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.
Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.
Jana
J
Chtchetinin
Wes D
WD
Gifford
Sichen
S
Li
William A
WA
Paznekas
Ethylin Wang
EW
Jabs
Albert
A
Lai
D000015:Abnormalities, Multiple / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018031:Connexin 43 / Q000235:genetics* / Q000378:metabolism; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D021381:Protein Transport; D014443:Tyrosine / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D018031:Connexin 43; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D014443:Tyrosine