Role of the cytoplasmic loop domain of Cx43 in its intracellular localization and function: possible interaction with cadherin.
We have previously shown that intracellular trafficking and function of connexin (Cx) 26 and Cx43 are controlled by E-cadherin. In the present study, we attempted to determine which part of Cx43 is involved in this control mechanism. Since Cx26 has a very short C terminus in the cytoplasm, we hypothesized that the C-terminal domain may not be important for this process and, indeed, found that green fluorescence protein (GFP)-tagged Cx43DeltaC (deleted from the codon 239) moved to the plasma membrane both in P3/22(E), a mouse papilloma cell line which expresses E-cadherin, and HeLa cells only at high calcium culture conditions. We then found that the GFP-tagged Cx43(CL 26)DeltaC mutant, in which the cytoplasmic loop domain of Cx43 was exchanged with that of Cx26, remains in the cytoplasm in HeLa, HeLaCx43 and P3/22(E) cells, suggesting the importance of the cytoplasmic loop domain. In order to determine which part of the cytoplasmic domain plays a key role, we introduced four deletion mutations (deletion of codons 101-111 [mutant D1], 120-130 [D2], 131-137 [D3] or 146-159 [D4]) to the GFP-tagged Cx43DeltaC gene. When these mutants were transfected into HeLa cells, D1 and D4 mutants were localized in the cytoplasm, while D2 and D3 were found in the plasma membrane only in high Ca(2+) medium. However, none of these four mutants recovered gap junctional intercellular communication (GJIC). On the other hand, when these mutants were transfected into HeLaCx43 and P3/22(E) cells (which express functional Cx43), D1, D2 and D3, but not D4, moved to the plasma membrane and colocalized with endogenous Cx43 in high Ca(2+) medium; all of these mutants showed a dominant negative effect on GJIC in HeLaCx43 cells. Further deletion studies indicated that the critical amino acids involved in this intracellular trafficking of Cx43 lie between codons 100 and 102.
Nambara
Chika
C
Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 1-2, Gakuen, Sanda, 669-1337, Hyogo, Japan.
Kawasaki
Yumi
Y
Yamasaki
Hiroshi
H
D000818:Animals; D015820:Cadherins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002450:Cell Communication / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D000072259:Connexin 26; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D017630:Connexins; D003593:Cytoplasm / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007365:Intercellular Junctions / Q000378:metabolism; D051379:Mice; D010212:Papilloma / Q000378:metabolism* / Q000473:pathology; D017434:Protein Structure, Tertiary; D012878:Skin Neoplasms / Q000378:metabolism* / Q000473:pathology
D015820:Cadherins; D018031:Connexin 43; D017630:Connexins; C000607042:GJB2 protein, human; D000072259:Connexin 26; D002118:Calcium