title: Cx50 requires an intact PDZ-binding motif and ZO-1 for the formation of functional intercellular channels.
abstract: The three connexins expressed in the ocular lens each contain PDZ domain-binding motifs directing a physical association with the scaffolding protein ZO-1, but the significance of the interaction is unknown. We found that Cx50 with PDZ-binding motif mutations did not form gap junction plaques or induce cell-cell communication in HeLa cells, whereas the addition of a seven-amino acid PDZ-binding motif restored normal function to Cx50 lacking its entire C-terminal cytoplasmic domain. C-Terminal deletion had a similar although weaker effect on Cx46 but little if any effect on targeting and function of Cx43. Furthermore, small interfering RNA knockdown of ZO-1 completely inhibited the formation of gap junctions by wild-type Cx50 in HeLa cells. Thus both a PDZ-binding motif and ZO-1 are necessary for Cx50 intercellular channel formation in HeLa cells. Knock-in mice expressing Cx50 with a PDZ-binding motif mutation phenocopied Cx50 knockouts. Furthermore, differentiating lens fibers in the knock-in displayed extensive intracellular Cx50, whereas plaques in mature fibers contained only Cx46. Thus normal Cx50 function in vivo also requires an intact PDZ domain-binding motif. This is the first demonstration of a connexin-specific requirement for a connexin-interacting protein in gap junction assembly.
authors.affiliation: Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Zhifang
authors.initials: Z
authors.lastname: Chai
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Goodenough
authors.forename: David L
authors.initials: DL
authors.lastname: Paul
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D002450:Cell Communication / Q000235:genetics; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism*; D055786:Gene Knockout Techniques; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering; D062826:Zonula Occludens-1 Protein; D000099264:Connexin 50
chemical_list: D017630:Connexins; D005136:Eye Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D062826:Zonula Occludens-1 Protein; D000099264:Connexin 50; C569042:TJP1 protein, human; C569043:Tjp1 protein, mouse; C082917:GJA3 protein, human