The cytoplasmic tail of CD44 is required for basolateral localization in epithelial MDCK cells but does not mediate association with the detergent-insoluble cytoskeleton of fibroblasts.
A number of recent reports on the trafficking of receptor proteins in MDCK epithelial cells have provided evidence that delivery to the basolateral domain requires a specific targeting sequence and that deletion of this sequence results in constitutive expression on the apical surface. To date, these studies have concentrated on receptors which are competent for internalization via the clathrin coated pits. We have examined the localization of a resident plasma membrane protein by transfecting human CD44 into MDCK cells. Using human specific and cross-species reactive antibodies, we show that in MDCK cells both the endogenous and transfected wild-type CD44 are found on the basolateral surface where they are restricted to the lateral domain. Deletion of the CD44 cytoplasmic tail reduces the half life of this mutant protein and causes it to be expressed both on the apical surface and to a significant extent within the cell. We have also used biochemical and morphological analysis to investigate the interaction of CD44 with the cytoskeleton in detergent extracted cells. Strikingly different extraction results were obtained between epithelial and fibroblast cells. However, there is no difference in the Triton X-100 solubility of the transfected wild-type and tail-less CD44 in fibroblasts and both forms of the protein remain associated with the cortical cytoskeleton after Triton X-100 extraction. These results demonstrate that the sequence present in the cytoplasmic domain of CD44 responsible for its distribution in epithelial cells is functionally and spatially separate from the ability of this protein to associate with the cytoskeleton.
Neame
S J
SJ
Department of Biology, Imperial College of Science, Technology and Medicine, London, United Kingdom.
Isacke
C M
CM
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002460:Cell Line; D016764:Cell Polarity; D003599:Cytoskeleton / Q000378:metabolism; D004285:Dogs; D004848:Epithelium / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D016201:Receptors, Lymphocyte Homing / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship
D016201:Receptors, Lymphocyte Homing