Connexin43 Forms Supramolecular Complexes through Non-Overlapping Binding Sites for Drebrin, Tubulin, and ZO-1.
Gap junctions are membrane specialization domains identified in most tissue types where cells abut each other. The connexin channels found in these membrane domains are conduits for direct cell-to-cell transfer of ions and molecules. Connexin43 (Cx43) is the most ubiquitous connexin, with critical roles in heart, skin, and brain. Several studies described the interaction between Cx43 and the cytoskeleton involving the actin binding proteins Zonula occludens (ZO-1) and drebrin, as well as with tubulin. However, a direct interaction has not been identified between drebrin and Cx43. In this study, co-IP and NMR experiments were used to demonstrate that the Cx43-CT directly interacts with the highly conserved N-terminus region of drebrin. Three Cx43-CT areas were found to be involved in drebrin binding, with residues 264-275 being critical for the interaction. Mimicking Src phosphorylation within this region (Y265) significantly disrupted the interaction between the Cx43-CT and drebrin. Immunofluorescence showed colocalization of Cx43, drebrin, and F-actin in astrocytes and Vero cells membrane, indicating that Cx43 forms a submembrane protein complex with cytoskeletal and scaffolding proteins. The co-IP data suggest that Cx43 indirectly interacts with F-actin through drebrin. Along with the known interaction of the Cx43-CT with ZO-1 and tubulin, the data presented here for drebrin indicate non-overlapping and separated binding sites for all three proteins for which simultaneous binding could be important in regulating cytoskeleton rearrangements, especially for neuronal migration during brain development.
National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California La Jolla, La Jolla, California, United States of America.
Cinzia
C
Ambrosi
National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California La Jolla, La Jolla, California, United States of America.
Cynthia
C
Ren
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.
Gaelle
G
Spagnol
National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California La Jolla, La Jolla, California, United States of America.
Gabriel
G
Cavin
National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California La Jolla, La Jolla, California, United States of America.
Angela
A
Cone
Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California, United States of America.
Elena E
EE
Grintsevich
National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California La Jolla, La Jolla, California, United States of America.|Department of Neurosciences, University of California La Jolla, La Jolla, California, United States of America.
Gina E
GE
Sosinsky
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.
Paul L
PL
Sorgen
D008841:Actin Cytoskeleton; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D001665:Binding Sites; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D002465:Cell Movement; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D018031:Connexin 43 / Q000378:metabolism*; D005260:Female; D017629:Gap Junctions; D006801:Humans; D046912:Multiprotein Complexes / Q000378:metabolism*; D009479:Neuropeptides / Q000378:metabolism*; D054731:PDZ Domains; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D014404:Tubulin / Q000378:metabolism*; D062826:Zonula Occludens-1 Protein / Q000378:metabolism*
D018031:Connexin 43; D046912:Multiprotein Complexes; D009479:Neuropeptides; D014404:Tubulin; D062826:Zonula Occludens-1 Protein; C053386:drebrins