Zonula occludens-1 is a scaffolding protein for signaling molecules. Galpha(12) directly binds to the Src homology 3 domain and regulates paracellular permeability in epithelial cells.
Zonula occludens proteins are multidomain proteins usually localized at sites of intercellular junctions, yet little is known about their role in regulating junctional properties. Multiple signaling proteins regulate the junctional complex, and several (including G proteins) have been co-localized with zonula occludens-1 (ZO-1) in the tight junction of epithelial cells. However, evidence for direct interactions between signaling proteins and tight junction proteins has been lacking. In these studies, we constructed Galpha-glutathione S-transferase (GST) fusion proteins and tested for interactions with [(35)S]methionine-labeled in vitro translated ZO-1 and ZO-2. Only Galpha(12) directly interacted with in vitro translated ZO-1 and ZO-2. Using a series of ZO-1 domains expressed as GST fusion proteins and in vitro translated [(35)S]methionine-labeled Galpha(12), we found that Galpha(12) and constitutively active (Q229L) alpha(12) (QLalpha(12)) bind to the Src homology 3 (SH3) domain of ZO-1. This binding was not detected with SH3 domains from other proteins. Inducible expression of wild-type alpha(12) and QLalpha(12) in Madin-Darby canine kidney (MDCK) cells was established using the Tet-Off system. In Galpha(12)-expressing cells, we found that ZO-1 and Galpha(12) co-localize by confocal microscopy and co-immunoprecipitate. Galpha(12) from MDCK cell lysates bound to the GST-ZO-1-SH3 domain, and expression of QLalpha(12) in MDCK cells reversibly increased paracellular permeability. These studies indicated that ZO-1 directly interacts with Galpha(12) and that Galpha(12) regulates barrier function of MDCK cells.
Meyer
Tobias N
TN
Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Schwesinger
Catherine
C
Denker
Bradley M
BM
D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000502:physiology*; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism*; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D010750:Phosphoproteins / Q000378:metabolism*; D015398:Signal Transduction; D062826:Zonula Occludens-1 Protein; D018909:src Homology Domains
D008565:Membrane Proteins; D010750:Phosphoproteins; C569043:Tjp1 protein, mouse; D062826:Zonula Occludens-1 Protein; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020962:Heterotrimeric GTP-Binding Proteins