G J Strous

The ubiquitin conjugation system is required for ligand-induced endocytosis and degradation of the growth hormone receptor.

The ubiquitin‐dependent protein degradation system has recently been implicated in downregulation of signal transducing receptors. Growth hormone receptor (GHR) cDNA was transfected into Chinese hamster ovary cells, which exhibit a temperature‐sensitive defect in ubiquitin conjugation (CHO‐ts20), as well as into wild‐type cells (CHO‐E36). Upon binding of growth hormone (GH), two GHR polypeptides dimerize and initiate signal transduction. In CHO‐E36 and in CHO‐ts20 at the permissive temperature the GHR was ubiquitinated and degraded in a GH‐dependent fashion. However, at the non‐permissive temperature in CHO‐ts20 cells, neither GH‐dependent uptake nor degradation of the GHR was observed, while in CHO‐E36 cells both GHR uptake and degradation were accelerated. Incubation of CHO‐E36 cells with inhibitors of endosomal/lysosomal function (NH4Cl, bafilomycin A1) markedly reduced ligand‐induced GHR degradation. Our results indicate that a functional ubiquitin conjugating system is required for GH‐induced endocytosis and that degradation of both the exoplasmic and cytoplasmic portions of the GHR occurs within the endosomal/lysosomal compartment.

Immunoelectron microscopic localization of acidic intracellular compartments in hepatoma cells.

Using protein A‐colloidal gold immunoelectron microscopy and monospecific antibodies to the weak base primaquine, we have delineated acidic intracellular compartments in the human hepatoma cell line, HepG2. Primaquine specifically accumulated within endocytotic compartments (including CURL vesicles, multivesicular bodies and lysosomes). In addition, the Golgi cisternae were positive. However, the CURL tubules, which contain recycling asialoglycoprotein receptor, did not accumulate primaquine. Thus, there may be a gradient of acidification within the endocytotic pathway.

A cycloheximide-resistant pool of receptors for asialoglycoproteins and mannose 6-phosphate residues in the Golgi complex of hepatocytes.

Following in vivo administration of cycloheximide (20 mg/kg body weight i.p.) protein synthesis was completely inhibited (99%) in rat liver. No newly synthesized asialoglycoprotein receptor (ASGP‐R) could be detected by metabolic labeling. Fluorescence immunocytochemistry of several secretory proteins and plasma membrane proteins, including the receptors for polymeric IgA (IgA‐R), demonstrated a rapid loss from the Golgi complex following cycloheximide administration. On the other hand, two membrane proteins, the receptors for ASGP‐R and mannose 6‐phosphate (MP‐R), were not altered in their cellular localization including the Golgi. Using quantitative immunoelectron microscopy with colloidal gold, we found that 2 h and 4 h after cycloheximide administration, the densities of ASGP‐R and MP‐R in the membranes of the Golgi complex were unaltered compared with control liver. Similarly, there was no significant effect of cycloheximide on the receptor labeling in coated vesicles and compartment of uncoupling receptors and ligands (CURL). These observations are consistent with an involvement of the Golgi and CURL pools of the receptors in intracellular trafficking, endocytosis and receptor recycling.

Recycling of 5'-nucleotidase in a rat hepatoma cell line.

Intracellular movement of cell surface 5′‐nucleotidase was studied in H4S cells, a rat hepatoma cell line. Surface labelled cells were incubated for various periods at 37 degrees C and treated with neuraminidase at 0 degrees C. Removal of sialic acid residues from glycoproteins results in a change of their isoelectric points. Analysis with isoelectric focusing was then used to distinguish between cell surface and intracellular 5′‐nucleotidase. Incubation of 125I‐surface‐labelled cells at 37 degrees C resulted in a gradual decrease of labelled 5′‐nucleotidase at the plasma membrane until, at 60 to 90 min, a steady state was reached with 52% of the label on the cell surface and 48% intracellular. Pretreatment of the cells with the weak base primaquine had no influence on this distribution while at the same time uptake of iron via the transferrin receptor was inhibited. Using immunoelectron microscopy 5′‐nucleotidase was found on the cell surface, in multivesicular endosomes and the Golgi complex. Preincubation of the cells in the presence of cycloheximide caused a reduction of labelling in the Golgi complex, whereas the label in the other compartments was retained. These results lead to the conclusion that 5′‐nucleotidase does not recycle through the Golgi complex and that in contrast to the transferrin receptor the recycling of 5′‐nucleotidase is not inhibited by primaquine.

Ubiquitin System-Dependent Regulation of Growth Hormone Receptor Signal Transduction

The growth hormone (GH) receptor is a key regulator of cellular metabolism. Unlike most growth factor receptors, its downregulation is not initiated by its ligand. Like many growth factor receptors, specific molecular mechanisms guarantee that a receptor can signal only once in its lifetime. Three features render the GH receptor unique: (a) an active ubiquitination system is required for both uptake (endocytosis) and degradation in the lysosomes; (b) uptake of the receptor is a continuous process, independent of both GH binding and Jak2 signal transduction; (c) only the cell surface expression of dimerised GH receptors is controlled by the ubiquitin system. This system enables two independent regulatory mechanisms for the endocrinology of the GH/GHR axis: the pulsatile secretion of GH by the pituitary and the GH sensitivity of individual cells of the body by the effects of the ubiquitin system on GH receptor availability.

