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.

Beta-arrestin and Mdm2, unsuspected partners in signaling from the cell surface.

Mdm2 is a ubiquitin-protein ligase known to ubiquitinate p53, promoting its degradation by the ubiquitin-proteasome system. Shenoy and co-workers showed that Mdm2 can act as a key factor in the sequestration of the cell surface beta(2)-adrenergic receptor (beta-AR) through interactions with beta-arrestin. Strous and Schantl discuss how Mdm2 may be a switch connecting extracellular signals mediated through G protein-coupled receptors (GPCRs) to p53 and its functions in apoptosis and cell cycle progression.

The ubiquitin-dependent endocytosis motif is required for efficient incorporation of growth hormone receptor in clathrin-coated pits, but not clathrin-coated lattices.

Endocytosis of the growth hormone receptor (GHR) requires an active ubiquitin-conjugation system. In addition, it depends on a 10 amino acid residues motif in the GHR-cytoplasmic tail, the ubiquitin dependent-endocytosis or UbE-motif. To gain insight into the role of ubiquitination in the early steps of endocytosis, we performed an ultrastructural analysis of GH-uptake in Chinese hamster cells expressing wild-type or mutant GHRs. In wild-type GHR cells, GH was found to be exclusively taken up via clathrin-coated pits. In early endosomes it was efficiently sorted from recycling transferrin and targeted to the degradative pathway. Mutation of all lysine residues of a truncated GHR (GHR-399K-) precludes ubiquitination of the receptor, but internalization of GHR-399K- still depends on an active ubiquitin system. We found that GHR-399K- incorporates GH into clathrin-coated vesicles with the same efficiency as wild-type GHR. By contrast, a mutation in the UbE-motif (GHR-F327A) largely abolished incorporation of GH into clathrin-coated vesicles. Notably, access of GH to clathrin-coated lattices was not affected in GHR-F327A cells. These data corroborate and extend previous data that the UbE-motif but not ubiquitination of the receptor itself recruits GHR into clathrin-coated vesicles. Moreover, they suggest that incorporation of GHR into clathrin-coated lattices is differentially regulated from incorporation into clathrin-coated pits.

Proteasome inhibitors block a late step in lysosomal transport of selected membrane but not soluble proteins.

The ubiquitin-proteasome pathway acts as a regulator of the endocytosis of selected membrane proteins. Recent evidence suggests that it may also function in the intracellular trafficking of membrane proteins. In this study, several models were used to address the role of the ubiquitin-proteasome pathway in sorting of internalized proteins to the lysosome. We found that lysosomal degradation of ligands, which remain bound to their receptors within the endocytic pathway, is blocked in the presence of specific proteasome inhibitors. In contrast, a ligand that dissociates from its receptor upon endosome acidification is degraded under the same conditions. Quantitative electron microscopy showed that neither the uptake nor the overall distribution of the endocytic marker bovine serum albumin-gold is substantially altered in the presence of a proteasome inhibitor. The data suggest that the ubiquitin-proteasome pathway is involved in an endosomal sorting step of selected membrane proteins to lysosomes, thereby providing a mechanism for regulated degradation.

The ubiquitin-proteasome pathway regulates lysosomal degradation of the growth hormone receptor and its ligand.

The growth hormone (GH) receptor (GHR) is a mammalian plasma membrane protein whose internalization is mediated by the ubiquitin-proteasome pathway. GH internalization and degradation are inhibited when cells are treated with proteasome inhibitors. Here we show that a GHR truncated at residue 369 can enter the cells in the presence of a proteasome inhibitor, but that the subsequent lysosomal degradation of GH is blocked. Lysosomal inhibitors prolong the half-life of both receptor and ligand. Experiments with antibodies against different receptor tail sections show that degradation of the GHR cytosolic domain precedes degradation of the extracellular GH-binding domain. A possible role for the ubiquitin-proteasome pathway in the degradation of the receptor and ligand is discussed.

Growth hormone receptor ubiquitination, endocytosis, and degradation are independent of signal transduction via Janus kinase 2.

The ubiquitin-proteasome system is required in growth hormone receptor (GHR) endocytosis. For cytokine receptors, which lack intrinsic tyrosine kinase activity, signal transduction is initiated by the activation of a member of the Janus kinase (JAK) family. Previously, we have shown that GHR and JAK2 tyrosine (de) phosphorylation are regulated via the ubiquitin system. In this study, we examined the role of JAK2-mediated signal transduction in GHR internalization and down-regulation. Mutation of the attachment site for JAK2, box-1, in the GHR cytoplasmic tail resulted in the complete absence of GHR and JAK2 phosphorylation. This modification did not alter the rate and extent of receptor-bound growth hormone internalization as compared with a functional GHR, nor did it change its turnover and transport to the plasma membrane. In addition, the receptor was still normally ubiquitinated and remained dependent on both an intact ubiquitin system and proteasomal action for its internalization. Thus, GHR ubiquitination, endocytosis, and degradation occur independently of GHR signal transduction via JAK2. We conclude that whereas endocytosis and degradation require the ubiquitin system, they are independent of GHR signal transduction.

Identification of a major cyclic AMP-dependent protein kinase A phosphorylation site within the cytoplasmic tail of the low-density lipoprotein receptor-related protein: implication for receptor-mediated endocytosis.

The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multiligand endocytic receptor that belongs to the LDL receptor family. Recently, studies have revealed new roles of LDL receptor family members as transducers of extracellular signals. Our previous studies have demonstrated LRP phosphorylation within its cytoplasmic tail, but the nature of LRP phosphorylation and its potential function was unknown. In the present study using both in vivo and in vitro analysis, we found that LRP phosphorylation is mediated by the cAMP-dependent protein kinase A (PKA). Using site-directed mutagenesis and LRP minireceptor constructs, we further identified the predominant LRP phosphorylation site at serine 76 of its cytoplasmic tail. Finally, we demonstrated that mutations of serine 76, which abolish LRP phosphorylation by PKA, result in a decrease in the initial endocytosis rate of LRP and a lower efficiency in delivery of ligand for degradation. Thus, the role of PKA phosphorylation of LRP in receptor-mediated endocytosis may provide a mechanism by which the endocytic function of LRP can be regulated by external signals.

The signal transduction of the growth hormone receptor is regulated by the ubiquitin/proteasome system and continues after endocytosis.

The growth hormone receptor (GHR) intracellular domain contains all of the information required for signal transduction as well as for endocytosis. Previously, we showed that the proteasome mediates the clathrin-mediated endocytosis of the GHR. Here, we present evidence that the proteasomal inhibitor MG132 prolongs the GH-induced activity of both GHR and JAK2, presumably through stabilization of GHR and JAK2 tyrosine phosphorylation. If proteasomal inhibitor was combined with ligand in an endocytosis-deficient GHR mutant, the same phenomenon occurred indicating that proteasomal action on tyrosine dephosphorylation is independent of endocytosis. Experiments with a GHR-truncated tail mutant (GHR-(1-369)) led to a prolonged JAK2 phosphorylation caused by the loss of a phosphatase-binding site. This raised the question of what happens to the signal transduction of the GHR after its internalization. Co-immunoprecipitation of GH.GHR complexes before and after endocytosis showed that JAK2 as well as other activated proteins are bound to the GHR not only at the cell surface but also intracellularly, suggesting that the GHR signal transduction continues in endosomes. Additionally, these results provide evidence that GHR is present in endosomes both in its full-length and truncated form, indicating that the receptor is down-regulated by the proteasome.

Growth hormone receptor ubiquitination coincides with recruitment to clathrin-coated membrane domains.

