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

Yeaman C, Le Gall AH, Baldwin AN, Monlauzeur L, Le Bivic A, Rodriguez-Boulan E - J. Cell Biol. (1997)

Bottom Line: 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.Basolateral sorting stalk-minus p75(NTR) does not occur by default, but requires sequences present in the cytoplasmic domain.However, the single N-glycan present on p75(NTR) is not required for apical sorting of either transmembrane or secreted forms.

View Article: PubMed Central - PubMed

Affiliation: Dyson Vision Research Institute, Department of Ophthalmology, and Department of Cell Biology, Cornell University Medical College, New York 10021, USA.

ABSTRACT
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.

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Amino acids 193–215 can be deleted from the juxtamembrane stalk domain without compromising apical sorting  of p75NTR. (A) Stably transfected MDCK clones expressing  Δ193–215 p75NTR were grown on filters for 5 d and then subjected  to domain-selective biotinylation with NHS-SS-biotin as described in Materials and Methods. Biotinylated proteins were  precipitated from cell lysates with avidin-agarose and resolved by  SDS-PAGE. Electrophoretic protein transfers were probed with  rabbit polyclonal antibodies against the cytoplasmic domain of  p75NTR and detected with 125I-anti–rabbit IgG. Blots were analyzed with a Molecular Dynamics phosphorimager. Bars represent the mean ± SD of triplicate determinations from a representative experiment. (B) Stably transfected MDCK cultures  expressing Δ193–215 p75NTR were starved in cysteine-free medium for 30 min and then pulse labeled with [35S]cysteine for 20  min. After the pulse, cultures were chased in medium containing  an excess of unlabeled cysteine. At indicated chase times, filters  were placed on ice and incubated with NHS-SS-biotin either in  the apical or basolateral medium. Δ193–215 p75NTR was immunoprecipitated from cell lysates with an antibody against the cysteine-rich domain (ME 20.4) and surface-labeled protein was recovered on streptavidin-agarose.
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Figure 7: Amino acids 193–215 can be deleted from the juxtamembrane stalk domain without compromising apical sorting of p75NTR. (A) Stably transfected MDCK clones expressing Δ193–215 p75NTR were grown on filters for 5 d and then subjected to domain-selective biotinylation with NHS-SS-biotin as described in Materials and Methods. Biotinylated proteins were precipitated from cell lysates with avidin-agarose and resolved by SDS-PAGE. Electrophoretic protein transfers were probed with rabbit polyclonal antibodies against the cytoplasmic domain of p75NTR and detected with 125I-anti–rabbit IgG. Blots were analyzed with a Molecular Dynamics phosphorimager. Bars represent the mean ± SD of triplicate determinations from a representative experiment. (B) Stably transfected MDCK cultures expressing Δ193–215 p75NTR were starved in cysteine-free medium for 30 min and then pulse labeled with [35S]cysteine for 20 min. After the pulse, cultures were chased in medium containing an excess of unlabeled cysteine. At indicated chase times, filters were placed on ice and incubated with NHS-SS-biotin either in the apical or basolateral medium. Δ193–215 p75NTR was immunoprecipitated from cell lysates with an antibody against the cysteine-rich domain (ME 20.4) and surface-labeled protein was recovered on streptavidin-agarose.

Mentions: We reported previously that deletion of amino acids 203–215 within the stalk domain did not affect apical sorting of human p75NTR (Le Bivic et al., 1991). Although it is true that deletion of residues 203–215 removes a cluster of potentially glycosylated amino acids, the mutant receptor (BE p75NTR) still retains nine serine/threonine residues within the region spanning residues 168–218 (Table I). To further define the size of the domain involved in apical sorting, a third mutant was constructed, in which amino acids 193–215 were deleted (Fig. 1). This protein retains 10 of the 16 serine/threonine residues present in the native receptor and contains O-linked oligosaccharides (Table I). Cell surface biotinylation revealed that the steady state distributions of Δ193–215 p75NTR in two independently isolated clones were 80–85% apical and 15–20% basolateral (Fig. 7 A). These distributions reflect direct delivery from the TGN, because targeting of metabolically labeled protein occurs primarily to the apical surface domain (Fig. 7 B). Therefore, apical sorting information is likely contained within a region spanning residues 168–193 or 216–218.


