Biomedical subjects

Camilla Raiborg

Publications and source records attributed to Camilla Raiborg.

16 recordsLinked to original sources

PubMed2006

A new side to ubiquitin.

Mono-ubiquitination is a common mechanism of protein regulation, and more than ten ubiquitin-interacting domains that recognize the hydrophobic region centered on Ile44 of ubiquitin have been characterized. Two recent reports describe the crystal structure of the Rab5 guanine-nucleotide-exchange factor Rabex-5 and show that it contains two novel ubiquitin-binding domains. One of these is an A20 zinc finger that binds to a polar interaction interface of ubiquitin centered on Asp58. The discovery of an alternative interaction face of ubiquitin opens new avenues for understanding how this small protein regulates protein function.

Animals↗

PubMed2006

Both clathrin-positive and -negative coats are involved in endosomal sorting of the EGF receptor.

Sorting of endocytosed EGF receptor (EGFR) to internal vesicles of multivesicular bodies (MVBs) depends on sustained activation and ubiquitination of the EGFR. Ubiquitination of EGFR is mediated by the ubiquitin ligase Cbl, being recruited to the EGFR both directly and indirectly through association with Grb2. Endosomal sorting of ubiquitinated proteins further depends on interaction with ubiquitin binding adaptors like Hrs. Hrs localizes to flat, clathrin-coated domains on the limiting membrane of endosomes. In the present study, we have investigated the localization of EGFR, Cbl and Grb2 with respect to coated and non-coated domains of the endosomal membrane and to vesicles within MVBs. Both EGFR, Grb2, and Cbl were concentrated in coated domains of the limiting membrane before translocation to inner vesicles of MVBs. While almost all Hrs was in clathrin-positive coats, EGFR and Grb2 in coated domains only partially colocalized with Hrs and clathrin. The extent of colocalization of EGFR and Grb2 with Hrs and clathrin varied with time of incubation with EGF. These results demonstrate that both clathrin-positive and clathrin-negative electron dense coats exist on endosomes and are involved in endosomal sorting of the EGFR.

Cells, Cultured↗

PubMed2006

Flat clathrin coats on endosomes mediate degradative protein sorting by scaffolding Hrs in dynamic microdomains.

Endocytosed membrane proteins that are destined for degradation in lysosomes are ubiquitylated and recognised by sorting complexes on endosome membranes. The ubiquitin-binding sorting component Hrs as well as ubiquitylated cargo are enriched in a characteristic flat clathrin coat on the endosome membrane. The function of clathrin within this coat has not been investigated. Here, we show that both clathrin and the clathrin-box motif of Hrs are required for the clustering of Hrs into restricted microdomains. The C-terminus of Hrs, which contains the clathrin-box, is sufficient to redirect a phosphatidylinositol(3)-phosphate-binding protein into the Hrs- and clathrin-containing microdomains. Although these microdomains show little lateral diffusion in the membrane, they are dynamic structures that exchange Hrs and clathrin with similar kinetics, and acquire the downstream sorting component Tsg101. The clathrin-mediated clustering is essential for the function of Hrs in degradative protein sorting. We conclude that clathrin is responsible for concentrating Hrs in endosomal microdomains specialised for recognition of ubiquitylated membrane proteins, thus enabling efficient sorting of cargo into the degradative pathway.

Cell Line↗

PubMed2006

The ESCRT-III subunit hVps24 is required for degradation but not silencing of the epidermal growth factor receptor.

The endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are thought to mediate the biogenesis of multivesicular endosomes (MVEs) and endosomal sorting of ubiquitinated membrane proteins. Here, we have compared the importance of the ESCRT-I subunit tumor susceptibility gene 101 (Tsg101) and the ESCRT-III subunit hVps24/CHMP3 for endosomal functions and receptor signaling. Like Tsg101, endogenous hVps24 localized mainly to late endosomes. Depletion of hVps24 by siRNA showed that this ESCRT subunit, like Tsg101, is important for degradation of the epidermal growth factor (EGF) receptor (EGFR) and for transport of the receptor from early endosomes to lysosomes. Surprisingly, however, whereas depletion of Tsg101 caused sustained EGF activation of the mitogen-activated protein kinase pathway, depletion of hVps24 had no such effect. Moreover, depletion of Tsg101 but not of hVps24 caused a major fraction of internalized EGF to accumulate in nonacidified endosomes. Electron microscopy of hVps24-depleted cells showed an accumulation of EGFRs in MVEs that were significantly smaller than those in control cells, probably because of an impaired fusion with lyso-bisphosphatidic acid-positive late endosomes/lysosomes. Together, our results reveal functional differences between ESCRT-I and ESCRT-III in degradative protein trafficking and indicate that degradation of the EGFR is not required for termination of its signaling.

