Subcellular localization of polycystin in human renal epithelial cell lines

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Abstract

The predicted protein product of the gene, PKD I, which is mutated in type 1 autosomal dominant polycystic kidney disease (ADPKD 1) has been named polycystin, is of large molecular weight (~ 460 kDa) and is of unknown distribution and function. Using a panel of polyclonal rabbit antisera raised against synthetic peptide sequences from its unique portion, we have observed enhanced expression of polycystin by immunohistochemistry in renal and biliary epithelium and hepatocytes in fetal tissue and tissue from individuals with type 1 autosomal dominant polycystic kidney disease (ADPKD 1 ) as compared to normal adult kidney and liver. To further clarify the intracellular localization of polycystin we have examined proliferating human cell lines of renal epithelial origin with one selected antiserum by Western blot and immunoprecipitation techniques, immunofluorescent laser scanning confocal microscopy (LSCM) and immunogold electron microscopy (IEM). The cell lines used were G401 (derived from a human childhood renal tumor) and normal human proximal tubular epithelial cells (RPTEC). Cells were grown as polarized monolayers on collagen treated membranes for LSCM and IEM. LSCM with three dimensional reconstruction demonstrated discrete localization in a speckled pattern at or just below the apical membrane in confluent and subconfluent cells. IEM showed discrete localization to subcellular structures which were predominantly close to the apical membrane. A group of two to three high molecular weight bands (Mr > 200kDa) were strongly detected by Western analysis in both cell lines and by immunoprecipitation in G401 cells, but not by control antisera. We conclude that polycystin is strongly expressed during proliferation by polarized cells of human renal epithelial origin and is localized to discrete structures at or just below the apical membrane. It may have alternate forms or be partly degraded within these structures.

Original languageEnglish (US)
JournalJournal of Investigative Medicine
Volume44
Issue number3
StatePublished - 1996

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TRPP Cation Channels
Autosomal Dominant Polycystic Kidney
Confocal microscopy
Epithelial Cells
Cells
Electron microscopy
Membranes
Kidney
Immune Sera
Cell Line
Confocal Microscopy
Scanning
Electron Microscopy
Lasers
Immunoprecipitation
Molecular weight
Tissue
Molecular Weight
Liver
Tumors

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

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title = "Subcellular localization of polycystin in human renal epithelial cell lines",
abstract = "The predicted protein product of the gene, PKD I, which is mutated in type 1 autosomal dominant polycystic kidney disease (ADPKD 1) has been named polycystin, is of large molecular weight (~ 460 kDa) and is of unknown distribution and function. Using a panel of polyclonal rabbit antisera raised against synthetic peptide sequences from its unique portion, we have observed enhanced expression of polycystin by immunohistochemistry in renal and biliary epithelium and hepatocytes in fetal tissue and tissue from individuals with type 1 autosomal dominant polycystic kidney disease (ADPKD 1 ) as compared to normal adult kidney and liver. To further clarify the intracellular localization of polycystin we have examined proliferating human cell lines of renal epithelial origin with one selected antiserum by Western blot and immunoprecipitation techniques, immunofluorescent laser scanning confocal microscopy (LSCM) and immunogold electron microscopy (IEM). The cell lines used were G401 (derived from a human childhood renal tumor) and normal human proximal tubular epithelial cells (RPTEC). Cells were grown as polarized monolayers on collagen treated membranes for LSCM and IEM. LSCM with three dimensional reconstruction demonstrated discrete localization in a speckled pattern at or just below the apical membrane in confluent and subconfluent cells. IEM showed discrete localization to subcellular structures which were predominantly close to the apical membrane. A group of two to three high molecular weight bands (Mr > 200kDa) were strongly detected by Western analysis in both cell lines and by immunoprecipitation in G401 cells, but not by control antisera. We conclude that polycystin is strongly expressed during proliferation by polarized cells of human renal epithelial origin and is localized to discrete structures at or just below the apical membrane. It may have alternate forms or be partly degraded within these structures.",
author = "Griffin, {M. D.} and Vicente Torres and Grande, {Joseph Peter} and Rajiv Kumar",
year = "1996",
language = "English (US)",
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journal = "Journal of Investigative Medicine",
issn = "1081-5589",
publisher = "Lippincott Williams and Wilkins",
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TY - JOUR

T1 - Subcellular localization of polycystin in human renal epithelial cell lines

AU - Griffin, M. D.

