Regulation of the vasopressin V2 receptor by vasopressin in polarized renal collecting duct cells

Binding of arginine-vasopressin (AVP) to its V2 receptor (V2R) in the basolateral membrane of principal cells induces Aquaporin-2-mediated water reabsorption in the kidney. To study the regulation of the V2R by dDAVP in a proper model, a polarized renal cell line stably-expressing V2R-GFP was generated. Labeled AVP-binding studies revealed an equal basolateral vs. apical membrane distribution for V2R-GFP and endogenous V2R. In these cells, GFP-V2R was expressed in its mature form and localized for 75% in the basolateral membrane and for 25% to late endosomes/lysosomes. dDAVP caused a dose- and time-dependent internalization of V2R-GFP, which was completed within 1 h with 100 nM dDAVP, was prevented by coincubation with a V2R antagonist, and which reduced its half-life from 11.5 to 2.8 h. Semiquantification of the V2R-GFP colocalization with E-cadherin (basolateral membrane), early endosomal antigen-1 (EEA-1) and lysosome-associated membrane protein-2 (LAMP-2) in time revealed that most dDAVP-bound V2R was internalized via early endosomes to late endosomes/lysosomes, where it was degraded. The dDAVP-internalized V2R did not recycle to the basolateral membrane. In conclusion, we established the itinerary of the V2R in a polarized cell model that likely resembles the in vivo V2R localization and regulation by AVP to a great extent.     

O-Glycosylation of the V2 vasopressin receptor.

The human V2 vasopressin receptor contains one consensus site for N-linked glycosylation at asparagine 22 in the predicted extracellular amino terminal segment of the protein. This segment also contains clusters of serines and threonines that are potential sites for O-glycosylation. Mutagenesis of asparagine 22 to glutamine abolished N-linked glycosylation of the V2 receptor (N22Q-V2R), without altering its function or level of expression. The N22Q-V2R expressed in transfected cells migrated in denaturing acrylamide gels as two protein bands with a difference of 7000 Da. Protein labeling experiments demonstrated that the faster band could be chase to the slower one suggesting the presence of O-linked sugars. Sialidase treatment of membranes from cells expressing the N22Q-V2R or of immunoprecipitated metabolically labeled V2R accelerated the migration of the protein in acrylamide gels demonstrating the existence of O-glycosylation, the first time this type of glycosylation has been found in a G protein coupled receptor. Synthesis of metabolically labeled receptor in the presence of 1 mM phenyl-N-acetyl-alpha-D-galactosaminide, a competitive inhibitor of N-acetyl-alpha-D-galactose and N-acetylneuraminic acid transferases, also produced a receptor that migrated faster in denaturing gels. Serines and threonines present in the amino terminus were analyzed by alanine scanning mutagenesis to identify the acceptor sites. O-glycosylation was found at most serines and threonines present in the amino terminus. Because the disappearance of a site opened the availability of others to the transferases, the exact identification of the acceptor sites was not feasible. The wild type V2R expressed in HEK 293, COS, or MDCK cells underwent N- and O-linked glycosylation. The mutant V2R bearing all serine/threonine substitutions by alanine at the amino terminus yielded a receptor functionally indistinguishable from the wild type protein, whose mobility in polyacrylamide gels was no longer affected by sialidase treatment.     

Regulation of LC3B levels by ubiquitination and proteasomal degradation

ABSTRACT

Like other biological processes, macroautophagy/autophagy must be tightly controlled for maintenance of cellular homeostasis and for proper response to changing cellular conditions. To gain insights into the regulation of autophagy, we recently conducted a genome-wide CRISPR-Cas9 knockout screen using cells expressing endogenous LC3B tagged with GFP-mCherry as a reporter. This approach allowed us to identify the ubiquitin-activating enzyme UBA6 and the hybrid ubiquitin-conjugating enzyme/ubiquitin ligase BIRC6 as novel autophagy regulators. We found that these enzymes cooperate to mediate monoubiquitination and proteasomal degradation of LC3B, thus limiting the pool of LC3B available for autophagy. Depletion of UBA6 or BIRC6 increased the level of cytosolic LC3B, enhancing the degradation of autophagy adaptors and the clearance of intracellular proteins aggregates. This finding could be the basis for the development of pharmacological inhibitors of UBA6 or BIRC6 for the treatment of protein aggregation disorders. Recent work by another group showed that BIRC6 itself is subject to ubiquitination and proteasomal degradation, highlighting the existence of a complex regulatory network for the control of LC3B levels.     

