Palmitoylation of the V2 vasopressin receptor carboxyl tail enhances beta-arrestin recruitment leading to efficient receptor endocytosis and ERK1/2 activation

A large number of G protein-coupled receptors are palmitoylated on cysteine residues located in their carboxyl tail, but the general role of this post-translational modification remains poorly understood. Here we show that preventing palmitoylation of the V2 vasopressin receptor, by site-directed mutagenesis of cysteines 341 and 342, significantly delayed and decreased both agonist-promoted receptor endocytosis and mitogen-activated protein kinase activation. Pharmacological blockade of receptor endocytosis is without effect on the vasopressin-stimulated mitogen-activated protein kinase activity, excluding the possibility that the reduced kinase activation mediated by the palmitoylation-less mutant could result from altered receptor endocytosis. In contrast, two dominant negative mutants of beta-arrestin which inhibit receptor endocytosis also attenuated vasopressin-stimulated mitogen-activated protein kinase activity, suggesting that the scaffolding protein, beta-arrestin, represents the common link among receptor palmitoylation, endocytosis, and kinase activation. Coimmunoprecipitation and bioluminescence resonance energy transfer experiments confirmed that inhibiting receptor palmitoylation considerably reduced the vasopressin-stimulated recruitment of beta-arrestin to the receptor. Interestingly, the changes in beta-arrestin recruitment kinetics were similar to those observed for vasopressin-stimulated receptor endocytosis and mitogen-activated protein kinase activation. Taken together the results indicate that palmitoylation enhances the recruitment of beta-arrestin to the activated V2 vasopressin receptor thus facilitating processes requiring the scaffolding action of beta-arrestin.     

MAPK signalling: ERK5 versus ERK1/2

Extracellular-signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and, similar to ERK1/2, has the Thr-Glu-Tyr (TEY) activation motif. Both ERK5 and ERK1/2 are activated by growth factors and have an important role in the regulation of cell proliferation and cell differentiation. Moreover, both the ERK5 and the ERK1/2 pathways are sensitive to PD98059 and U0126, which are two well-known inhibitors of the ERK pathway. Despite these similarities, recent studies have revealed distinctive features of the ERK5 pathway: ERK5 has a key role in cardiovascular development and neural differentiation; ERK5 nuclear translocation is controlled by its own nuclear localizing and nuclear export activities; and the carboxy-terminal half of ERK5, which follows its kinase catalytic domain, has a unique function.     

Insulin-like growth factors mediate heterotrimeric G protein-dependent ERK1/2 activation by transactivating sphingosine 1-phosphate receptors

Although several studies have shown that a subset of insulin-like growth factor (IGF) signals require the activation of heterotrimeric G proteins, the molecular mechanisms underlying IGF-stimulated G protein signaling remain poorly understood. Here, we have studied the mechanism by which endogenous IGF receptors activate the ERK1/2 mitogen-activated protein kinase cascade in HEK293 cells. In these cells, treatment with pertussis toxin and expression of a Galpha(q/11)-(305-359) peptide that inhibits G(q/11) signaling additively inhibited IGF-stimulated ERK1/2 activation, indicating that the signal was almost completely G protein-dependent. Treatment with IGF-1 or IGF-2 promoted translocation of green fluorescent protein (GFP)-tagged sphingosine kinase (SK) 1 from the cytosol to the plasma membrane, increased endogenous SK activity within 30 s of stimulation, and caused a statistically significant increase in intracellular and extracellular sphingosine 1-phosphate (S1P) concentration. Using a GFP-tagged S1P1 receptor as a biological sensor for the generation of physiologically relevant S1P levels, we found that IGF-1 and IGF-2 induced GFP-S1P receptor internalization and that the effect was blocked by pretreatment with the SK inhibitor, dimethylsphingosine. Treating cells with dimethylsphingosine, silencing SK1 expression by RNA interference, and blocking endogenous S1P receptors with the competitive antagonist VPC23019 all significantly inhibited IGF-stimulated ERK1/2 activation, suggesting that IGFs elicit G protein-dependent ERK1/2 activation by stimulating SK1-dependent transactivation of S1P receptors. Given the ubiquity of SK and S1P receptor expression, S1P receptor transactivation may represent a general mechanism for G protein-dependent signaling by non-G protein-coupled receptors.     

