Direct association of connexin36 with zonula occludens-2 and zonula occludens-3.
The gap junction protein connexin36 (Cx36) is widely expressed in neurons and was previously shown to interact with the PDZ domain-containing protein zonula occludens-1 (ZO-1). We investigated whether Cx36 is also able to interact with other members of zonula occludens family of proteins, namely, ZO-2 and ZO-3, the former of which was reported to be co-localized with Cx36 at gap junctions in mouse retina. HeLa cells transfected with Cx36 and cultured betaTC-3 cells were found to express ZO-2 and ZO-3, and both of these ZO proteins were co-localized with Cx36 at gap junctional cell-cell contacts. In lysates of Cx36-transfected HeLa cells, ZO-2 and ZO-3 were shown to co-immunoprecipitate with Cx36, whereas Cx36/ZO-2 association was absent in cells transfected with truncated Cx36 lacking its C-terminus SAYV PDZ interaction motif. In vitro pull-down assays revealed that Cx36 interacts with the PDZ1, but not with the other two PDZ domains in ZO-2 or ZO-3. Truncated Cx36 lacking its PDZ binding motif failed to bind the PDZ1 domain of either ZO-2 or ZO-3. A 14 amino acid peptide corresponding to the C-terminus of Cx36 was also shown to interact with the PDZ1 domains of ZO-2 and ZO-3, and this peptide inhibited the association of Cx36 with the PDZ1 domains of these ZO proteins. These results indicate that Cx36 associates with the first PDZ domain of ZO-2 and ZO-3 and that this association requires the C-terminus SAYV sequence in Cx36. These findings, together with the known association of ZO-2 with a variety of proteins, including transcription factors, suggest that ZO-2 may serve to anchor regulatory proteins at gap junctions composed of Cx36.
Department of Physiology, University of Manitoba, Winnipeg, Canada.
Xinbo
X
Li
Shijun
S
Lu
James I
JI
Nagy
D020816:Amino Acid Motifs / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism; D002450:Cell Communication / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D019108:Tight Junctions / Q000378:metabolism; D062825:Zonula Occludens Proteins; D062827:Zonula Occludens-2 Protein; D000097004:Gap Junction delta-2 Protein
D002352:Carrier Proteins; D017630:Connexins; D008565:Membrane Proteins; D010455:Peptides; D062825:Zonula Occludens Proteins; D062827:Zonula Occludens-2 Protein; D000097004:Gap Junction delta-2 Protein; C569046:TJP2 protein, human; C111873:TJP3 protein, human; C569047:Tjp2 protein, mouse