Structural requirements for V2 vasopressin receptor proteolytic cleavage.

The ligand-induced proteolytic cleavage of the V2 vasopressin receptor transiently expressed in COS cells was investigated. After incubation of the cell membranes with a photoreactive ligand possessing full agonistic properties for V2 receptors, approximately 90% of the porcine and bovine V2 vasopressin receptors were cleaved in the upper part of transmembrane helix 2 at a heptapeptide sequence conserved in both vasopressin and oxytocin receptors. The oxytocin receptor was completely resistant to proteolysis after binding the same photoreactive ligand, which is only a partial agonist for this receptor. Chimeric V2/oxytocin receptors obtained by transfer of extracellular domains of the oxytocin receptor into the V2 receptor showed an increase in binding affinity for oxytocin versus vasopressin and a diminished cleavage. The proteolysis-resistant chimeric V2/oxytocin receptor, which contains the first three extracellular domains of the oxytocin receptor, stimulated cAMP accumulation to a larger extent in response to vasopressin than the wild-type receptor and showed impaired desensitization of the adenylate cyclase system. Our data indicate that the proteolytic cleavage of the V2 receptor requires a defined conformation, especially of the first two extracellular domains that is induced by agonist binding. Furthermore, the results suggest that the proteolytic V2 receptor cleavage might play a role in signal termination at elevated hormone concentrations.     

Mdm2 is involved in the ubiquitination and degradation of G-protein-coupled receptor kinase 2

G-protein-coupled receptor kinase 2 (GRK2) is a central regulator of G-protein-coupled receptor signaling. We report that Mdm2, an E3-ubiquitin ligase involved in the control of cell growth and apoptosis, plays a key role in GRK2 degradation. Mdm2 and GRK2 association is enhanced by beta(2)-adrenergic receptor stimulation and beta-arrestin. Increased Mdm2 expression accelerates GRK2 proteolysis and promotes kinase ubiquitination at defined residues, whereas GRK2 turnover is markedly impaired in Mdm2-deficient cells. Moreover, we find that activation of the PI3K/Akt pathway by insulin-like growth factor-1 alters Mdm2-mediated GRK2 degradation, leading to enhanced GRK2 stability and increased kinase levels. These data put forward a novel mechanism for controlling GRK2 expression in physiological and pathological conditions.     

Rescue of vasopressin V2 receptor mutants by chemical chaperones: specificity and mechanism

Because missense mutations in genetic diseases of membrane proteins often result in endoplasmic reticulum (ER) retention of functional proteins, drug-induced rescue of their cell surface expression and understanding the underlying mechanism are of clinical value. To study this, we tested chemical chaperones and sarco(endo)plasmic reticulum Ca2+ ATPase pump inhibitors on Madin-Darby canine kidney cells expressing nine ER-retained vasopressin type-2 receptor (V2R) mutants involved in nephrogenic diabetes insipidus. Of these nine, only V2R-V206D showed improved maturation and plasma membrane rescue with glycerol, dimethyl sulfoxide (DMSO), thapsigargin/curcumin, and ionomycin but not with other osmolytes or growth at 27 degrees C. This revealed that rescue is mutant specific and that this mutant is prone to rescue by multiple compounds. Rescue did not involve changed expression of molecular chaperones calnexin, heat-shock protein (HSP) 70, or HSP90. V2R antagonist SR121463B treatment revealed that V2R-V206D and V2R-S167T were rescued and matured to a greater extent, suggesting that the rescuing activity of a pharmacological versus chemical chaperone is broader and stronger. Calcium measurements showed that rescue of V2R-V206D by thapsigargin, curcumin, and ionomycin was because of increased cytosolic calcium level, rather than decreased endoplasmic reticulum calcium level. The molecular mechanism underlying rescue by DMSO, glycerol, and SR121463B is different, because with these compounds intracellular calcium levels were unaffected.     

Rescue of a nephrogenic diabetes insipidus-causing vasopressin V2 receptor mutant by cell-penetrating peptides

Mutant membrane proteins are frequently retained in the early secretory pathway by a quality control system, thereby causing disease. An example are mutants of the vasopressin V(2) receptor (V(2)R) leading to nephrogenic diabetes insipidus. Transport-defective V(2)Rs fall into two classes: those retained exclusively in the endoplasmic reticulum (ER) and those reaching post-ER compartments such as the ER/Golgi intermediate compartment. Although numerous chemical or pharmacological chaperones that rescue the transport of ER-retained membrane proteins are known, substances acting specifically in post-ER compartments have not been described as yet. Using the L62P (ER-retained) and Y205C (reaching post-ER compartments) mutants of the V(2)R as a model, we show here that the cell-penetrating peptide penetratin and its synthetic analog KLAL rescue the transport of the Y205C mutant. In contrast, the location of the L62P mutant is not influenced by either peptide because the peptides are unable to enter the ER. We also show data indicating that the peptide-mediated transport rescue is associated with an increase in cytosolic Ca(2+) concentrations. Thus, we describe a new class of substances influencing protein transport specifically in post-ER compartments.     

c-Cbl mediates ubiquitination, degradation, and down-regulation of human protease-activated receptor 2

Mechanisms that arrest G-protein-coupled receptor (GPCR) signaling prevent uncontrolled stimulation that could cause disease. Although uncoupling from heterotrimeric G-proteins, which transiently arrests signaling, is well described, little is known about the mechanisms that permanently arrest signaling. Here we reported on the mechanisms that terminate signaling by protease-activated receptor 2 (PAR(2)), which mediated the proinflammatory and nociceptive actions of proteases. Given its irreversible mechanism of proteolytic activation, PAR(2) is a model to study the permanent arrest of GPCR signaling. By immunoprecipitation and immunoblotting, we observed that activated PAR(2) was mono-ubiquitinated. Immunofluorescence indicated that activated PAR(2) translocated from the plasma membrane to early endosomes and lysosomes where it was degraded, as determined by immunoblotting. Mutant PAR(2) lacking intracellular lysine residues (PAR(2)Delta14K/R) was expressed at the plasma membrane and signaled normally but was not ubiquitinated. Activated PAR(2) Delta14K/R internalized but was retained in early endosomes and avoided lysosomal degradation. Activation of wild type PAR(2) stimulated tyrosine phosphorylation of the ubiquitin-protein isopeptide ligase c-Cbl and promoted its interaction with PAR(2) at the plasma membrane and in endosomes in an Src-dependent manner. Dominant negative c-Cbl lacking the ring finger domain inhibited PAR(2) ubiquitination and induced retention in early endosomes, thereby impeding lysosomal degradation. Although wild type PAR(2) was degraded, and recovery of agonist responses required synthesis of new receptors, lysine mutation and dominant negative c-Cbl impeded receptor ubiquitination and degradation and allowed PAR(2) to recycle and continue to signal. Thus, c-Cbl mediated ubiquitination and lysosomal degradation of PAR(2) to irrevocably terminate signaling by this and perhaps other GPCRs.     

Dynamics of the G protein-coupled vasopressin V2 receptor signaling network revealed by quantitative phosphoproteomics

G protein-coupled receptors (GPCRs) regulate diverse physiological processes, and many human diseases are due to defects in GPCR signaling. To identify the dynamic response of a signaling network downstream from a prototypical G(s)-coupled GPCR, the vasopressin V2 receptor, we have carried out multireplicate, quantitative phosphoproteomics with iTRAQ labeling at four time points following vasopressin exposure at a physiological concentration in cells isolated from rat kidney. A total of 12,167 phosphopeptides were identified from 2,783 proteins, with 273 changing significantly in abundance with vasopressin. Two-dimensional clustering of phosphopeptide time courses and Gene Ontology terms revealed that ligand binding to the V2 receptor affects more than simply the canonical cyclic adenosine monophosphate-protein kinase A and arrestin pathways under physiological conditions. The regulated proteins included key components of actin cytoskeleton remodeling, cell-cell adhesion, mitogen-activated protein kinase signaling, Wnt/β-catenin signaling, and apoptosis pathways. These data suggest that vasopressin can regulate an array of cellular functions well beyond its classical role in regulating water and solute transport. These results greatly expand the current view of GPCR signaling in a physiological context and shed new light on potential roles for this signaling network in disorders such as polycystic kidney disease. Finally, we provide an online resource of physiologically regulated phosphorylation sites with dynamic quantitative data (http://helixweb.nih.gov/ESBL/Database/TiPD/index.html).     

Inhibition of renal cystic disease development and progression by a vasopressin V2 receptor antagonist

The polycystic kidney diseases (PKDs) are a group of genetic disorders causing significant renal failure and death in children and adults. There are no effective treatments. Two childhood forms, autosomal recessive PKD (ARPKD) and nephronophthisis (NPH), are characterized by collecting-duct cysts. We used animal models orthologous to the human disorders to test whether a vasopressin V2 receptor (VPV2R) antagonist, OPC31260, would be effective against early or established disease. Adenosine-3',5'-cyclic monophosphate (cAMP) has a major role in cystogenesis, and the VPV2R is the major cAMP agonist in the collecting duct. OPC31260 administration lowered renal cAMP, inhibited disease development and either halted progression or caused regression of established disease. These results indicate that OPC31260 may be an effective treatment for these disorders and that clinical trials should be considered.     

The splice variant of the V2 vasopressin receptor adopts alternative topologies

The V2 receptor gene encodes two receptor variants by alternative splicing, the canonical V2 receptor (V2a receptor) and V2b. The V2b variant has an amino acid sequence identical to that of the V2a receptor up to the sixth transmembrane domain, but the V2b sequences corresponding to the putative seventh transmembrane domain and the carboxyl terminus are different from those of the V2a receptor. Here we investigate the topology and subcellular distribution of the V2b variant. We found that, in contrast to the V2a receptor, the V2b adopted two topologies: one with six transmembrane segments with the C-terminus on the extracellular side of the membrane and another with seven transmembrane segments with the C-terminus on the intracellular side, similar to typical G-protein-coupled receptors. Furthermore, we observed that both topological isoforms oligomerized with the V2a canonical receptor. Unlike the V2a receptor, V2b did not move to the plasma membrane, but it is retained in the ER--Golgi compartments. These findings indicate that the C-terminal sequence beyond the sixth transmembrane of the V2a is required for the stabilization of the seven-transmembrane topology of the receptor and is also essential for the trafficking of the receptor to the plasma membrane.     

Regulation of Hedgehog signaling by ubiquitination

The Hedgehog (Hh) signaling pathway plays crucial roles both in embryonic development and in adult stem cell function. The timing, duration and location of Hh signaling activity need to be tightly controlled. Abnormalities of Hh signal transduction lead to birth defects or malignant tumors. Recent data point to ubiquitination-related posttranslational modifications of several key Hh pathway components as an important mechanism of regulation of the Hh pathway. Here we review how ubiquitination regulates the localization, stability and activity of the key Hh signaling components.     

A comparative study of plasma vasopressin levels and V1 and V2 vasopressin receptor properties in congenital hypothyroid rat under thyroxine or vasopressin therapy

The effects of propylthiouracil (PTU) treatment on the plasma vasopressin level, on the number of hepatic (V1) or renal (V2) vasopressin receptors and on the hormone-sensitive adenylate cyclase activity in the kidney of developing rats were studied in parallel. In addition, we investigated the corrective effects of thyroxine therapy on the plasma vasopressin level and parameters related to the liver, and the effects of vasopressin therapy on the parameters related to the kidney. As already reported in the case of the number of V2 receptors and adenylate cyclase activity in the kidney, the deficient plasma vasopressin level in hypothyroid rats was completely corrected by two daily physiological doses of thyroxine given from birth to the age of sacrifice (1 month). Unlike the V1 receptors, the V2 receptors are known to be highly dependent on their specific circulating ligand. Since, first of all, the deficit was similar in the numbers of V1 and V2 receptors in hypothyroid rats, and, secondly, the treatment of hypothyroid rats by two daily physiological doses of long lasting vasopressin was found ineffective to recover the deficit in the number of V2 receptors, it can be concluded that thyroid deficiency directly alters vasopressin receptor biosynthesis in both liver and kidney, instead of acting via the depressed plasma vasopressin level.     

