Dominant negative mutation in cell surface beta 1,4-galactosyltransferase inhibits cell-cell and cell-matrix interactions.

Evans, Susan C.; Lopez, Linda C.; Shur, Barry D.

doi:10.1083/jcb.120.4.1045

Cited by 73 publications

(72 citation statements)

References 47 publications

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“…As a control, the Xba I/Ban I fragment (728 bp) containing the coding sequence of CAT was purified from the pCAT-Basic vector. The gel-purified fragments were treated with T4 DNA polymerase to generate blunt ends, then each was cloned back into either Xba I-cut, T4 polymeraseblunted EV142 vector [containing the metallothionein I (MT-1)-inducible promoter], or Pst I-cut, blunted pKJ vector [containing the phosphoglycerokinase (PGK) constitutive promoter (Evans et al, 1993)]. Orientation of the fragments was confirmed by restriction endonuclease digestion analysis and by sequencing.…”

Section: Cell Culturementioning

confidence: 99%

“…Construction of the truncated long (TLGT) and truncated short (TSGT) GalT I constructs has been previously described (Evans et al, 1993). The TL-CAT and TS-CAT fusions were generated by removing TLGT and TSGT from the EV142 vector (Evans et al, 1993), followed by digestion with Xba I, and cloning into the Xba I site of the pCAT-Basic vector (Promega). The TL-CAT and TS-CAT fusions were removed from pCAT-Basic by digestion with Hind III and Nar I followed by Ban I.…”

Section: Cell Culturementioning

confidence: 99%

“…This indirectly illustrates the ability of the long cytoplasmic domain to function at the cell surface (Evans et al, 1993). In this study, we took a more direct approach by asking if the cytoplasmic and transmembrane domains of long GalT I are able to target a reporter construct to the cell surface, and if so, if mutating specific amino acid residues within the cytoplasmic domain abrogates cell surface expression and/or function.…”

mentioning

confidence: 99%

“…In this regard, previous results show that both GalT I isoforms can function biosynthetically in the Golgi complex. However, only the long GalT I isoform can function as a receptor on the cell surface because of its association with the detergent-insoluble cytoskeleton (Evans et al, 1993;Wassler et al, 2001;Wassler and Shur, 2000). The long cytoplasmic domain may influence GalT I subcellular distribution because of its increased length (Munro, 1995), or specific sequences within the long cytoplasmic domain may override putative Golgi retention signals thought to reside in the transmembrane domain (Colley, 1997).…”

mentioning

confidence: 99%

“…However, a portion of GalT I is also found on the cell surface where it functions as a cell adhesion molecule during a variety of cellular interactions by binding N-acetylglucosamine (GlcNAc)-containing oligosaccharide substrates, or ligands, in the extracellular matrix (Shur et al, 1998). Similar to other cell adhesion molecules and matrix receptors, GalT I requires association with the detergent-insoluble pool in order to function as a receptor for extracellular glycoside ligands (Appeddu and Shur, 1994a;Appeddu and Shur, 1994b;Eckstein and Shur, 1992;Evans et al, 1993). Furthermore, ligand-induced GalT I aggregation activates cell-specific intracellular signaling cascades, including transient activation of focal adhesion kinase leading to cytoskeletal reorganization during fibroblast migration (Wassler and Shur, 2000), and heterotrimeric G-protein activation leading to vesicle exocytosis in sperm and Xenopus oocytes (Gong et al, 1995;Shi et al, 2001).…”

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confidence: 99%

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Mutational analysis of the cytoplasmic domain of β1,4-galactosyltransferase I: influence of phosphorylation on cell surface expression

Hathaway

Evans

Dubois

et al. 2003

Journal of Cell Science

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β1,4-Galactosyltransferase I (GalT I) exists in two subcellular compartments where it performs two distinct functions. The majority of GalT I is localized in the Golgi complex where it participates in glycoprotein biosynthesis; however, a small portion of GalT I is expressed on the cell surface where it functions as a matrix receptor by binding terminal N-acetylglucosamine residues on extracellular glycoside ligands. The GalT I polypeptide occurs in two alternate forms that differ only in the length of their cytoplasmic domains. It is thought that the longer cytoplasmic domain is responsible for GalT I function as a cell surface receptor because of its ability to associate with the detergent-insoluble cytoskeleton. In this study, we demonstrate that the long GalT I cytoplasmic and transmembrane domains are capable of targeting a reporter protein to the plasma membrane, whereas the short cytoplasmic and transmembrane domains do not have this property. The surface-localized GalT I reporter protein partitions with the detergent-insoluble pool, a portion of which co-fractionates with caveolin-containing lipid rafts. Site-directed mutagenesis of the cytoplasmic domain identified a requirement for serine and threonine residues for cell surface expression and function. Replacing either the serine or threonine with aspartic acid reduces surface expression and function, whereas substitution with neutral alanine has no effect on surface expression or function. These results suggest that phosphorylation negatively regulates GalT I function as a surface receptor. Consistent with this, phosphorylation of the endogenous, full-length GalT I inhibits its stable expression on the cell surface. Thus, the 13 amino acid extension unique to the long GalT I isoform is required for GalT I expression on the cell surface, the function of which is regulated by phosphorylation.

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Section: Cell Culturementioning

confidence: 99%

Section: Cell Culturementioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Mutational analysis of the cytoplasmic domain of β1,4-galactosyltransferase I: influence of phosphorylation on cell surface expression

Hathaway

Evans

Dubois

et al. 2003

Journal of Cell Science

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Translation of genome to glycome: role of the Golgi apparatus

et al. 2019

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Glycans are one of the four biopolymers of the cell and they play important roles in cellular and organismal physiology. They consist of both linear and branched structures and are synthesized in a nontemplated manner in the secretory pathway of mammalian cells with the Golgi apparatus playing a key role in the process. In spite of the absence of a template, the glycans synthesized by a cell are not a random collection of possible glycan structures but a distribution of specific glycans in defined quantities that is unique to each cell type (Cell type here refers to distinct cell forms present in an organism that can be distinguished based on morphological, phenotypic and/or molecular criteria.) While information to produce cell type‐specific glycans is encoded in the genome, how this information is translated into cell type‐specific glycome (Glycome refers to the quantitative distribution of all glycan structures present in a given cell type.) is not completely understood. We summarize here the factors that are known to influence the fidelity of glycan biosynthesis and integrate them into known glycosylation pathways so as to rationalize the translation of genetic information to cell type‐specific glycome.

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