beta-arrestin-dependent, G protein-independent ERK1/2 activation by the beta2 adrenergic receptor

Physiological effects of beta adrenergic receptor (beta2AR) stimulation have been classically shown to result from G(s)-dependent adenylyl cyclase activation. Here we demonstrate a novel signaling mechanism wherein beta-arrestins mediate beta2AR signaling to extracellular-signal regulated kinases 1/2 (ERK 1/2) independent of G protein activation. Activation of ERK1/2 by the beta2AR expressed in HEK-293 cells was resolved into two components dependent, respectively, on G(s)-G(i)/protein kinase A (PKA) or beta-arrestins. G protein-dependent activity was rapid, peaking within 2-5 min, was quite transient, was blocked by pertussis toxin (G(i) inhibitor) and H-89 (PKA inhibitor), and was insensitive to depletion of endogenous beta-arrestins by siRNA. beta-Arrestin-dependent activation was slower in onset (peak 5-10 min), less robust, but more sustained and showed little decrement over 30 min. It was insensitive to pertussis toxin and H-89 and sensitive to depletion of either beta-arrestin1 or -2 by small interfering RNA. In G(s) knock-out mouse embryonic fibroblasts, wild-type beta2AR recruited beta-arrestin2-green fluorescent protein and activated pertussis toxin-insensitive ERK1/2. Furthermore, a novel beta2AR mutant (beta2AR(T68F,Y132G,Y219A) or beta2AR(TYY)), rationally designed based on Evolutionary Trace analysis, was incapable of G protein activation but could recruit beta-arrestins, undergo beta-arrestin-dependent internalization, and activate beta-arrestin-dependent ERK. Interestingly, overexpression of GRK5 or -6 increased mutant receptor phosphorylation and beta-arrestin recruitment, led to the formation of stable receptor-beta-arrestin complexes on endosomes, and increased agonist-stimulated phospho-ERK1/2. In contrast, GRK2, membrane translocation of which requires Gbetagamma release upon G protein activation, was ineffective unless it was constitutively targeted to the plasma membrane by a prenylation signal (CAAX). These findings demonstrate that the beta2AR can signal to ERK via a GRK5/6-beta-arrestin-dependent pathway, which is independent of G protein coupling.     

Cell cycle-dependent coupling of the vasopressin V1a receptor to different G proteins

Arginine vasopressin (AVP) regulates biological processes by binding to G protein-coupled receptors. In Swiss 3T3 fibroblasts, expressing the V(1a) subtype of vasopressin receptors, AVP mobilizes calcium from intracellular stores. In proliferating cells, the AVP-induced increase in intracellular calcium concentration ([Ca(2+)](i)) was mediated by G proteins of the G(q) family, which are insensitive to pertussis toxin (PTX) pretreatment of the cells. In quiescent cells, the AVP-induced increase in [Ca(2+)](i) was partially PTX-sensitive, suggesting an involvement of G(i) proteins. We confirmed this by photoaffinity labeling of G proteins in Swiss 3T3 cell membranes activated by AVP. In Swiss 3T3 cells arrested in the G(0)/G(1) phase of the cell cycle, the AVP-induced increase in [Ca(2+)](i) was also partially PTX-sensitive but was PTX-insensitive in cells arrested in other phases of the cell cycles. The blocking effect of PTX pretreatment in G(0)/G(1) cells was mimicked by microinjection of antisense oligonucleotides suppressing the expression of the Galpha(i3) subunits. These results were confirmed by microinjection of antibodies directed against the C terminus of G protein alpha-subunits. The data presented indicate that in Swiss 3T3 fibroblasts synchronized in the G(0)/G(1) phase of the cell cycle the V(1a) receptor couples to G(q/11) and G(i3) to activate the phospholipase C-beta, leading to release of intracellular calcium.     

