Different functional recognition of basolateral signals in Caco-2 and MDCK cells.

Using the basolateral mutant PS of the normally apical neurotrophin receptor p75 (p75NTR) we have identified two cytoplasmic determinants responsible for this reversed localization in the human intestinal cell line, Caco2. These signals are based on two consecutive leucines (322-323) and a tyrosine (Y308). Truncation of the cytoplasmic tail removing the two leucines or their replacement by alanines led to a nonpolarized expression of the resulting mutants in Caco2 cells. To our surprise, the same mutations had no effect on the basolateral localization of the mutant PS in MDCK cells. In MDCK cells, the basolateral localization was entirely dependent on a cytoplasmic tyrosine Y308, while in Caco-2 cells this tyrosine signal was functional as a basolateral signal only when the cytoplasmic domain of PS was truncated shortly after it. These data indicate for the first time that there is a differential recognition of basolateral signals between MDCK and Caco-2 cells.

[Identification of signals and mechanisms of sorting of plasma membrane proteins in intestinal epithelial cells]. / Identification de signaux et de mécanismes de tri des protéines de la membrane plasmique dans les cellules épithéliales intestinales.

In epithelial cells the plasma membrane is divided into domains that are biochemically and functionally different. In intestinal cells for example the apical domain is facing the intestinal lumen and is involved in the uptake of nutriments while the basolateral domain is mediating cell-cell adhesion and signalisation. We are interested in deciphering the mechanisms underlying the creation and maintenance of such specialized domains. As an epithelial model we have used the intestinal cell line Caco-2 and we have studied the transport and sorting of the human neurotrophin receptor (p75 NTR) in these cells. Newly synthesized p75 NTR is first transported to the basolateral membrane and then is accumulated on the apical membrane after transcytosis. This final apical localization is controlled by the presence of a membrane anchor and a cluster of O-glycosylation sites located in the part of the ectodomain close to the membrane. Among the mechanisms likely to be involved in the sorting of apical components we have looked for a role of lipid-protein microdomain formation in the Golgi apparatus. These membrane microdomains are highly enriched in glycosylphosphatidyl inositol (GPI) anchored proteins, glycosphingolipids and apical proteins such as sucrase isomaltase (SI). Such a composition is also found for endocytic structures called caveolae which are made of caveolin 1. We have expressed caveolin 1 in Caco-2 cells which do not express it and also caveolin 2, a related protein of unknown function. Expression of caveolin 1 led to formation of caveolae indicating that this protein is necessary for caveolae formation while caveolin 2 is restricted to the Golgi apparatus and has no effect on caveolae formation. However Caveolin 2 increased the amount of SI incorporated in microdomains suggesting a role in recruitment into the apical pathway. The choice for a site of fusion for transport vesicles is the last step of control during exocytosis. To identify proteins involved in that step we have cloned and characterized two members of the t-SNARE family, namely syntaxin 3 and SNAP23. Syntaxin 3 is present on the apical membrane and forms a complex with SNAP23 which is also localized on the basolateral membrane where it forms a complex with syntaxin 4. Overexpression of syntaxin 3 in Caco-2 led to a decrease of SI exocytosis towards the apical membrane confirming that syntaxin 3 is involved in targeting the fusion of apical transport vesicles to the apical pole of the cells.

Putative O-glycosylation sites and a membrane anchor are necessary for apical delivery of the human neurotrophin receptor in Caco-2 cells.

We have expressed the human neurotrophin receptor p75 (p75(NTR)) in the intestinal epithelial cell line Caco-2 as a model to study intracellular transport and subcellular sorting signals in intestinal cells. p75(NTR) was localized at the apical membrane of Caco-2 cells and reached this membrane mainly via an indirect pathway. Apical localization, intracellular routing, and basolateral to apical transcytosis were not affected by truncation of the cytoplasmic domain or replacement of the transmembrane domain by a glycosyl phosphatidylinositol anchor. Removal of membrane anchoring resulted in basolateral secretion of the ectodomain of p75(NTR) in Caco-2 cells but in apical secretion in Madin-Darby canine kidney (MDCK) cells. Substitution of potential O-glycosylation sites present in the stalk of p75(NTR) led to intracellular cleavage and secretion of the ectodomain into the basolateral medium both in Caco-2 and MDCK cells. These results suggest that the stalk of p75(NTR) carries an apical sorting information that is recognized efficiently by Caco-2 cells only when attached to the membrane. This apical sorting information is linked to the presence of predicted O-glycosylation sites in that region. These putative O-glycosylation sites also play a role in the regulation of p75(NTR) transport to the cell surface and in the prevention of rapid degradation by cleavage of the stalk domain.