AU - Torres, Vicente

AU - Grande, Joseph Peter

AU - Kumar, Rajiv

PY - 1996

Y1 - 1996

N2 - The predicted protein product of the gene, PKD I, which is mutated in type 1 autosomal dominant polycystic kidney disease (ADPKD 1) has been named polycystin, is of large molecular weight (~ 460 kDa) and is of unknown distribution and function. Using a panel of polyclonal rabbit antisera raised against synthetic peptide sequences from its unique portion, we have observed enhanced expression of polycystin by immunohistochemistry in renal and biliary epithelium and hepatocytes in fetal tissue and tissue from individuals with type 1 autosomal dominant polycystic kidney disease (ADPKD 1 ) as compared to normal adult kidney and liver. To further clarify the intracellular localization of polycystin we have examined proliferating human cell lines of renal epithelial origin with one selected antiserum by Western blot and immunoprecipitation techniques, immunofluorescent laser scanning confocal microscopy (LSCM) and immunogold electron microscopy (IEM). The cell lines used were G401 (derived from a human childhood renal tumor) and normal human proximal tubular epithelial cells (RPTEC). Cells were grown as polarized monolayers on collagen treated membranes for LSCM and IEM. LSCM with three dimensional reconstruction demonstrated discrete localization in a speckled pattern at or just below the apical membrane in confluent and subconfluent cells. IEM showed discrete localization to subcellular structures which were predominantly close to the apical membrane. A group of two to three high molecular weight bands (Mr > 200kDa) were strongly detected by Western analysis in both cell lines and by immunoprecipitation in G401 cells, but not by control antisera. We conclude that polycystin is strongly expressed during proliferation by polarized cells of human renal epithelial origin and is localized to discrete structures at or just below the apical membrane. It may have alternate forms or be partly degraded within these structures.

AB - The predicted protein product of the gene, PKD I, which is mutated in type 1 autosomal dominant polycystic kidney disease (ADPKD 1) has been named polycystin, is of large molecular weight (~ 460 kDa) and is of unknown distribution and function. Using a panel of polyclonal rabbit antisera raised against synthetic peptide sequences from its unique portion, we have observed enhanced expression of polycystin by immunohistochemistry in renal and biliary epithelium and hepatocytes in fetal tissue and tissue from individuals with type 1 autosomal dominant polycystic kidney disease (ADPKD 1 ) as compared to normal adult kidney and liver. To further clarify the intracellular localization of polycystin we have examined proliferating human cell lines of renal epithelial origin with one selected antiserum by Western blot and immunoprecipitation techniques, immunofluorescent laser scanning confocal microscopy (LSCM) and immunogold electron microscopy (IEM). The cell lines used were G401 (derived from a human childhood renal tumor) and normal human proximal tubular epithelial cells (RPTEC). Cells were grown as polarized monolayers on collagen treated membranes for LSCM and IEM. LSCM with three dimensional reconstruction demonstrated discrete localization in a speckled pattern at or just below the apical membrane in confluent and subconfluent cells. IEM showed discrete localization to subcellular structures which were predominantly close to the apical membrane. A group of two to three high molecular weight bands (Mr > 200kDa) were strongly detected by Western analysis in both cell lines and by immunoprecipitation in G401 cells, but not by control antisera. We conclude that polycystin is strongly expressed during proliferation by polarized cells of human renal epithelial origin and is localized to discrete structures at or just below the apical membrane. It may have alternate forms or be partly degraded within these structures.

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