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Posttranslational modification by ubiquitin controls multiple cellular functions and is counteracted by the activities of deubiquitinating enzymes. UBPy (USP8) is a growth-regulated ubiquitin isopeptidase that interacts with the HRS-STAM complex. Using Cre-loxP-mediated gene targeting in mice, we show that lack of UBPy results in embryonic lethality, whereas its conditional inactivation in adults causes fatal liver failure. The defect is accompanied by a strong reduction or absence of several growth factor receptor tyrosine kinases (RTKs), like epidermal growth factor receptor, hepatocyte growth factor receptor (c-met), and ERBB3. UBPydeficient cells exhibit aberrantly enlarged early endosomes colocalizing with enhanced ubiquitination and have reduced levels of HRS and STAM2. Congruently immortalized cells gradually stop proliferation upon induced deletion of UBPy. These results unveil a central and nonredundant role of UBPy in growth regulation, endosomal sorting, and the control of RTKs in vivo.Posttranslational modification of proteins by mono-or polyubiquitination represents a central mechanism for modulating a wide range of cellular functions, like protein stability, intracellular transport, protein interactions, and transcriptional activity. Ubiquitin is covalently bound to substrates by the activity of E1, E2, and E3 conjugating enzymes (17,44,53). Analogous to other posttranslational modifications, ubiquitination is a reversible process counteracted by deubiquitinating enzymes (DUBs), which cleave the isopeptide linkage between the protein substrate and the ubiquitin residue (14). While the processes and biological consequences of ubiquitin conjugation have been intensively studied, the role of DUBs is just beginning to emerge. Based on structural predictions, the human genome contains more than 90 putative DUBs. These fall into the subclasses of ubiquitin C-terminal hydrolases, ubiquitin-specific proteases (USPs), Machado Joseph disease protein domain proteases, ovarian tumor proteases, and JAMM motif proteases (1, 37, 42). The USPs, with more than 50 members, comprise the largest class of DUBs. Members of the USP family have been associated with the regulation of different cellular pathways. USP7 (HAUSP) regulates p53 stability by deubiquitination of p53 and Mdm2 (32, 33), USP2a was reported to regulate the stability of fatty acid synthase (12), and USP1 has been shown to deubiquitinate the monoubiquitinated proteins Fanconia anemia protein FANCD2 (41) and DNA replication processivity factor PCNA (19).USP8 (UBPy/HUMORF8) was first described as a growthregulated ubiquitin isopeptidase that accumulates upon growth stimulation. Protein levels of UBPy decrease, when cells undergo growth arrest by contact inhibition, suggesting a possible role in the control of mammalian-cell proliferation (40). An oncogenic fusion product of the 5Ј end of phosphatidylinositol 3-kinase p85␤ fused to the 3Ј end of UBPy, which contains the catalytic domain, was isolated from a patient with chronic myelogenous leukemia (21). B...

Objective-Angiotensin peptides play a central role in cardiovascular physiology and pathology. Among these peptides, angiotensin II (Ang II) has been investigated most intensively. However, further angiotensin peptides such as Ang 1-7, Ang III, and Ang IV also contribute to vascular regulation, and may elicit additional, different, or even opposite effects to Ang II. Here, we describe a novel Ang II-related, strong vasoconstrictive substance in plasma from healthy humans and end-stage renal failure patients. ]-Ang II, in the following named Angiotensin A (Ang A), is most likely generated enzymatically. In the presence of mononuclear leukocytes, Ang II is converted to Ang A by decarboxylation of Asp 1 . Ang A has the same affinity to the AT 1 receptor as Ang II, but a higher affinity to the AT 2 receptor. In the isolated perfused rat kidney, Ang A revealed a smaller vasoconstrictive effect than Ang II, which was not modified in the presence of the AT2 receptor antagonist PD 123319, suggesting a lower intrinsic activity at the AT 1 receptor. Ang II and Ang A concentrations in plasma of healthy subjects and end-stage renal failure patients were determined by matrix-assisted laser desorption/ionisation mass-analysis, because conventional enzyme immunoassay for Ang II quantification did not distinguish between Ang II and Ang A. In healthy subjects, Ang A concentrations were less than 20% of the Ang II concentrations, but the ratio Ang A / Ang II was higher in end-stage renal failure patients. Conclusion-Ang

Little is known concerning the intracellular transport of the G protein-coupled receptors (GPCRs). Previous studies suggested a functional role for those residues immediately preceding the conserved palmitoylated cysteine residues in the intracellular carboxyl termini of some GPCRs in cell surface transport. For the human vasopressin V2 receptor, we assessed the significance of a dileucine sequence with an upstream glutamate residue (ELRSLLCC) in mediating cell surface delivery. A series of deletion and point mutants in this region were constructed, and the mutant receptors were expressed in transiently transfected COS.M6 cells. By using [3H]arginine vasopressin binding assays to intact cells and immunofluorescence studies with intact and permeabilized cells, we show that residues E335 (mutant E335Q) and L339 (mutant L339T) are obligatory for receptor transport to the plasma membrane. Residue L340 has a minor but significant influence. [3H]Arginine vasopressin binding experiments on membranes of lysed cells failed to detect any intracellular binding sites for the transport-deficient mutant receptors, suggesting that residues E335 and L339 participate in receptor folding. Studies with green fluorescent protein-tagged receptors demonstrate that the bulk of the mutant receptors E335Q and L339T are trapped in the endoplasmic reticulum. Complex glycosylation was absent in these mutant receptors, supporting this conclusion. These data demonstrate that the glutamate/dileucine motif of the vasopressin V2 receptor is critical for the escape of the receptor from the endoplasmic reticulum, most presumably by establishing a functional and transport-competent folding state. A databank analysis revealed that these residues are part of a conserved region in the GPCR family.

Some membrane-permeable antagonists restore cell surface expression of misfolded receptors retained in the endoplasmic reticulum (ER) and are therefore termed pharmacochaperones. Whether pharmacochaperones increase protein stability, thereby preventing rapid degradation, or assist folding via direct receptor interactions or interfere with quality control components remains elusive. We now show that the cell surface expression and function (binding of the agonist) of the mainly ER-retained wild-type murine vasopressin V 2 receptor GFP fusion protein (mV 2 R⅐GFP) is restored by the vasopressin receptor antagonists SR49059 and SR121463B with EC 50 values similar to their K D values. This effect was preserved when protein synthesis was abolished. In addition, SR121463B rescued eight mutant human V 2 Rs (hV 2 Rs, three are responsible for nephrogenic diabetes insipidus) characterized by amino acid exchanges at the C-terminal end of transmembrane helix TM I and TM VII. In contrast, mutants with amino acid exchanges at the interface of TM II and IV were not rescued by either antagonist. The mechanisms involved in successful rescue of cell surface delivery are explained in a three-dimensional homology model of the antagonist-bound hV 2 R.Water homeostasis in mammals is regulated through arginine-vasopressin (AVP), 1 acting through the vasopressin V 2 receptor (V 2 R) expressed in the renal collecting duct (1). In Xlinked nephrogenic diabetes insipidus (NDI), the kidney shows a resistance to the action of AVP, caused by inactivating mutations of the human V 2 R (hV 2 R) gene (2). More than 150 different mutations have been described (for review, see Ref.3), 50% of which are missense mutations resulting in the substitution of a single amino acid. Most of the hV 2 R mutants with a single amino acid exchange are retained within the ER and not transported to the cell surface (for review, see Ref.3). Most likely, the amino acid exchanges result in improper folding of the mutant hV 2 Rs and subsequently prolonged association with molecular chaperones. For example, for the NDI mutant hR337X, a prolonged association with the ER-chaperone calnexin has been observed (4). Chaperone association prevents the aggregation of misfolded proteins, but also inhibits the exit of improperly folded proteins from the ER until correct folding is established.Recently, it has been found that membrane-permeable antagonists not only inhibit receptor activation, but also promote cell surface expression of misfolded, ER-retained G proteincoupled receptors (GPCRs). This concept represents an intriguing new approach for the therapy of congenital diseases caused by mutations in genes encoding GPCRs. For the ER-retained rhodopsin mutant P23H (a frequent cause of autosomal-dominant retinitis pigmentosa), it has been shown in vitro that the inverse agonist 9-cis-retinal or the non-hydrolyzable inverse agonist 11-cis-7-ring-retinal promoted cell surface expression (5,6). Restoration of cell surface expression by antagonists or inverse agonists has also been...

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Endothelin-1 (ET-1) is a potent vasoactive peptide that acts on endothelin A (ETEndothelins (ET-1, ET-2, and ET-3) 1 are important regulators in the vascular system. They act via two receptors: the endothelin A (ET A ) and endothelin B (ET B ) receptors (1, 2). Although human ET A and ET B receptors share 59% amino acid sequence identity (exceeding 75% at the cytoplasmic face), both receptor subtypes couple to different G proteins and differ in their ligand-induced internalization and intracellular trafficking. Whereas the ET A receptor stimulates G proteins of the G q/11 and G 12/13 families, the ET B receptor activates mainly G proteins of the G i and G q/11 families (3, 4). Whether ET B receptors also stimulate proteins of the G 12/13 family is still controversial and may depend on expression levels or cell types investigated (5, 6). Upon ligand binding, both receptor subtypes are rapidly desensitized by phosphorylation through the G protein-coupled receptor kinase type 2 (7). Following internalization via caveolae and/or clathrin-coated pits, the ET A receptor is recycled back to the cell surface (8, 9). In contrast, the ET B receptor is exclusively internalized via a clathrin-dependent pathway and transported to late endosomes and lysosomes (9, 10).The ET A receptor is mainly expressed in vascular smooth muscle cells. Its activation elicits a long-lasting contraction via an increase in cytosolic Ca 2ϩ concentrations and activation of Rho proteins (11,12). The ET B receptor is predominantly expressed in endothelial cells and stimulates the release of NO and prostacyclin, thereby causing relaxation of vascular smooth muscle cells (13). In addition, ET A and ET B receptors are co-expressed in numerous cells, e.g. astrocytes, cardiomyocytes, epithelial cells of the choroid plexus and the anterior pituitary, and certain vascular smooth muscle cells (14 -16). In disease states, such as atherosclerosis and hypercholesterolemia, vascular smooth muscle cells co-express ET A and ET B receptors (17). Because atypical ligand binding was observed for cells co-expressing ET A and ET B receptors, e.g. astrocytes, epithelial cells of the anterior pituitary, or vascular smooth muscle cells, it was suggested that the two receptor subtypes form heterodimers (15,16,18). For example, in epithelial cells of the anterior pituitary, ET B receptor-selective ligands such as sarafotoxin 6c, ET-3, and IRL1620 were competitors of 125 I-ET-1 binding only in the presence of the ET A receptor-selective antagonist BQ123 (16). In astrocytes, ET A and ET B receptors cooperatively control ET-1 clearance, because only the combi-