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

Yeaman C, Le Gall AH, Baldwin AN, Monlauzeur L, Le Bivic A, Rodriguez-Boulan E - J. Cell Biol. (1997)

Amino acids 193–215 can be deleted from the juxtamembrane stalk domain without compromising apical sorting  of p75NTR. (A) Stably transfected MDCK clones expressing  Δ193–215 p75NTR were grown on filters for 5 d and then subjected  to domain-selective biotinylation with NHS-SS-biotin as described in Materials and Methods. Biotinylated proteins were  precipitated from cell lysates with avidin-agarose and resolved by  SDS-PAGE. Electrophoretic protein transfers were probed with  rabbit polyclonal antibodies against the cytoplasmic domain of  p75NTR and detected with 125I-anti–rabbit IgG. Blots were analyzed with a Molecular Dynamics phosphorimager. Bars represent the mean ± SD of triplicate determinations from a representative experiment. (B) Stably transfected MDCK cultures  expressing Δ193–215 p75NTR were starved in cysteine-free medium for 30 min and then pulse labeled with [35S]cysteine for 20  min. After the pulse, cultures were chased in medium containing  an excess of unlabeled cysteine. At indicated chase times, filters  were placed on ice and incubated with NHS-SS-biotin either in  the apical or basolateral medium. Δ193–215 p75NTR was immunoprecipitated from cell lysates with an antibody against the cysteine-rich domain (ME 20.4) and surface-labeled protein was recovered on streptavidin-agarose.
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Related In: Results  -  Collection

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Figure 7: Amino acids 193–215 can be deleted from the juxtamembrane stalk domain without compromising apical sorting of p75NTR. (A) Stably transfected MDCK clones expressing Δ193–215 p75NTR were grown on filters for 5 d and then subjected to domain-selective biotinylation with NHS-SS-biotin as described in Materials and Methods. Biotinylated proteins were precipitated from cell lysates with avidin-agarose and resolved by SDS-PAGE. Electrophoretic protein transfers were probed with rabbit polyclonal antibodies against the cytoplasmic domain of p75NTR and detected with 125I-anti–rabbit IgG. Blots were analyzed with a Molecular Dynamics phosphorimager. Bars represent the mean ± SD of triplicate determinations from a representative experiment. (B) Stably transfected MDCK cultures expressing Δ193–215 p75NTR were starved in cysteine-free medium for 30 min and then pulse labeled with [35S]cysteine for 20 min. After the pulse, cultures were chased in medium containing an excess of unlabeled cysteine. At indicated chase times, filters were placed on ice and incubated with NHS-SS-biotin either in the apical or basolateral medium. Δ193–215 p75NTR was immunoprecipitated from cell lysates with an antibody against the cysteine-rich domain (ME 20.4) and surface-labeled protein was recovered on streptavidin-agarose.
Mentions: We reported previously that deletion of amino acids 203–215 within the stalk domain did not affect apical sorting of human p75NTR (Le Bivic et al., 1991). Although it is true that deletion of residues 203–215 removes a cluster of potentially glycosylated amino acids, the mutant receptor (BE p75NTR) still retains nine serine/threonine residues within the region spanning residues 168–218 (Table I). To further define the size of the domain involved in apical sorting, a third mutant was constructed, in which amino acids 193–215 were deleted (Fig. 1). This protein retains 10 of the 16 serine/threonine residues present in the native receptor and contains O-linked oligosaccharides (Table I). Cell surface biotinylation revealed that the steady state distributions of Δ193–215 p75NTR in two independently isolated clones were 80–85% apical and 15–20% basolateral (Fig. 7 A). These distributions reflect direct delivery from the TGN, because targeting of metabolically labeled protein occurs primarily to the apical surface domain (Fig. 7 B). Therefore, apical sorting information is likely contained within a region spanning residues 168–193 or 216–218.

Bottom Line: 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.Basolateral sorting stalk-minus p75(NTR) does not occur by default, but requires sequences present in the cytoplasmic domain.However, the single N-glycan present on p75(NTR) is not required for apical sorting of either transmembrane or secreted forms.

View Article: PubMed Central - PubMed

Affiliation: Dyson Vision Research Institute, Department of Ophthalmology, and Department of Cell Biology, Cornell University Medical College, New York 10021, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus