Cystin, a lipid raft and cilia-associated protein in PKD

Summary

Principal Investigator: Lisa M Guay-Woodford
Abstract: [unreadable] DESCRIPTION (provided by applicant): Primary cilia are dynamic, complex structures that contain >250 proteins, including several polycystic kidney disease (PKD)-related proteins. In renal epithelial cells, the primary apical cilium appears to be a major effector of differentiation signals and to play a critical role in PKD pathogenesis. Recent in vitro studies demonstrate that the primary cilium acts as a cellular sensor, transducing apical mechanical signals through a polycystin-1/polycystin-2-dependent Ca++ signaling pathway. However, the precise mechanisms involved in cilia formation, stabilization, and signal transduction are not well-defined and even less is known about how these cilia-associated proteins are targeted to cilia and functionally assembled. We have identified Cys1 as the disease-gene in cpk mice; demonstrated that its novel protein product, cystin, localizes to the primary apical cilium; and determined that cystin fractionates with lipid rafts through an N-terminal domain, probably the predicted N-myristoylation/ polybasic motif. We hypothesize that cystin traffics to the primary cilium via lipid raft-mediated mechanisms, associates with the cilial membrane, and serves as part of the molecular framework that stabilizes the microtubular scaffold of the ciliary axoneme. Using a suite of stably transfected cell lines that express wild-type cystin and various truncation mutants as GFP-tagged fusion proteins, we have determined that the N-terminal domain is necessary but not sufficient for targeting cystin to cilia and a second, novel signal is required. Since cystin is expressed at low levels and no functional assays currently exist, we have developed an innovative set of strategies to further characterize this novel protein and its intracellular trafficking itinerary as first steps toward defining its function. Specifically, in this proposal, we will: 1) Determine whether cystin tagged with green fluorescent protein (cystin-GFP) rescues the cpk phenotype and targets correctly to the primary cilium of renal epithelia in vivo; 2) Characterize cystin with respect to the predicted N-myristoylation site, putative cilia-targeting signals, and putative interacting partners; and 3) Examine the dynamics of cystin intracellular trafficking to the primary apical cilium. The central hypotheses underlying the proposed studies are that defects in primary cilia function impair the terminal phases of renal tubulo-epithelial differentiation and the epithelial response to this developmental arrest is cyst formation. Therefore, primary apical cilium represents a new focal point for dissecting the complex mechanisms involved in renal cystic disease and ultimately, perhaps a new target for therapeutic interventions. [unreadable] [unreadable] [unreadable] [unreadable]
Funding Period: 2000-03-15 - 2010-06-30
more information: NIH RePORT

Top Publications

  1. ncbi Overexpression of innate immune response genes in a model of recessive polycystic kidney disease
    M Mrug
    Division of Genetic and Translational Medicine, Department of Medicine, The University of Alabama at Birmingham, 720 20th Street South, Birmingham, AL 35294, USA
    Kidney Int 73:63-76. 2008
  2. pmc Cystin localizes to primary cilia via membrane microdomains and a targeting motif
    Binli Tao
    Departments of Medicine and Genetics, University of Alabama at Birmingham, 720 20th Street South, Birmingham, AL 35294, USA
    J Am Soc Nephrol 20:2570-80. 2009
  3. pmc Telomerase immortalization of principal cells from mouse collecting duct
    Stacy L Steele
    Ralph H Johnson Veterans Affairs Medical Center, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29403, USA
    Am J Physiol Renal Physiol 299:F1507-14. 2010
  4. ncbi A classification of ductal plate malformations based on distinct pathogenic mechanisms of biliary dysmorphogenesis
    Peggy Raynaud
    de Duve Institute, Universite Catholique de Louvain, Brussels, Belgium
    Hepatology 53:1959-66. 2011

Detail Information

Publications4

  1. ncbi Overexpression of innate immune response genes in a model of recessive polycystic kidney disease
    M Mrug
    Division of Genetic and Translational Medicine, Department of Medicine, The University of Alabama at Birmingham, 720 20th Street South, Birmingham, AL 35294, USA
    Kidney Int 73:63-76. 2008
    ..We also found elevated complement component 3 activation in two other mouse-recessive models and human-recessive PKD. Our results suggest that abnormal complement component 3 activation is a key element of progression in PKD...
  2. pmc Cystin localizes to primary cilia via membrane microdomains and a targeting motif
    Binli Tao
    Departments of Medicine and Genetics, University of Alabama at Birmingham, 720 20th Street South, Birmingham, AL 35294, USA
    J Am Soc Nephrol 20:2570-80. 2009
    ..Analysis of deletion and chimeric constructs identified an AxEGG motif that is necessary to target and retain cystin in the cilium. Derangement of these localization motifs may lead to cystic kidney disease...
  3. pmc Telomerase immortalization of principal cells from mouse collecting duct
    Stacy L Steele
    Ralph H Johnson Veterans Affairs Medical Center, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29403, USA
    Am J Physiol Renal Physiol 299:F1507-14. 2010
    ..This technique should be valuable in generating cell lines from genetically engineered mouse models...
  4. ncbi A classification of ductal plate malformations based on distinct pathogenic mechanisms of biliary dysmorphogenesis
    Peggy Raynaud
    de Duve Institute, Universite Catholique de Louvain, Brussels, Belgium
    Hepatology 53:1959-66. 2011
    ..In HNF1β-deficient liver, maturation of the primitive ductal structures was impaired. Normal differentiation and maturation but abnormal duct expansion was apparent in cpk mouse livers and in human fetal ARPKD...