Calcium-sensing receptor ubiquitination and degradation mediated by the E3 ubiquitin ligase dorfin

Calcium-sensing receptors (CaR) contribute to regulation of systemic calcium homeostasis by activation of G(q)- and G(i)-linked signaling pathways in the parathyroids, kidney, and intestine. Little is known about the mechanisms regulating CaR synthesis and degradation. Screening of a human kidney yeast two-hybrid library identified the E3 ubiquitin ligase dorfin as a binding partner for the intracellular carboxyl terminus of CaR. Interaction between CaR and dorfin was confirmed by coimmunoprecipitation from HEK293 cells. Ubiquitination of CaR was observed in the presence of the proteasomal inhibitor MG132; mutation of all putative intracellular loop and carboxyl-terminal lysine residues abolished ubiquitination of CaR. Coexpression with dorfin decreased the amount of total CaR protein and increased CaR ubiquitination, whereas a dominant negative fragment of dorfin had opposite effects. The AAA-ATPase p97/valosin-containing protein associates with both CaR and dorfin in HEK293 cells. Treatment with tunicamycin, an inhibitor of N-linked glycosylation, induced the appearance of the unglycosylated 115-kDa CaR form, which was further increased by exposure to MG132, or upon transfection with a dorfin dominant negative construct, suggesting that dorfin-mediated proteasomal degradation of immature CaR occurs from the endoplasmic reticulum. Because endogenous CaR in Madin-Darby canine kidney cells is also subject to degradation from the endoplasmic reticulum, dorfin-mediated ubiquitination may contribute to a general mechanism for CaR quality control during biosynthesis.     

Molecular basis of ligand recognition and activation of human V2 vasopressin receptor

Dear Editor, Vasopressin type 2 receptor(V2R)belongs to the vasopressin(VP)/oxytocin(OT)receptor subfamily of G protein-coupled receptors(GPCRs),which comprises...     

MDM2 promotes ubiquitination and degradation of MDMX

The p53 tumor suppressor is regulated by MDM2-mediated ubiquitination and degradation. Mitogenic signals activate p53 by induction of ARF expression, which inhibits p53 ubiquitination by MDM2. Recent studies showed that the MDM2 homolog MDMX is also an important regulator of p53. We present evidence that MDM2 promotes MDMX ubiquitination and degradation by the proteasomes. This effect is stimulated by ARF and correlates with the ability of ARF to bind MDM2. Promotion of MDM2-mediated MDMX ubiquitination requires the N-terminal domain of ARF, which normally inhibits MDM2 ubiquitination of p53. An intact RING domain of MDM2 is also required, both to interact with MDMX and to provide E3 ligase function. Increase of MDM2 and ARF levels by DNA damage, recombinant ARF adenovirus infection, or inducible MDM2 expression leads to proteasome-mediated down-regulation of MDMX levels. Therefore, MDMX and MDM2 are coordinately regulated by stress signals. The ARF tumor suppressor differentially regulates the ability of MDM2 to promote p53 and MDMX ubiquitination and activates p53 by targeting both members of the MDM2 family.     

TRIM27 negatively regulates NOD2 by ubiquitination and proteasomal degradation

NOD2, the nucleotide-binding domain and leucine-rich repeat containing gene family (NLR) member 2 is involved in mediating antimicrobial responses. Dysfunctional NOD2 activity can lead to severe inflammatory disorders, but the regulation of NOD2 is still poorly understood. Recently, proteins of the tripartite motif (TRIM) protein family have emerged as regulators of innate immune responses by acting as E3 ubiquitin ligases. We identified TRIM27 as a new specific binding partner for NOD2. We show that NOD2 physically interacts with TRIM27 via the nucleotide-binding domain, and that NOD2 activation enhances this interaction. Dependent on functional TRIM27, ectopically expressed NOD2 is ubiquitinated with K48-linked ubiquitin chains followed by proteasomal degradation. Accordingly, TRIM27 affects NOD2-mediated pro-inflammatory responses. NOD2 mutations are linked to susceptibility to Crohn's disease. We found that TRIM27 expression is increased in Crohn's disease patients, underscoring a physiological role of TRIM27 in regulating NOD2 signaling. In HeLa cells, TRIM27 is partially localized in the nucleus. We revealed that ectopically expressed NOD2 can shuttle to the nucleus in a Walker A dependent manner, suggesting that NOD2 and TRIM27 might functionally cooperate in the nucleus.We conclude that TRIM27 negatively regulates NOD2-mediated signaling by degradation of NOD2 and suggest that TRIM27 could be a new target for therapeutic intervention in NOD2-associated diseases.