Down-Regulation↗

PubMed2006

Double-sided ubiquitin binding of Hrs-UIM in endosomal protein sorting.

Hrs has an essential role in sorting of monoubiquitinated receptors to multivesicular bodies for lysosomal degradation, through recognition of ubiquitinated receptors by its ubiquitin-interacting motif (UIM). Here, we present the structure of a complex of Hrs-UIM and ubiquitin at 1.7-A resolution. Hrs-UIM forms a single alpha-helix, which binds two ubiquitin molecules, one on either side. These two ubiquitin molecules are related by pseudo two-fold screw symmetry along the helical axis of the UIM, corresponding to a shift by two residues on the UIM helix. Both ubiquitin molecules interact with the UIM in the same manner, using the Ile44 surface, with equal binding affinities. Mutational experiments show that both binding sites of Hrs-UIM are required for efficient degradative protein sorting. Hrs-UIM belongs to a new subclass of double-sided UIMs, in contrast to its yeast homolog Vps27p, which has two tandem single-sided UIMs.

Amino Acid Motifs↗

PubMed2006

Regulation of ubiquitin-binding proteins by monoubiquitination.

Proteins containing ubiquitin-binding domains (UBDs) interact with ubiquitinated targets and regulate diverse biological processes, including endocytosis, signal transduction, transcription and DNA repair. Many of the UBD-containing proteins are also themselves monoubiquitinated, but the functional role and the mechanisms that underlie this modification are less well understood. Here, we demonstrate that monoubiquitination of the endocytic proteins Sts1, Sts2, Eps15 and Hrs results in intramolecular interactions between ubiquitin and their UBDs, thereby preventing them from binding in trans to ubiquitinated targets. Permanent monoubiquitination of these proteins, mimicked by the fusion of ubiquitin to their carboxyl termini, impairs their ability to regulate trafficking of ubiquitinated receptors. Moreover, we mapped the in vivo monoubiquitination site in Sts2 and demonstrated that its mutation enhances the Sts2-mediated effects of epidermal-growth-factor-receptor downregulation. We propose that monoubiquitination of ubiquitin-binding proteins inhibits their capacity to bind to and control the functions of ubiquitinated targets in vivo.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Eap45 in mammalian ESCRT-II binds ubiquitin via a phosphoinositide-interacting GLUE domain.

Ubiquitination serves as a key sorting signal in the lysosomal degradation of endocytosed receptors through the ability of ubiquitinated membrane proteins to be recognized and sorted by ubiquitin-binding proteins along the endocytic route. The ESCRT-II complex in yeast contains one such protein, Vps36, which harbors a ubiquitin-binding NZF domain and is required for vacuolar sorting of ubiquitinated membrane proteins. Surprisingly, the presumptive mammalian ortholog Eap45 lacks the ubiquitin-binding module of Vps36, and it is thus not clear whether mammalian ESCRT-II functions to bind ubiquitinated cargo. In this paper, we provide evidence that Eap45 contains a novel ubiquitin-binding domain, GLUE (GRAM-like ubiquitin-binding in Eap45), which binds ubiquitin with similar affinity and specificity as other ubiquitin-binding domains. The GLUE domain shares similarities in its primary and predicted secondary structures to phosphoinositide-binding GRAM and PH domains. Accordingly, we find that Eap45 binds to a subset of 3-phosphoinositides, suggesting that ubiquitin recognition could be coordinated with phosphoinositide binding. Furthermore, we show that Eap45 colocalizes with ubiquitinated proteins on late endosomes. These results are consistent with a role for Eap45 in endosomal sorting of ubiquitinated cargo.

Amino Acid Sequence↗

PubMed2004

The growth-regulatory protein HCRP1/hVps37A is a subunit of mammalian ESCRT-I and mediates receptor down-regulation.

The biogenesis of multivesicular bodies and endosomal sorting of membrane cargo are driven forward by the endosomal sorting complexes required for transport, ESCRT-I, -II, and -III. ESCRT-I is characterized in yeast as a complex consisting of Vps23, Vps28, and Vps37. Whereas mammalian homologues of Vps23 and Vps28 (named Tsg101 and hVps28, respectively) have been identified and characterized, a mammalian counterpart of Vps37 has not yet been identified. Here, we show that a regulator of proliferation, hepatocellular carcinoma related protein 1 (HCRP1), interacts with Tsg101, hVps28, and their upstream regulator Hrs. The ability of HCRP1 (which we assign the alternative name hVps37A) to interact with Tsg101 is conferred by its mod(r) domain and is shared with hVps37B and hVps37C, two other mod(r) domain-containing proteins. HCRP1 cofractionates with Tsg101 and hVps28 by size exclusion chromatography and colocalizes with hVps28 on LAMP1-positive endosomes. Whereas depletion of Tsg101 by siRNA reduces cellular levels of both hVps28 and HCRP1, depletion of HCRP1 has no effect on Tsg101 or hVps28. Nevertheless, HCRP1 depletion strongly retards epidermal growth factor (EGF) receptor degradation. Together, these results indicate that HCRP1 is a subunit of mammalian ESCRT-I and that its function is essential for lysosomal sorting of EGF receptors.

