Cell-cell interactions in the kidney: inducible expression of mutant G protein alpha-subunits in Madin-Darby canine kidney cells for studies of epithelial cell junction structure and function

Methods Mol Biol. 2006;341:61-72. doi: 10.1385/1-59745-113-4:61.

Abstract

Disrupted epithelial cell junctions are a hallmark of numerous disease processes. The signaling mechanisms regulating barrier function and re-establishment of intact junctions after injury and during development are complex and tightly regulated. We have shown that heterotrimeric G proteins regulate assembly and maintenance of epithelial cell barrier in cultured Madin-Darby canine kidney (MDCK) cells. The inducible expression of mutant signaling molecules (constitutively active or dominant negative) in polarized cells is a useful strategy for elucidating the role(s) for specific proteins. Using tetracycline-off inducible expression of wild-type and constitutively active Galpha12, we have demonstrated a fundamental role for Galpha12 in regulating the junction of MDCK cells. Inducible expression permits the comparison of the identical cell line in the presence and absence of the protein of interest and minimizes variation arising from distinct clones. The methods described here are applicable to virtually any protein that may regulate maintenance or assembly of the epithelial cell junctional complex.

Publication types

  • Comparative Study
  • Review

MeSH terms

  • Animals
  • Anti-Bacterial Agents / pharmacology
  • Cell Line
  • Cell Membrane Permeability / drug effects
  • Cell Membrane Permeability / genetics
  • Cell Polarity / drug effects
  • Cell Polarity / genetics
  • Dogs
  • Epithelial Cells / metabolism*
  • GTP-Binding Protein alpha Subunit, Gi2 / genetics
  • GTP-Binding Protein alpha Subunit, Gi2 / metabolism*
  • Gene Expression / drug effects
  • Gene Expression / genetics
  • Kidney / embryology
  • Kidney / injuries
  • Kidney / metabolism*
  • Mutation*
  • Signal Transduction* / drug effects
  • Signal Transduction* / genetics
  • Tetracycline / pharmacology
  • Tight Junctions / genetics
  • Tight Junctions / metabolism*

Substances

  • Anti-Bacterial Agents
  • GTP-Binding Protein alpha Subunit, Gi2
  • Tetracycline