title: Ubiquitin-mediated internalization of connexin43 is independent of the canonical endocytic tyrosine-sorting signal.
abstract: Gap junctions are specialized cell-cell contacts that provide direct intercellular communication between eukaryotic cells. The tyrosine-sorting signal (YXXØ), present at amino acids 286-289 of Cx43 (connexin43), has been implicated in the internalization of the protein. In recent years, ubiquitination of Cx43 has also been proposed to regulate gap junction intercellular communication; however, the underlying mechanism and molecular players involved remain elusive. In the present study, we demonstrate that ubiquitinated Cx43 is internalized through a mechanism that is independent of the YXXØ signal. Indeed, expression of a Cx43-Ub (ubiquitin) chimaera was shown to drive the internalization of a mutant Cx43 in which the YXXØ motif was eliminated. Immunofluorescence, cycloheximide-chase and cell-surface-protein biotinylation experiments demonstrate that oligomerization of Cx43-Ub into hemichannels containing wild-type Cx43 or mutant Cx43Y286A is sufficient to drive the internalization of the protein. Furthermore, the internalization of Cx43 induced by Cx43-Ub was shown to depend on its interaction with epidermal growth factor receptor substrate 15.
authors.affiliation: Centre of Ophthalmology and Vision Sciences, Faculty of Medicine, University of Coimbra, Coimbra, Portugal.
authors.forename: Steve
authors.initials: S
authors.lastname: Catarino
authors.forename: José S
authors.initials: JS
authors.lastname: Ramalho
authors.forename: Carla
authors.initials: C
authors.lastname: Marques
authors.forename: Paulo
authors.initials: P
authors.lastname: Pereira
authors.forename: Henrique
authors.initials: H
authors.lastname: Girão
mesh_terms: D000818:Animals; D019556:COS Cells; D002450:Cell Communication / Q000502:physiology; D002522:Chlorocebus aethiops; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D018031:Connexin 43; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin