Expanding role of G proteins in tight junction regulation: Galpha(s) stimulates TJ assembly.
Multiple signaling mechanisms regulate epithelial cell tight junction (TJ) assembly and maintenance. Several G proteins are likely to regulate these processes, but only G(i/o) have been specifically tested. Treatment of MDCK cells with cholera toxin, a Galpha(s) activator, accelerated TJ development in the calcium switch as measured by the time to half-maximal [T(50) (H)] transepithelial resistance (TER). Galpha(s) was predominantly localized in the lateral membrane, but a fraction colocalizes with ZO-1 in the TJ. MDCK cell lines expressing epitope-tagged Galpha(s) and constitutively active (R201Calpha(s)) showed a similar localization. TJ assembly was significantly faster in R201Calpha(s)-MDCK cell lines (T(50) (H) of 1.7 versus 3.3 h for controls) without detectable differences in cAMP levels. Confocal studies showed R201Calpha(s)-MDCK cells more rapidly localized ZO-1 and occludin into the developing TJ without affecting E-cadherin or Na(+)/K(+) ATPase localization. Endogenous Galpha(s) and R201Calpha(s) were immunoprecipitated with ZO-1 at baseline and during TJ assembly. The data supports a model of multiple Galpha subunits interacting with TJ proteins to regulate the assembly and maintenance of the TJ.
Saha
C
C
Renal Division, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Nigam
S K
SK
Denker
B M
BM
D019943:Amino Acid Substitution; D000818:Animals; D015820:Cadherins / Q000032:analysis / Q000502:physiology; D002118:Calcium / Q000502:physiology; D002460:Cell Line; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004285:Dogs; D004847:Epithelial Cells / Q000502:physiology / Q000648:ultrastructure; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000502:physiology*; D007700:Kinetics; D008565:Membrane Proteins / Q000032:analysis / Q000502:physiology; D018613:Microscopy, Confocal; D062793:Occludin; D010750:Phosphoproteins / Q000032:analysis / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D019108:Tight Junctions / Q000502:physiology* / Q000648:ultrastructure; D014162:Transfection; D062826:Zonula Occludens-1 Protein
D015820:Cadherins; D008565:Membrane Proteins; D062793:Occludin; D010750:Phosphoproteins; D011994:Recombinant Proteins; D062826:Zonula Occludens-1 Protein; D000242:Cyclic AMP; D019205:GTP-Binding Protein alpha Subunits, Gs; D002118:Calcium