Endocytosis of the growth hormone receptor (GHR) depends on a functional ubiquitin conjugation system. A 10-amino acid residue motif within the GHR cytosolic tail (the ubiquitin-dependent endocytosis motif) is involved in both GHR ubiquitination and endocytosis. As shown previously, ubiquitination of the receptor itself is not required. In this paper ubiquitination of the GHR was used as a tool to address the question of at which stage the ubiquitin conjugation system acts in the process of GHR endocytosis. If potassium depletion was used to interfere with early stages of coated pit formation, both GHR endocytosis and ubiquitination were inhibited. Treatment of cells with methyl-beta-cyclodextrin inhibited endocytosis at the stage of coated vesicle formation. Growth hormone addition to methyl-beta-cyclodextrin-treated cells resulted in an accumulation of ubiquitinated GHR at the cell surface. Using immunoelectron microscopy, the GHR was localized in flattened clathrin-coated membranes. In addition, when clathrin-mediated endocytosis was inhibited in HeLa cells expressing a temperature-sensitive dynamin mutant, ubiquitinated GHR accumulated at the cell surface. Together, these data show that the GHR is ubiquitinated at the plasma membrane, before endocytosis occurs, and indicate that the resident time of the GHR at the cell surface is regulated by the ubiquitin conjugation system together with the endocytic machinery.

The YXXL motif, but not the two NPXY motifs, serves as the dominant endocytosis signal for low density lipoprotein receptor-related protein.

All members of the low density lipoprotein (LDL) receptor family contain at least one copy of the NPXY sequence within their cytoplasmic tails. For the LDL receptor, it has been demonstrated that the NPXY motif serves as a signal for rapid endocytosis through coated pits. Thus, it is generally believed that the NPXY sequences function as endocytosis signals for all the LDL receptor family members. The primary aim of this study is to define the endocytosis signal(s) within the cytoplasmic tail of LDL receptor-related protein (LRP). By using LRP minireceptors, which mimic the function and trafficking of full-length endogenous LRP, we demonstrate that the YXXL motif, but not the two NPXY motifs, serves as the dominant signal for LRP endocytosis. We also found that the distal di-leucine motif within the LRP tail contributes to its endocytosis, and its function is independent of the YXXL motif. Although the proximal NPXY motif and the proximal di-leucine motif each play a limited role in LRP endocytosis in the context of the full-length tail, these motifs were functional within the truncated receptor tail. In addition, we show that LRP minireceptor mutants defective in endocytosis signal(s) accumulate at the cell surface and are less efficient in delivery of ligand for degradation.

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.

The ubiquitin-proteasome system and endocytosis.

Internalization of membrane proteins has been studied for more than three decades without solving all the underlying mechanisms. Our knowledge of clathrin-mediated endocytosis is certainly sufficient to understand the basic principles. However, more detailed insight is required to recognize why different proteins enter clathrin-coated pits with different rates and affinities. In addition to clathrin coat components, at least two adaptor systems and even more accessory proteins have been described to preselect membrane proteins before they can enter cells. Recent experimental data have identified the ubiquitin-proteasome system as a regulatory system for endocytosis. This system is well-known for its basic regulatory function in protein degradation, and controls a magnitude of key events. The ubiquitin-proteasome system is now identified as a regulator of the endocytosis of selected membrane proteins. In this review, we will discuss the complexity and implications of this mechanism for receptor-mediated endocytosis.

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 alpha-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.

Di-leucine-mediated internalization of ligand by a truncated growth hormone receptor is independent of the ubiquitin conjugation system.

The growth hormone receptor (GHR) is a member of the cytokine receptor family. Its function is to mediate cellular responses upon binding of growth hormone. Ligand binding induces dimerization and activation of the GHR. One mechanism by which the GHR is rapidly inactivated involves the ubiquitin conjugation system, a system implicated in the degradation of cytosolic and nuclear proteins. We have shown previously that the ubiquitin-conjugating system mediates internalization of the GHR. Here, we present evidence that in addition to the ubiquitin-dependent endocytosis signal, the cytosolic tail of the GHR contains a di-leucine motif. Upon truncation of the GHR at amino acid residue 349, this di-leucine motif is activated and mediates ubiquitin-independent internalization of the receptor. Di-leucine-mediated GHR internalization requires functional clathrin-coated pits and results in GHR transport to the lysosome. Although the full-length GHR internalizes independent of the di-leucine motif, this motif may function in internalization of GHR isoforms.

