Characterization of mouse polycystic kidney disease and receptor for egg jelly gene and protein in heterologous and native system

The mammalian polycystic kidney disease (PKD) gene family comprises eight members whose role in cell physiology is still poorly understood. Two of the founding members of the family, PKD1 and PKD2, are responsible for the majority of cases of autosomal dominant polycystic kidney disease. While PKD1...

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Bibliographic Details
Main Author: Butscheid, Yulia
Contributors: Gudermann, Thomas (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2006
Pharmakologisches Institut
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Summary:The mammalian polycystic kidney disease (PKD) gene family comprises eight members whose role in cell physiology is still poorly understood. Two of the founding members of the family, PKD1 and PKD2, are responsible for the majority of cases of autosomal dominant polycystic kidney disease. While PKD1 is considered to be a cell-surface receptor, PKD2 functions as a cation channel and has been described both at the plasma membrane and intracellularly. The present study focuses on PKDREJ – polycystic kidney disease and receptor for egg jelly, a protein that contains both a large receptor-like extracellular part and a putative ion channel region. PKDREJ homology to suREJ proteins, which seem to play a role in the induction of the sperm acrosome reaction in sea urchin, gives a reason to propose the same scenario for PKDREJ in mammals. Although the PKDREJ gene was identified in 1999, its features and protein characteristics remained elusive up to now. The present study investigates mouse PKDREJ gene and protein and provides the first data about its structure, expression and localization. It demonstrates that PKDREJ is highly expressed in testicular tissue and its expression there is restricted to germ cells and spermatozoa. Upon heterologous expression in HEK 293, COS7, GC-1, GC-2 cells and Xenopus laevis oocytes PKDREJ is retained intracellularly in endoplasmatic compartments. Since many known receptors and ion channels can not be properly trafficked without special partner or adaptor proteins, while expressed in a heterologous system, co-expression experiments with mouse HspA2, PKD2L2 and human PKD1 and PKD2 were done. In all cases PKDREJ localization remained intracellular irrespective of whether it was co-expressed with other proteins or not. The presence of the GPS domain in the PKDREJ amino acid structure gave a reason to speculate about the proteolytical cleavage within this site. The present work provides not only the biochemical characterisation of the PKDREJ protein, but also the first evidence about lack of the GPS processing. It shows that PKDREJ unlike PKD1 or suREJ3 does not undergo cleavage within G-protein-coupled receptor proteolytic site while being expressed in HEK293 cells or Xenopus laevis oocytes. Moreover, the explanation of this phenomenon, based on the structure of the GPS domain in PKDREJ and the autocatalytic mechanism of the cleavage, is proposed. To investigate the PKDREJ protein in native system PKDREJ-specific polyclonal antibodies were developed and characterised. By means of indirect immunofluorescence it was possible to demonstrate PKDREJ localization in the acrosomal region and on the inner aspect of the falciform-shaped mouse sperm head. Additional immunoelectron microscopy with mouse sperm revealed the plasma membrane localization of the PKDREJ protein and confirmed the predicted protein topology with the N-terminus located extracellularly. To examine the functional relevance of PKDREJ in terms of the induction of the sperm acrosome reaction capacitated mouse sperm was incubated with antibodies directed against the PKDREJ N-terminus. We have not observed antibody-induced Ca2+ influx or a significant increase in the fraction of acrosome-reacted sperm. Also, anti-PKDREJ antibodies failed to block ZP-induced acrosome reaction. These observations do not completely exclude a role of PKDREJ for the induction of the acrosome reaction, since the antibodies may neither be able to stabilize an active protein conformation required for transmembrane signaling nor to block relevant epitopes needed for ZP signaling. However, it may be also the first hint for another role of PKDREJ in sperm physiology. Although all other PKD family members are widely expressed, PKDREJ expression was considered to be testis-specific. Investigating non-testicular tissue distribution of PKDREJ we were able to detect the protein in cardiomyocytes and smooth muscles of pulmonary blood vessels; the staining pattern of PKDREJ subcellular localization in both cell types corresponded to transverse tubules (t-tubules). Another site of non-testicular expression was discovered in epithelial cells of kidney and epididymis. In kidney epithelium PKDREJ was co-localized with acetylated -tubulin, a marker of primary cilia. These results open a new direction for the further investigation of PKDREJ that will provide deeper insight into its function and physiological relevance.
DOI:https://doi.org/10.17192/z2006.0239