More insights into the CCN3/Connexin43 interaction complex and its role for signaling |
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Authors: | Alexandra Gellhaus Christoph Wotzlaw Teresa Otto Joachim Fandrey Elke Winterhager |
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Affiliation: | 1. Institute of Molecular Biology, University of Duisburg‐Essen, Hufelandstrasse 55, D‐45122 Essen, Germany;2. Institute of Physiology, University of Duisburg‐Essen, Hufelandstrasse 55, D‐45122 Essen, Germany |
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Abstract: | Connexin43 (Cx43) forms gap junction channels but also serves as a signaling center by binding to proteins via its C‐terminus. We have previously demonstrated that transfection of Cx43 leads to significantly reduced proliferation of placental tumor cells through upregulating and binding of the growth regulator CCN3 (NOV) at the C‐terminus of Cx43. Here, we combined fluorescence resonance energy transfer (FRET), co‐immunoprecipitation and proliferation and expression assays to characterize the interaction complex of Cx43 and CCN3. FRET measurements confirmed the interaction of CCN3 with wild‐type Cx43 (amino acids 1‐382) and with mutants of Cx43 truncated at the C‐terminus resulting in Cx43 proteins of amino acids 1‐374, 1‐273, 1‐264, 1‐257 in 293T cells. These results matched the co‐immunoprecipitation data. Interestingly, although FRET revealed distinct efficiencies in interaction of Cx43 with CCN3 for all deletion constructs only wild‐type Cx43 and one deletion construct (1‐374) led to increased CCN3 expression. Only these interactions which were associated with increased CCN3 expression resulted in a reduced cell proliferation. Our study provides evidence that only defined binding properties between Cx43 and CCN3 leading to an upregulation of CCN3 are needed for signaling. Furthermore, the data obtained by FRET analysis allowed us to model the 3D structure of the C‐terminus of Cx43 interacting with CCN3. J. Cell. Biochem. 110: 129–140, 2010. © 2010 Wiley‐Liss, Inc. |
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Keywords: | CCN3 Cx43 gap junction fluorescence resonance energy transfer (FRET) protein interaction cell proliferation |
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