaR is a G protein-coupled receptor that responds to Ca and other polycations to regulate PTH secretion by the parathyroid glands and Ca, NaCI, KCl, and H20 transport in the kidney. The CaR has unique signaling properties that are not fully explained by its activation of Gi- and Gq-dependent signaling pathways. In an effort to identify potential scaffolding proteins for the signaling systems controlled by the CaR and identify new signaling pathways through which it might act, we used the CaR intracellular C-terminus as "bait" to screen a human kidney cDNA library using the yeast two hybrid approach. We found that the CaR interacts with filamin (ABP-280), a 280 kDa actin-binding cytoskeletal protein. Filamin also binds to variety of other signaling molecules including SEKI (MKK4), caveolin, the dopamine 02 receptor, Rho GTPases, SMADs and a- and Beta integrins, suggesting that it can act as a scaffold. The goals of this application are to define the interaction of the CaR and filamin and to understand how this interaction affects the signaling functions of the CaR in renal epithelial cells. The specific aims are: Aim 1) Characterize the interaction of the C-terminus of the CaR with fliamin using the yeast two hybrid system and mammalian cells; Aim 2) Determine the functional significance of the CaR-filamin interaction by analysis of CaR-stimulated signals in cells that lack filamin and by specific disruption of CaR-filamin association; Aim 3) Establish the subcellular pattern of distribution of the CaR in renal epithelial cells and determine if the interaction of the CaR with filamin contributes to the localization of the CaR in renal epithelial cells; Aim 4) Identify the smallest portion of filamin that can reconstitute signaling by the CaR in filamin-null cells, and then identify additional proteins that interact with that fragment. These studies will test the hypothesis that the interaction of filamin and the CaR is important for appropriate cell signaling by the CaR and that filamin acts as a scaffolding protein to organize the signaling pathway(s) regulated by the CaR.