Ger J. Strous

Mucin-type glycoproteins

Considerable advances have been made in recent years in our understanding of the biochemistry of mucin-type glycoproteins. This class of compounds is characterized mainly by a high level of O-linked oligosaccharides. Initially, the glycoproteins were solely known as the major constituents of mucus. Recent studies have shown that mucins from the gastrointestinal tract, lungs, salivary glands, sweat glands, breast, and tumor cells are structurally related to high-molecular-weight glycoproteins, which are produced by epithelial cells as membrane proteins. During mucin synthesis, an orchestrated sequence of events results in giant molecules of Mr 4 to 6 x 10(6), which are stored in mucous granules until secretion. Once secreted, mucin forms a barrier, not only to protect the delicate epithelial cells against the extracellular environment, but also to select substances for binding and uptake by these epithelia. This review is designed to critically examine relations between structure and function of the different compounds categorized as mucin glycoproteins.

Intracellular site of asialoglycoprotein receptor-ligand uncoupling: Double-label immunoelectron microscopy during receptor-mediated endocytosis

In rats infused with asialoglycoprotein for 60 min, receptor-mediated endocytosis of the ligand occurred exclusively in hepatic parenchymal cells. We have used double-label immunoelectron microscopy on ultrathin cryosections of rat liver to identify the site at which the asialoglycoprotein receptor and its ligand dissociate following their common endocytosis. Asialoglycoprotein receptor, ligand and clathrin were identified and quantitated by the use of monospecific antibodies followed by gold-protein A complexes. Both receptor and ligand were found associated with the membrane of clathrin-coated vesicles close to the cell surface. We identified other vesicles that contained ligand accumulated within the lumen. The membranes of these latter vesicles contained little receptor, but receptor was concentrated in tubular extensions that were largely free of ligand. We call this organelle CURL (compartment of uncoupling of receptor and ligand). CURL vesicles appear to transform into secondary lysosomes, wherein the ligand is degraded. The tubular vesicles are, we propose, an intermediate in recycling the receptor to the cell surface.

Intracellular receptor sorting during endocytosis: Comparative immunoelectron microscopy of multiple receptors in rat liver

Using double-label quantitative immunoelectron microscopy on ultrathin cryosections of rat liver, we have compared the endocytotic pathways of the receptors for asialoglycoprotein (ASGP-R), mannose-6-phosphate ligands (MP-R), and polymeric IgA (IgA-R). All three were found within the Golgi complex, along the entire plasma membrane, in coated pits and vesicles, and within a compartment of uncoupling of receptors and ligand ( CURL ). The receptors occurred randomly at the cell surface, in coated pits and vesicles. Within CURL tubules ASGP-R and MP-R were colocalized , but IgA-R and ASGP-R displayed dramatic microheterogeneity. Thus, in addition to its role in uncoupling and sorting recycling receptor from ligand, CURL serves as a compartment to segregate recycling receptor (e.g. ASGP-R) from receptor involved in transcytosis (e.g. IgA-R).

Wnt signals are transmitted through N‐terminally dephosphorylated β‐catenin

β‐catenin mediates Wnt signaling by acting as the essential co‐activator for TCF transcription factors. Wnt signaling increases the half‐life and therefore the absolute level of β‐catenin in responding cells. The current model states that these changes in β‐catenin stability set the threshold for Wnt signaling. However, we find that pharmacological inhibition of proteasome activity by ALLN leads to accumulation of cytosolic β‐catenin but not to increased TCF‐mediated transcription. In addition, in temperature‐sensitive ubiquitylation mutant CHO cells inhibition of ubiquitylation increases β‐catenin levels, but does not induce transcriptional activation of TCF reporter genes. Using an antibody specific for β‐catenin dephosphorylated at residues Ser37 and Thr41, we show that Wnt signals specifically increase the levels of dephosphorylated β‐catenin, whereas ALLN does not. We conclude that changes in the phosphorylation status of the N‐terminus of β‐catenin that occur upon Wnt signaling independently affect the signaling properties and half‐life of β‐catenin. Hence, Wnt signals are transduced via N‐terminally dephosphorylated β‐catenin.

Hepatoma secretory proteins migrate from rough endoplasmic reticulum to Golgi at characteristic rates

In eukaryotic cells, secretory proteins and glycoproteins migrate from the rough endoplasmic reticulum, their site of synthesis, through Golgi vesicles before being released from the cell1–9. Cellular and viral integral plasma membrane glycoproteins are co-translationally inserted into the rough endoplasmic reticulum membrane and follow a similar pathway to the cell surface2,3,10–15. Previous studies using endoglycosidase H (Endo H) suggested that in rat hepatoma cells the vesicular stomatitis virus (VSV) G protein, albumin and transferrin migrate from the rough endoplasmic reticulum to the Golgi apparatus at different rates16. Here we show directly that in human hepatoma HepG2 cells, five secreted proteins mature from the rough endoplasmic reticulum to Golgi vesicles at characteristic rates which differ at least threefold. The results are incompatible with bulk-phase movement of the luminal contents of the endoplasmic reticulum, and suggest that there is a membrane-bound receptor that selectively mediates the transport of secretory proteins from the rough endoplasmic reticulum to the Golgi.

39 kDa receptor-associated protein is an ER resident protein and molecular chaperone for LDL receptor-related protein.

