Polycystic kidney disease (PKD) is a systemic hereditary disease characterized by renal and hepatic cysts. There is no known cure for this disease and it results in end-stage renal failure in approximately 50% of affected individuals. Mutations in either the PKD1 or the PKD2 gene, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively, are seen in over 95% of PKD cases. An inability of PC1 or PC2 to function normally is highly correlated with cystogenesis in PKD. There are currently, however, serious deficiencies in the molecular-level descriptions of PC1 and PC2, and in understanding how alterations in these primary amino acid sequences affect three-dimensional structure, in vitro function and in vivo phenotypes. Our plan is to use a multi-disciplinary approach to significantly improve the description of PC2. We will conduct studies that fall along a structure-function-phenotype axis that will describe the consequences of PC2 mutation in PKD.
In Aim 1 we will concentrate on describing the role Ca2+ binding to the EF-hand motif in the C-terminal cytoplasmic region of PC2. This region is critical for normal PC2 Ca2+ channel function and is often truncated in PKD. We will determine the structure of the C-terminal cytoplasmic region of PC2 in the presence of Ca2+ using NMR techniques. This structure will allow us to rationally design mutations we expect to alter Ca2+ binding and therefore PC2 channel function. We will then investigate the functional relevance of this region to PC2 Ca2+ signaling by conducting single channel measurements of PC2 channels in bilayers and by monitoring intracellular Ca2+ changes after agonist addition.
In Aim 2 we will investigate the role of phosphorylation of PC2 at serine 812, a residue located between the EF-hand and coiled-coil domains of PC2 that has been shown to be important for channel regulation and is often disrupted in PKD. We will investigate the effect of phosphorylation mimics on Ca2+ binding, on the global structure of the PC2 C-terminal tail, and on the direct C- terminal tail mediated PC1-PC2 interaction. The functional consequences of phosphorylation will be studied using single channel techniques and imaging of fluorescent Ca2+-sensitive dyes in intact cells.
In Aim 3 we will investigate the functional consequences of the interactions described in aims 1 and 2, the roles of Ca2+ binding and phosphorylation of PC2. For wild-type and mutant PC2 we will examine flow induced changes in Ca2+ signals, in cells obtained by transfection and in cells obtained by isolation from transgenic mice. We will generate transgenic mice that express wild-type or mutant PC2 and examine the hallmarks of PKD, the right- left axis and cyst formation. The studies proposed will allow us to correlate atomic-level molecular structures of PC2 with biophysical functional analyses and PKD phenotypes in mice. Or results will significantly enhance our molecular, functional and physiological understanding of PC2 in normal kidney function and the consequences of their dysregulation in PKD.
The project aims to enhance our molecular, functional and physiological understanding of the two proteins most often mutated in polycystic kidney disease, polycystin-1 and polycystin-2. We will determine the structure of a region important for regulation of this complex, will conduct functional studies and will describe the effects of mutations in mice for both polycystin-2. The proposed studies will help us understand the role of polycystin-1 and polycystin-2 in normal kidneys and in those affected by polycystic kidney disease.
Showing the most recent 10 out of 11 publications