The O-glycosylated stalk domain is required for apical sorting of neurotrophin receptors in polarized MDCK cells.

Delivery of newly synthesized membrane-spanning proteins to the apical plasma membrane domain of polarized MDCK epithelial cells is dependent on yet unidentified sorting signals present in the luminal domains of these proteins. In this report we show that structural information for apical sorting of transmembrane neurotrophin receptors (p75(NTR)) is localized to a juxtamembrane region of the extracellular domain that is rich in O-glycosylated serine/threonine residues. An internal deletion of 50 amino acids that removes this stalk domain from p75(NTR) causes the protein to be sorted exclusively of the basolateral plasma membrane. Basolateral sorting stalk-minus p75(NTR) does not occur by default, but requires sequences present in the cytoplasmic domain. The stalk domain is also required for apical secretion of a soluble form of p75(NTR), providing the first demonstration that the same domain can mediate apical sorting of both a membrane-anchored as well as secreted protein. However, the single N-glycan present on p75(NTR) is not required for apical sorting of either transmembrane or secreted forms.

Detergent-resistant membrane microdomains from Caco-2 cells do not contain caveolin.

In this study we analyzed the relationship between detergent-resistant microdomains and caveolae in Caco-2 cells. Caveolin was not detected on Western blots or Northern blots or by immunoprecipitation in these cells, in contrast to A 431 cells. Triton X-100-resistant membranes from Caco-2 and A 431 cells showed the same morphological aspect by electron microscopy and peaked at the same isopycnic density on sucrose gradients. Detergent-resistant microdomains from Caco-2 cells were enriched in glycosyl phosphatidylinositol (GPI)-anchored proteins, in sucrase-isomaltase, an apical marker, and in most of the proteins found in caveolin-rich membranes such as src-like proteins, fimbrin, ezrin, and Gi alpha. Caveolae-like structures were present in A 431 but absent from Caco-2 cells at the electron microscopic level. Detergent-resistant microdomains from Caco-2 cells resemble caveolin-rich microdomains in their molecular composition but do not seem to derive from morphologically identified caveolae. Our results also indicate that caveolin is not necessary for sorting of GPI-linked proteins to the apical membrane of Caco-2 cells.

A cytoplasmic tyrosine is essential for the basolateral localization of mutants of the human nerve growth factor receptor in Madin-Darby canine kidney cells.

Deletion of 58 internal amino acids from the C-terminal cytoplasmic domain of p75 human nerve growth factor receptor (hNGFR) changes its localization from apical to basolateral in transfected Madin-Darby Canine Kidney (MDCK) cells (Le Bivic, A., Sambuy, Y., Patzak, A., Patil, N., Chao, M., and Rodriguez-Boulan, E. (1991) J. Cell Biol. 115, 607-618). The mutant protein, PS-NGFR, also shows a dramatic increase in its ability to endocytose NGF and to recycle through basolateral endosomes. We report here the site-directed mutagenesis analysis of PS-NGFR to localize and characterize its basolateral and endocytic sorting signals. Both signals reside in the proximal part of the PS cytoplasmic tail, between positions 306 and 314. Transferring the cytoplasmic tail (19 residues) and transmembrane domain of a truncated PS mutant to the ectodomain of the placental alkaline phosphatase, an apical glypiated ectoenzyme, redirected it to the basolateral membrane and the endocytic compartments. A tyrosine at position 308, present in this short cytoplasmic segment, was mutated into phenylalanine or alanine. The resulting mutants were expressed predominantly on the apical membrane of MDCK cells. Their ability to endocytose NGF was reduced with the alanine mutant showing the stronger diminution. The PS mutant contains a short cytoplasmic sequence necessary both for basolateral targeting and endocytosis, and the requirement for tyrosine at position 308 is crucial for basolateral targeting.