Tyrosine‐dependent basolateral targeting of human connexin43–eYFP in Madin–Darby canine kidney cells can be disrupted by the oculodentodigital dysplasia mutation L90V

Polarized membrane sorting of connexin 43 (Cx43) has not been well‐characterized. Based on the presence of a putative sorting signal, YKLV(286–289), within its C‐terminal cytoplasmic domain, we hypothesized that Cx43 is selectively expressed on the basolateral surface of Madin–Darby canine kidney (MDCK) cells in a tyrosine‐dependent manner. We generated stable MDCK cell lines expressing human wild‐type and mutant Cx43–eYFP, and analyzed the membrane localization of Cx43–eYFP within polarized monolayers using confocal microscopy and selective surface biotinylation. We found that wild‐type Cx43–eYFP was selectively targeted to the basolateral membrane domain of MDCK cells. Substitution of alanine for Y286 disrupted basolateral targeting of Cx43–eYFP. Additionally, substitution of a sequence containing the transferrin receptor internalization signal, LSYTRF, for PGYKLV(284–289) also disrupted basolateral targeting. Taken together, these results indicate that Y286 in its native amino acid sequence is necessary for targeting Cx43–eYFP to the basolateral membrane domain of MDCK cells. To determine whether the F52dup or L90V oculodentodigital dysplasia ‐associated mutations could affect polarized sorting of Cx43–eYFP, we analyzed the expression of these Cx43–eYFP mutant constructs and found that the L90V mutation disrupted basolateral expression. These findings raise the possibility that some oculodentodigitial dysplasia‐associated mutations contribute to disease by altering polarized targeting of Cx43.