A murine and a porcine coronavirus are released from opposite surfaces of the same epithelial cells

Abstract Epithelial cells are important target cells for coronavirus infection. Earlier we have shown that transmissible gastroenteritis coronavirus (TGEV) and mouse hepatitis coronavirus (MHV) are released from different sides of porcine and murine epithelial cells, respectively. To study the release of these viruses from the same cells, we constructed a porcine LLC-PK1 cell line stably expressing the recombinant MHV receptor cDNA (LMR cells). The MHV and TGEV receptor glycoproteins were shown by immunofluorescence to appear at the surface of the cells and to be functional so that the cells were susceptible to both MHV and TGEV infection. Both coronaviruses entered polarized LMR cells only through the apical surface. Remarkably, while the cells remained susceptible to TGEV for long periods, infectability by MHV decreased with time after plating of the cells onto filters. This was not due to a lack of expression of the MHV receptor, since this glycoprotein was still abundant on the apical surface of these cells. TGEV and MHV appeared to exit LMR cells from opposite sides. Whereas TGEV was released preferentially at the apical membrane, MHV was released preferentially at the basolateral surface. These results show that vesicles containing the two coronaviruses are targeted differently in LMR cells. We propose that the viruses are sorted at the Golgi complex into different transport vesicles that carry information directing them to one of the two surface domains. The apical release of TGEV and the basolateral release of MHV might be factors contributing to the difference in virus spread found between TGEV and MHV in their respective natural hosts, the former causing mainly a localized enteric infection, the latter spreading through the body to other organs.

Mouse hepatitis virus strain A59 is released from opposite sides of different epithelial cell types

Coronaviruses infect humans and animals through epithelial cells of the gastrointestinal and respiratory tracts that serve as their primary target. When studying infections in cultured polarized epithelial cells, we found previously that coronaviruses are released from specific plasma-membrane domains; thus, mouse hepatitis virus (strain A59; MHV-A59) leaves murine epithelial kidney cells from the basolateral surface, whereas release of transmissible gastroenteritis virus from porcine epithelial kidney cells is confined to the apical membrane. This observation begged the question whether a particular coronavirus is consistently shed through the same membrane, irrespective of the nature of the epithelial cell. We therefore extended our studies with MHV-A59 to Madin-Darby canine kidney (MDCK) strain I and human colon carcinoma (Caco-2) cells, both of which are naturally refractory to MHV-A59 but were made susceptible to infection by transfection with recombinant MHV receptor cDNA. The release of MHV-A59 from Caco(MHVR) cells occurred preferentially from the basolateral side, consistent with our previous observations. In contrast, release from MDCK(MHVR) cells occurred almost exclusively from the apical surface. Because of this difference, we studied MHV-A59 infection of MDCK(MHVR) cells in more detail. The virus entered the cells preferentially from the apical side, a situation similar to that in murine epithelial cells, where the highest density of MHV receptor glycoprotein was found. The results from this and previous studies show that targeting of vesicles containing MHV-A59 to a specific side of epithelial cells may vary in different epithelial cell types.

Identification of the ubiquitin ligase Triad1 as a regulator of endosomal transport.

Summary The ubiquitin system plays an important role in trafficking of signaling receptors from the plasma membrane to lysosomes. Triad1 is a ubiquitin ligase that catalyzes the formation of poly-ubiquitin chains linked via lysine-48 as well as lysine-63 residues. We show that depletion of Triad1 affects the sorting of both growth hormone and epidermal growth factor. Triad1-depleted cells accumulate both ligands in endosomes. While fluid phase transport to the lysosomes is reduced in the absence of Triad1, growth hormone receptor can recycle back to the plasma membrane together with transferrin. Using immune electron microscopy we show that Triad1 depletion results in enlarged endosomes with enlarged and irregular shaped intraluminal vesicles. The endosomes display prominent clathrin coats and show increased levels of growth hormone label. We conclude that Triad1 is required for the proper function of multivesicular bodies.

Coronaviruses in polarized epithelial cells

Coronaviruses have a marked tropism for epithelial cells. In this paper the interactions of the porcine transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV-A59) with epithelial cells are compared. Porcine (LLC-PK1) and murine (mTAL) epithelial cells were grown on permeable supports. By inoculation from the apical or basolateral side both TGEV and MHV-A59 were found to enter the polarized cells only through the apical membrane. The release of newly synthesized TGEV from LLC-PK1 cells occurred preferentially from the apical plasma membrane domain, as evidenced by the accumulation of viral proteins and infectivity in the apical culture fluid. In contrast, MHV was released preferentially from the basolateral membrane of mTAL cells. The apical release of TGEV and the basolateral release of MHV may explain the in vivo establishment of a local and systemic infection, respectively.