Gap junction protein connexin-43 interacts directly with microtubules.
Gap junctions are specialized cell-cell junctions that mediate intercellular communication. They are composed of connexin proteins, which form transmembrane channels for small molecules [1, 2]. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating by growth factors [3-5]. The Cx43 tail contains various protein interaction sites, but little is known about binding partners. To identify Cx43-interacting proteins, we performed pull-down experiments using the C-terminal tail of Cx43 fused to glutathione-S-transferase. We find that the Cx43 tail binds directly to tubulin and, like full-length Cx43, sediments with microtubules. Tubulin binding to Cx43 is specific in that it is not observed with three other connexins. We established that a 35-amino acid juxtamembrane region in the Cx43 tail, which contains a presumptive tubulin binding motif, is necessary and sufficient for microtubule binding. Immunofluorescence and immunoelectron microscopy studies reveal that microtubules extend to Cx43-based gap junctions in contacted cells. However, intact microtubules are dispensable for the regulation of Cx43 gap-junctional communication. Our findings suggest that, in addition to its well-established role as a channel-forming protein, Cx43 can anchor microtubule distal ends to gap junctions and thereby might influence the properties of microtubules in contacted cells.
Giepmans
B N
BN
Division of Cellular Biochemistry, The Netherlands Cancer Institute and Center for Biomedical Genetics, 121, 1066 CX Amsterdam, Plesmanlaan, The Netherlands.
Verlaan
I
I
Hengeveld
T
T
Janssen
H
H
Calafat
J
J
Falk
M M
MM
Moolenaar
W H
WH
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D006801:Humans; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism
D018031:Connexin 43; D011993:Recombinant Fusion Proteins; D014404:Tubulin