Bridged bicyclic vasopressin receptor antagonists with V(2)-selective or dual V(1a)/V(2) activity

The synthesis and biological testing of a novel series of nonpeptide vasopressin receptor antagonists, containing a bridged bicyclic nucleus, are reported. Variation of substituents (R(1)-R(3)) in general formula 3, and the configuration of the stereocenter, resulted in potent V(2)-selective (e.g., 5) and balanced dual V(1a)/V(2) (e.g., 10) compounds. Data from receptor binding, cell-based functional, and in vivo assays are presented [corrected]     

The heterotrimeric G protein G(i3) regulates hepatic autophagy downstream of the insulin receptor

Compelling evidence suggests that the heterotrimeric G protein G(i3) specifically transmits the antiautophagic effects of insulin and amino acids in the liver. This points to a previously unrecognized cross talk between the insulin receptor tyrosine kinase and G(i3). Interestingly, G(i3) is localized not only to plasma membranes but also to membranes of the autophagosomal compartment. Furthermore, as part of insulin's or phenylalanine's actions to inhibit autophagy, G(i3) is redistributed away from autophagosomes. Therefore, endomembrane-associated rather than plasma membrane-localized G(i3) may serve as the target of insulin's endocrine and metabolic actions. We therefore propose that the function and regulation of organelle-associated heterotrimeric G proteins may be different from their roles at the plasma membrane where they act as signal transducers of seven-transmembrane receptors. Here, we discuss recent findings and propose a function for G(i3) in mTOR-dependent signaling pathways. We hypothesize that G(i) family members may have tissue-specific roles in the regulation of autophagy under different physiological and pathological conditions.     

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.     

The ubiquitin-related protein PLIC-1 regulates heterotrimeric G protein function through association with Gbetagamma

PLIC-1, a newly described ubiquitin-related protein, inhibited both Jurkat migration toward SDF-1alpha and A431 wound healing, but the closely related PLIC-2 did not. PLIC-1 prevented the SDF-1alpha-induced activation of phospholipase C, decreased ligand-induced internalization of SDF-1alpha receptor CXCR4 and inhibited chemotaxis signaled by a transfected Gi-coupled receptor. However, PLIC-1 had no effect on Gs-mediated adenylyl cyclase activation, and inhibited only the Gbetagamma-dependent component of Gq-initiated increase in [Ca2+]i, which is consistent with selective inhibition of Gbetagamma function. PLIC-1 colocalized with G proteins in lamellae and pseudopods, and precipitated Gbetagamma in pull downs. Interaction with Gbetagamma did not require PLIC-1's ubiquitin-like or ubiquitin-associated domains, and proteasome inhibition had no effect on SDF-1alpha activation of phospholipase C, indicating that PLIC-1's inhibition of Gbetagamma did not result from effects on proteasome function. Thus, PLIC-1 inhibits Gi signaling by direct association with Gbetagamma; because it also interacts with CD47, a modulator of integrin function, it likely has a role integrating adhesion and signaling components of cell migration.     

The hyaluronan-binding protease upregulates ERK1/2 and PI3K/Akt signalling pathways in fibroblasts and stimulates cell proliferation and migration

The hyaluronan-binding protease (HABP) is a serine protease in human plasma which is structurally related to plasminogen activators, coagulation factor XII and hepathocyte growth factor activator. It can in vitro activate the coagulation factor FVII, kininogen and plasminogen activators. The present study was initiated to gain a more complete picture of the cell-associated activities of this fibrinolysis-related protease. Treatment of lung fibroblasts with HABP lead to a rapid activation of signalling pathways, including the mitogen-activated protein kinase (MAPK) pathway with c-Raf, MEK and ERK1/2. Additionally the activation of the PI3K/Akt pathway and of several translation-related proteins was found. Proliferation assays confirmed the assumption of a strong growth-stimulating effect of HABP on human lung and skin fibroblasts. Intracellular signalling and growth stimulation were strongly dependent on the proteolytic activity of HABP. Stimulation of signalling and proliferation by HABP involved the fibroblast growth factor receptor 1 (FGFR-1). HABP-stimulated proliferation of lung fibroblasts MRC-5 was accompanied by a significant intracellular increase in basic fibroblast growth factor (bFGF), the major ligand of FGFR-1; bFGF could however not be identified in the supernatant of HABP-treated cells. Though, the conditioned medium from HABP-treated cells showed a strong growth-promoting activity on quiescent fibroblasts, indicating the release of a yet unknown growth factor amplifying the initial growth stimulus. In a two-dimensional wound model HABP stimulated the invasion of fibroblasts into a scratch area, adding a strong pro-migratory activity to this plasma protease. In summary, HABP exhibits a significant growth factor-like activity on quiescent human lung and dermal fibroblasts. Our findings suggest that this fibrinolysis-related plasma protease may participate in physiologic or pathologic processes where cell proliferation and migration are pivotal, like tissue repair, vascular remodelling, wound healing or tumor development.     

