Association of connexin36 with zonula occludens-1 in HeLa cells, betaTC-3 cells, pancreas, and adrenal gland.
The PDZ domain-containing protein zonula occludens-1 (ZO-1), a well-established component of tight junctions, has recently been shown to interact with various connexin proteins that form gap junctions. We investigated the association of connexin36 (Cx36) with ZO-1 in various cultured cells and tissues. Punctate immunofluorescence labeling for Cx36 was detected in Cx36-transfected HeLa cells, betaTC-3 cells, pancreatic islets, and adrenal medulla. Immunofluorescence for ZO-1 was also punctate in cells and tissues, and was colocalized with Cx36 at points of cell-cell contact. Immunoprecipitation of either Cx36 or ZO-1 from cell lysates and tissue homogenates resulted in immunoblot detection of ZO-1 or Cx36, respectively, in immunoprecipitates. A 14-amino acid peptide corresponding to the carboxy-terminus of Cx36 showed binding capacity to the PDZ1 domain of ZO-1, which was eliminated after removal of the last 4 carboxy-terminus amino acids. Low micromolar concentrations of the 14-amino acid peptide produced up to 85% inhibition of Cx36 interaction with the PDZ1 domain of ZO-1. These results provide evidence for molecular interaction between Cx36 and ZO-1 in vitro, and in vivo, and suggest that the interference with Cx36/ZO-1 interaction by short carboxy-terminus peptides of Cx36 may be of value for functional studies of this interaction.
Li
Xinbo
X
Department of Physiology, Faculty of Medicine, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, R3E 3J7, Canada.
Olson
Carl
C
Lu
Shijun
S
Nagy
James I
JI
D000313:Adrenal Medulla / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D017630:Connexins / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D007515:Islets of Langerhans / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D010179:Pancreas / Q000166:cytology / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D062826:Zonula Occludens-1 Protein; D000097004:Gap Junction delta-2 Protein
D017630:Connexins; D005982:Glutathione Transferase; D008565:Membrane Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D062826:Zonula Occludens-1 Protein; D000097004:Gap Junction delta-2 Protein; C569042:TJP1 protein, human; C569043:Tjp1 protein, mouse