Amino Acid Sequence↗

PubMed2004

Cbl-dependent ubiquitination is required for progression of EGF receptors into clathrin-coated pits.

Ligand binding causes the EGF receptor (EGFR) to become ubiquitinated by Cbl upon association with the adaptor protein Grb2. We have investigated the role of ubiquitin and Grb2 in ligand-induced endocytosis of the EGFR. Incubation of cells with EGF on ice caused translocation of Grb2 and Cbl from the cytosol to the rim of coated pits. Grb2 with point mutations in both SH3 domains inhibited recruitment of the EGFR to clathrin-coated pits, in a Ras-independent manner. On overexpression of the Cbl-binding protein Sprouty, ubiquitination of the EGFR was inhibited, the EGFR was recruited only to the rim of coated pits, and endocytosis of the EGFR was inhibited. Conjugation-defective ubiquitin similarly inhibited recruitment of EGF-EGFR to clathrin-coated pits. Even though this does not prove that cargo must be ubiquitinated, this indicates the importance of interaction of ubiquitinated protein(s) with proteins harboring ubiquitin-interacting domains. We propose that Grb2 mediates transient anchoring of the EGFR to an Eps15-containing molecular complex at the rim of coated pits and that Cbl-induced ubiquitination of the EGFR allows relocation of EGFR from the rim to the center of clathrin-coated pits.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Phosphatidylinositol 3-phosphate is found in microdomains of early endosomes.

Phosphatidylinositol 3-phosphate [PI(3)P] is a phosphatidylinositol 3-kinase product whose localisation is restricted to the limiting membranes of early endosomes and to the internal vesicles of multivesicular bodies. In this study the intracellular distribution of PI(3)P was compared with those of another phosphoinositide and a number of endosomal proteins. Using a 2xFYVE probe specific for PI(3)P we found that PI(3)P is present in microdomains within the endosome membrane, whereas a phosphoinositide required for clathrin-mediated endocytosis, PI(4,5)P2, was only detected at the plasma membrane. The small GTPase Rab5 as well as the PI(3)P-binding proteins EEA1, SARA and CISK were found to be abundant within PI(3)P-containing endosomal microdomains. In contrast, another PI(3)P-binding protein, Hrs, was found concentrated in clathrin-coated endosomal microdomains with low levels of PI(3)P. While PI(3)P-containing microdomains could be readily distinguished on enlarged endosomes in cells transfected with a constitutively active Rab5 mutant, such domains could also be detected in endosomes of non-transfected cells. We conclude that the membranes of early endosomes consist of microdomains in which PI(3)P and specific proteins are concentrated. These microdomains may be necessary for the assembly of distinct multimolecular complexes that specify organelle identity, membrane trafficking and receptor signalling.

Endosomes↗

PubMed2003

STAM and Hrs are subunits of a multivalent ubiquitin-binding complex on early endosomes.

STAM1 and STAM2, which have been identified as regulators of receptor signaling and trafficking, interact directly with Hrs, which mediates the endocytic sorting of ubiquitinated membrane proteins. The STAM proteins interact with the same coiled-coil domain that is involved in the targeting of Hrs to endosomes. In this work, we show that STAM1 and STAM2, as well as an endocytic regulator protein, Eps15, can be co-immunoprecipitated with Hrs both from membrane and cytosolic fractions and that recombinant Hrs, STAM1/STAM2, and Eps15 form a ternary complex. We find that overexpression of Hrs causes a strong recruitment of STAM2 to endosome membranes. Moreover, STAM2, like Hrs and Eps15, binds ubiquitin, and Hrs, STAM2, and Eps15 colocalize with ubiquitinated proteins in clathrin-containing endosomal microdomains. The localization of Hrs, STAM2, Eps15, and clathrin to endosome membranes is controlled by the AAA ATPase mVps4, which has been implicated in multivesicular body formation. Depletion of cellular Hrs by small interfering RNA results in a strongly reduced recruitment of STAM2 to endosome membranes and an impaired degradation of endocytosed epidermal growth factor receptors. We propose that Hrs, Eps15, and STAM proteins function in a multivalent complex that sorts ubiquitinated proteins into the multivesicular body pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Protein sorting into multivesicular endosomes.

