Connexin43 associated with an N-cadherin-containing multiprotein complex is required for gap junction formation in NIH3T3 cells

Previous studies have indicated an intimate linkage between gap junction and adherens junction formation. It was suggested this could reflect the close membrane-membrane apposition required for junction formation. In NIH3T3 cells, we observed the colocalization of connexin43 (Cx43 alpha 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 alpha 1, N-cadherin, and N-cadherin-associated proteins were coimmunoprecipitated by antibodies to either Cx43 alpha 1, N-cadherin, or various N-cadherin-associated proteins. These findings suggest that Cx43 alpha 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 alpha 1 required N-cadherin, and conversely, N-cadherin cell surface expression required Cx43 alpha 1. Pulse-chase labeling and cell surface biotinylation experiments indicated that in the absence of N-cadherin, Cx43 alpha 1 cell surface trafficking is blocked. Surprisingly, siRNA knockdown of p120, an N-cadherin-associated protein known to modulate cell surface turnover of N-cadherin, reduced N-cadherin cell surface expression without altering Cx43 alpha 1 expression. These observations suggest that in contrast to the coregulated cell surface trafficking of Cx43 alpha 1 and N-cadherin, N-cadherin turnover at the cell surface may be regulated independently of Cx43 alpha 1. Functional studies showed gap junctional communication is reduced and cell motility inhibited with N-cadherin or Cx43 alpha 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.