Connexin43 Associated with an N-cadherin-containing Multiprotein Complex Is Required for Gap Junction Formation in NIH3T3 Cells

Abstract

Previous studies have indicated an intimate linkage between gap junction and adherens junction formation. It was suggested this could reflect the close membranemembrane apposition required for junction formation. In NIH3T3 cells, we observed the colocalization of connexin43 (Cx43␣1) gap junction protein with N-cadherin, p120, and other N-cadherin-associated proteins at regions of cell-cell contact. We also found that Cx43␣1, N-cadherin, and N-cadherin-associated proteins were coimmunoprecipitated by antibodies to either Cx43␣1, N-cadherin, or various N-cadherin-associated proteins. These findings suggest that Cx43␣1 and N-cadherin are coassembled in a multiprotein complex containing various N-cadherin-associated proteins. Studies using siRNA knockdown indicated that cell surface expression of Cx43␣1 required N-cadherin, and conversely, Ncadherin cell surface expression required Cx43␣1. Pulse-chase labeling and cell surface biotinylation experiments indicated that in the absence of N-cadherin, Cx43␣1 cell surface trafficking is blocked. Surprisingly, siRNA knockdown of p120, an N-cadherin-associated protein known to modulate cell surface turnover of Ncadherin, reduced N-cadherin cell surface expression without altering Cx43␣1 expression. These observations suggest that in contrast to the coregulated cell surface trafficking of Cx43␣1 and N-cadherin, N-cadherin turnover at the cell surface may be regulated independently of Cx43␣1. Functional studies showed gap junctional communication is reduced and cell motility inhibited with N-cadherin or Cx43␣1 knockdown, consistent with the observed loss of both gap junction and cadherin contacts with either knockdown. Overall, these studies indicate that the intracellular coassembly of connexin and cadherin is required for gap junction and adherens junction formation, a process that likely underlies the intimate association between gap junction and adherens junction formation.

N-cadherin ligation, but not Sonic hedgehog binding, initiates Cdo-dependent p38α/β MAPK signaling in skeletal myoblasts

“…The percentage of beads that clustered a given fluorescent protein in these assays was quantified. Eighty-five percent of Ncad beads clustered Ncad-DsRed, and 77% clustered Cdo, whether or not exogenous Ncad was expressed (exogenous Ncad is unnecessary as NIH 3T3 cells express abundant endogenous Ncad) (34), whereas only 20% of Ncad beads clustered GFP (Fig. 4C).…”

The Second PDZ Domain of Zonula Occludens-1 Is Dispensable for Targeting to Connexin43 Gap Junctions

“…Alternatively, the presence of low levels of ZO-1 at the edge of Cx43-GFP plaques could result indirectly via macromolecular associations between GJs and other cell-cell junctions that incorporate ZO-1. Adherens junctions are logical candidates in this respect as they are known to play a pivotal role in GJ stability (Hertig et al 1996;Jongen et al 1991;Li et al 2005;Meyer et al 1992;Musil et al 1990;Wei et al 2005) and cadherin-associated catenin complexes have been shown to bind both Cx43 (Ai et al 2000;Wei et al 2005) and ZO-1 (Itoh et al 1997).…”

N-cadherin haploinsufficiency affects cardiac gap junctions and arrhythmic susceptibility

“…Recent evidence indicates that N-cadherin and β-catenin control the targeting of Cx43 to adherens junctions by interacting with microtubule plus-end-tracking proteins (13). Studies in NIH3T3 cells showed that knockdown of N-cadherin affects trafficking of a N-cadherin/Cx43 multiprotein complex to the cell surface (14). Thus, N-cadherin may be involved in more than one step in the development of functionally mature gap junction plaques at the plasma membrane.…”