The low density lipoprotein receptor‐related protein (LRP) is a multifunctional endocytic receptor with the ability to bind and endocytose several structurally and functionally distinct ligands. A 39 kDa receptor‐associated protein (RAP) inhibits all ligand interactions with LRP in vitro. In the present study, we demonstrate that RAP is an endoplasmic reticulum (ER) resident protein. The tetrapepetide sequence HNEL at the C‐terminus of RAP is both necessary and sufficient for RAP retention within the ER. Metabolic labeling combined with cross‐linking studies show that RAP interacts with LRP in vivo. Pulse‐chase analysis reveals that this association is transient early in the secretory pathway and coincides with LRP aggregation and reduced ligand binding activity. Both internal triplicated LRP binding domains on RAP and multiple RAP binding domains on LRP appear to contribute to the aggregation of LRP and RAP. Dissociation of RAP from LRP results from the lower pH encountered later in the secretory pathway and correlates with an increase in LRP ligand binding activity. Taken together, our results thus suggest that RAP functions intracellularly as a molecular chaperone for LRP and regulates its ligand binding activity along the secretory pathway.

Identification of a novel ubiquitin conjugation motif, required for ligand-induced internalization of the growth hormone receptor.

In addition to its role in selective protein degradation, the conjugation of ubiquitin to proteins has also been implicated in the internalization of plasma membrane proteins, including the α‐factor receptor Ste2p, uracil permease Fur4p, epithelial sodium channel ENaC and the growth hormone receptor (GHR). Binding of GH to its receptor induces receptor dimerization, resulting in the activation of signal transduction pathways and an increase of GHR ubiquitination. Previously, we have shown that the ubiquitin conjugation system mediates GH‐induced GHR internalization. Here, we present evidence that a specific domain of the GHR regulates receptor endocytosis via the ubiquitin conjugation system. This ubiquitin‐dependent endocytosis (UbE) motif consists of the amino acid sequence DSWVEFIELD and is homologous to sequences in other proteins, several of which are known to be ubiquitinated. In addition, we show that GH internalization by a truncated GHR is independent of the presence of lysine residues in the cytosolic domain of this receptor, while internalization still depends on an intact ubiquitin conjugation system. Thus, GHR internalization requires the recruitment of the ubiquitin conjugation system to the GHR UbE motif rather than the conjugation of ubiquitin to the GHR itself.

Ligand-independent growth hormone receptor dimerization occurs in the endoplasmic reticulum and is required for ubiquitin system-dependent endocytosis

The regulatory effect of growth hormone (GH) on its target cells is mediated via the GH receptor (GHR). GH binding to the GHR results in the formation of a GH-(GHR)2 complex and the initiation of signal transduction cascades via the activation of the tyrosine kinase JAK2. Subsequent endocytosis and transport to the lysosome of the ligand-receptor complex is regulated via the ubiquitin system and requires the presence of an intact ubiquitin-dependent endocytosis (UbE) motif in the cytosolic tail of the GHR. Recently, the model of ligand-induced receptor dimerization has been challenged. In this study, ligand-independent GHR dimerization is demonstrated in the endoplasmic reticulum and at the cell surface by coimmunoprecipitation of an epitope-tagged truncated GHR with wild-type GHR. In addition, evidence is provided that the extracellular domain of the GHR is not required to maintain this interaction. Internalization of a chimeric receptor, which fails to dimerize, is independent of an intact UbE-motif. Therefore, we postulate that dimerization of GHR molecules is required for ubiquitin system-dependent endocytosis.

Endocytosis and Degradation of the Growth Hormone Receptor Are Proteasome-dependent

The ubiquitin conjugation system is involved in ligand-induced endocytosis of the growth hormone receptor (GHR) via a cytosolic 10-amino acid ubiquitin-dependent endocytosis motif. Herein, we demonstrate that the proteasome is also involved in growth hormone receptor down-regulation. Ligand-induced degradation was blocked in the presence of specific proteasomal inhibitors. In addition, growth hormone (GH) internalization was inhibited, whereas the transferrin receptor cycle remained unaffected. A truncated GHR entered the cells independent of proteasome action. In addition, we show that GH internalization is independent of the presence of lysine residues in the cytosolic domain of the receptor, whereas its internalization can still be inhibited by proteasomal inhibitors. Thus, GHR internalization requires proteasome action in addition to an active ubiquitin conjugation system, but ubiquitination of the GHR itself seems not to be required.

Epidermal Growth Factor Induces Ubiquitination of Eps15

Epidermal growth factor (EGF) receptor pathway substrate clone 15 (Eps15) has been described as a 142-kDa EGF receptor substrate. It has been shown to bind to the EGF receptor, adaptor protein-2, and clathrin and is present at clathrin-coated pits and vesicles. Upon stimulation of cells with EGF or transforming growth factor α, Eps15 becomes rapidly and transiently phosphorylated on tyrosine residues. This phosphorylation coincides with an increase of 8 kDa in molecular mass. Here we show that this increase in molecular mass is not due to tyrosine phosphorylation. Instead, we found both by Western blotting and protein sequencing that this EGF-induced increase in molecular mass is the result of monoubiquitination. Eps15 ubiquitination but not tyrosine phosphorylation was inhibited under conditions that blocked EGF-induced internalization of the EGF receptor. Our results establish ubiquitination as a second form of EGF-stimulated covalent modification of Eps15.