Multivesicular endosomes are important as compartments for receptor downregulation and as intermediates in the formation of secretory lysosomes. Work during the past year has shed light on the molecular mechanisms of protein sorting into multivesicular endosomes and yielded information about the machinery involved in multivesicular endosome formation. Monoubiquitination functions as a signal for sorting transmembrane proteins into intraluminal vesicles of multivesicular endosomes and subsequent delivery to lysosomes. A molecular machinery that contains the ubiquitin-binding protein Hrs/Vps27 appears to be central in this sorting process. Three conserved multisubunit complexes, ESCRT-I, -II and -III, are essential for both sorting and multivesicular endosomes formation. Enveloped RNA viruses such as HIV can redirect these complexes from multivesicular endosomes to the plasma membrane to facilitate viral budding.

Animals↗

PubMed2003

The E3 ubiquitin ligase AIP4 mediates ubiquitination and sorting of the G protein-coupled receptor CXCR4.

Ubiquitination of the chemokine receptor CXCR4 serves as a targeting signal for lysosomal degradation, but the mechanisms mediating ubiquitination and lysosomal sorting remain poorly understood. Here we report that the Nedd4-like E3 ubiquitin ligase AIP4 mediates ubiquitination of CXCR4 at the plasma membrane, and of the ubiquitin binding protein Hrs on endosomes. CXCR4 activation promotes CXCR4 colocalization with AIP4 and Hrs within the same region of endosomes. Endosomal sorting of CXCR4 is dependent on Hrs as well as the AAA ATPase Vps4, the latter involved in regulating the ubiquitination status of both CXCR4 and Hrs. We propose a model whereby AIP4, Hrs, and Vps4 coordinate a cascade of ubiquitination and deubiquitination events that sort CXCR4 to the degradative pathway.

Adenosine Triphosphatases↗

PubMed2002

Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of early endosomes.

After endocytosis, some membrane proteins recycle from early endosomes to the plasma membrane whereas others are transported to late endosomes and lysosomes for degradation. Conjugation with the small polypeptide ubiquitin is a signal for lysosomal sorting. Here we show that the hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, is involved in the endosomal sorting of ubiquitinated membrane proteins. Hrs contains a clathrin-binding domain, and by electron microscopy we show that Hrs localizes to flat clathrin lattices on early endosomes. We demonstrate that Hrs binds directly to ubiquitin by way of a ubiquitin-interacting motif (UIM), and that ubiquitinated proteins localize specifically to Hrs- and clathrin-containing microdomains. Whereas endocytosed transferrin receptors fail to colocalize with Hrs and rapidly recycle to the cell surface, transferrin receptors that are fused to ubiquitin interact with Hrs, localize to Hrs- and clathrin-containing microdomains and are sorted to the degradative pathway. Overexpression of Hrs strongly and specifically inhibits recycling of ubiquitinated transferrin receptors by a mechanism that requires a functional UIM. We conclude that Hrs sorts ubiquitinated membrane proteins into clathrin-coated microdomains of early endosomes, thereby preventing their recycling to the cell surface.

Animals↗

PubMed2002

Phosphorylation of Hrs downstream of the epidermal growth factor receptor.

The hepatocyte growth factor-regulated tyrosine kinase substrate Hrs is an early endosomal protein that is thought to play a regulatory role in the trafficking of growth factor/receptor complexes through early endosomes. Stimulation of cells with epidermal growth factor (EGF) rapidly leads to phosphorylation of Hrs, raising the question whether the receptor tyrosine kinase phosphorylates Hrs directly. Here, we present evidence that a downstream kinase, rather than the active receptor kinase is responsible. We show that the nonreceptor tyrosine kinase Src is able to phosphorylate Hrs in vitro and in vivo, but that Hrs is nevertheless phosphorylated in Src-, Yes- and Fyn-negative cells. Moreover, we show that only 10-20% of Hrs is phosphorylated following EGF stimulation, and that phosphorylation occurs at multiple tyrosines located in different parts of Hrs. These results suggest that Hrs is a substrate for several kinases downstream of the EGF receptor.

Animals↗

PubMed2002

Hrs and endocytic sorting of ubiquitinated membrane proteins.

Endocytosed receptors are either recycled to the plasma membrane or trapped within intralumenal vesicles of multi-vesicular bodies for subsequent degradation in lysosomes. How the cell is able to sort receptors in endosomes has so far been largely unknown. The hepatocyte growth factor regulated tyrosine kinase substrate, Hrs, is an essential protein that has been implicated in cell signalling and intracellular membrane trafficking. Very recently, several reports have demonstrated a role for Hrs in endocytic sorting of ubiquitinated membrane proteins. Here, we review current knowledge about how Hrs recognises ubiquitinated cargo that is destined for lysosomal degradation, and how Hrs may act as a key regulator of the molecular machinery involved in receptor sorting and multivesicular body formation.

Animals↗