Molecular cloning of human gastric mucin MUC5AC reveals conserved cysteine-rich D-domains and a putative leucine zipper motif.

To further clone the human gastric mucin MUC5AC cDNA, we screened a human gastric cDNA library with previously identified MUC5AC sequences. We obtained 32 independent clones encoding newly identified sequences comprising the entire N-terminal sequence of MUC5AC, up to 3024 bp upstream of the previously identified MUC5AC sequences. The N-terminus of MUC5AC shows high homology (43% identity) with the N-terminus of MUC2 and contains three domains homologous to the D-domains found in the pro-von Willebrand factor. Furthermore, the N-terminus of MUC5AC contains a putative leucine zipper motif not found in any other mucin identified so far. Moreover, a large central repetitive sequence was identified encoding approximately 2500 amino acids (7.5 kb). We were able to establish that the MUC5AC cDNA together with the previously identified 6.1 kb of MUC5AC cDNA sequence is about 16.6 kb, encoding 5525 amino acids. A model of the domain structure of MUC5AC is presented.

Linkage of the ubiquitin-conjugating system and the endocytic pathway in ligand-induced internalization of the growth hormone receptor.

The major function of the ubiquitin-conjugating system is the targeting of cytosolic and nuclear proteins for degradation by the proteasome. Recently, ubiquitin conjugation has been implicated in the downregulation of signalling receptors such as the mammalian growth hormone receptor (GHR) and the alpha-factor receptor in yeast. By examining truncated receptors, the internalization-deficient receptor mutant F327A and conditions under which clathrin-mediated GHR endocytosis is inhibited, we show here that GHR ubiquitination and ligand-induced GHR internalization are coupled events. Previously, we had shown that GHR endocytosis is dependent on an intact ubiquitination system. Here we present evidence that GHR ubiquitination depends on an intact endocytic pathway. Our data indicate that the ubiquitin-conjugating system and the endocytic pathway interact at the cytoplasmic tail of the GHR at the plasma membrane, where they cooperate to regulate internalization of the GHR.

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 alpha, 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.

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.

Growth hormone-induced signal tranduction depends on an intact ubiquitin system.

The growth hormone receptor (GHR) is a ubiquitinated cell surface protein. Ligand binding and receptor dimerization activate the cytosolic kinase Jak2. This event initiates signal transduction via STAT proteins. Expression of GHR in a Chinese hamster ovary (CHO) cell line, which exhibits a temperature-sensitive defect in ubiquitin conjugation (CHO-ts20), as well as in wild type cells (CHO-E36) has shown that endocytosis of the receptor requires an intact ubiquitin conjugation system (Strous G. J., van Kerkhof, P., Govers, R., Ciechanover A., and Schwartz, A. L. (1996) EMBO J. 15, 3806-3812). We have now examined the requirement for ubiquitin conjugation in growth factor-mediated signal transduction. In CHO-E36 and in CHO-ts20 cells at the permissive temperature, STAT proteins were activated in a growth factor-dependent fashion. However, no activation of STAT proteins was observed at the nonpermissive temperature in CHO-ts20 cells. Neither tyrosine phosphorylation of GHR nor of Jak2 was inhibited at the nonpermissive temperature. When tyrosine phosphorylation was inhibited following treatment with staurosporin, ubiquitination of the receptor proceeded normally. Furthermore, mutation of GHR phenylalanine-327, which prevents GHR endocytosis, inhibited receptor ubiquitination but allowed normal Jak/STAT-mediated signal transduction. Thus, these data provide evidence that the ubiquitin conjugation system is involved in the Jak/STAT signaling pathway, be it not at the initial stage(s) of Jak2 activity.