The C-terminus of connexin43 adopts different conformations in the Golgi and gap junction as detected with structure-specific antibodies.
The C-terminus of the most abundant and best-studied gap-junction protein, connexin43, contains multiple phosphorylation sites and protein-binding domains that are involved in regulation of connexin trafficking and channel gating. It is well-documented that SDS/PAGE of NRK (normal rat kidney) cell lysates reveals at least three connexin43-specific bands (P0, P1 and P2). P1 and P2 are phosphorylated on multiple, unidentified serine residues and are found primarily in gap-junction plaques. In the present study we prepared monoclonal antibodies against a peptide representing the last 23 residues at the C-terminus of connexin43. Immunofluorescence studies showed that one antibody (designated CT1) bound primarily to connexin43 present in the Golgi apparatus, whereas the other antibody (designated IF1) labelled predominately connexin43 present in gap junctions. CT1 immunoprecipitates predominantly the P0 form whereas IF1 recognized all three bands. Peptide mapping, mutational analysis and protein-protein interaction experiments revealed that unphosphorylated Ser364 and/or Ser365 are critical for CT1 binding. The IF1 paratope binds to residues Pro375-Asp379 and requires Pro375 and Pro377. These proline residues are also necessary for ZO-1 interaction. These studies indicate that the conformation of Ser364/Ser365 is important for intracellular localization, whereas the tertiary structure of Pro375-Asp379 is essential in targeting and regulation of gap junctional connexin43.
Sosinsky
Gina E
GE
National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California, San Diego, La Jolla, CA 92093-0608, USA. gsosinsky@ucsd.edu
Solan
Joell L
JL
Gaietta
Guido M
GM
Ngan
Lucy
L
Lee
Grace J
GJ
Mackey
Mason R
MR
Lampe
Paul D
PD
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002460:Cell Line; D018031:Connexin 43 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D017629:Gap Junctions / Q000737:chemistry*; D006056:Golgi Apparatus / Q000737:chemistry*; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011487:Protein Conformation; D051381:Rats
D000906:Antibodies; D018031:Connexin 43
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