KDR stimulates endothelial cell migration through heterotrimeric G protein Gq/11-mediated activation of a small GTPase RhoA

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) functions by activating two receptor tyrosine kinases, Flt-1 (VEGFR-1) and KDR (VEGFR-2), both of which are selectively expressed on the primary vascular endothelium. KDR is responsible for VPF/VEGF-stimulated endothelial cell (EC) proliferation and migration, whereas Flt-1 down-modulates KDR-mediated EC proliferation. Flt-1 mediates down-regulation of EC proliferation through pertussis toxin-sensitive G proteins, betagamma subunits, small GTPase CDC42, and partly by Rac-1. However, the molecular mechanism by which KDR mediates EC migration is not clear yet. Here we show for the first time that activation of RhoA and Rac1 is fully and partially required for KDR-mediated human umbilical vein endothelial cell (HUVEC) migration, respectively, and that CDC42, however, is not involved. Furthermore, overexpression of the RhoA dominant negative mutant RhoA-19N does not affect VPF/VEGF-stimulated KDR phosphorylation, intracellular Ca(2+) mobilization, and mitogen-activated protein kinase phosphorylation. Utilizing the receptor chimeras (EGDR and EGLT) in which the extracellular domain of the epidermal growth factor receptor (EGFR) was fused to the transmembrane domain and the intracellular domains of KDR and Flt-1, respectively, we demonstrate that RhoA activation is mediated by EGDR, not by EGLT, and that EGDR mediates activation of Rac1, not CDC42. Furthermore, the EGDR-mediated RhoA and Rac1 activation is regulated by G proteins Gq/11, Gbetagamma, and phospholipase C independent of phosphatidylinositol 3-kinase and intracellular Ca(2+) mobilization. Interestingly, the RhoA activation can be partially inhibited by overexpression of Rac1-17N, but overexpression of RhoA-19N has no effect on Rac1 activation. Finally, Gq/11 and Gbetagamma subunits are also required for VPF/VEGF-stimulated HUVEC migration. Taken together, our results indicate that KDR stimulates endothelial cell migration through a heterotrimeric G protein Gq/11 and Gbetagamma-mediated RhoA pathway.     

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).     

Activation of ERK1/2 and cPLA(2) by the p55 TNF receptor occurs independently of FAN

The generation of proinflammatory eicosanoids in response to tumor necrosis factor (TNF) involves the activation of cytosolic phospholipase A(2) (cPLA(2)), presumably by phosphorylation through extracellular signal-regulated kinases (ERK). Earlier results had suggested that a pathway involving the p55 TNF receptor (TNF-R55), neutral sphingomyelinase (N-SMase), and c-Raf-1 activates ERK and cPLA(2). We have previously shown that a cytoplasmic region of TNF-R55 distinct from the death domain regulates the activation of N-SMase through binding of the adapter protein FAN. Analysis of embryonal fibroblasts from FAN knockout mice revealed that TNF-induced activation of both ERK and cPLA(2) occurs without involvement of FAN. Furthermore, we provide evidence that the TNF-dependent activation of ERK and cPLA(2) requires the intact death domain of TNF-R55. Finally, we demonstrate that in murine fibroblasts cPLA(2) is phosphorylated in response to TNF solely by ERK, but not by p38 mitogen-activated protein kinase, suggesting a signaling pathway from TNF-R55 via the death domain to ERK and cPLA(2).     

The inhibitor of apoptosis protein fusion c-IAP2.MALT1 stimulates NF-kappaB activation independently of TRAF1 AND TRAF2

The inhibitors of apoptosis (IAPs) are a family of cell death inhibitors found in viruses and metazoans. All members of the IAP family have at least one baculovirus IAP repeat (BIR) motif that is essential for their anti-apoptotic activity. The t(11, 18)(q21;q21) translocation fuses the BIR domains of c-IAP2 with the paracaspase/MALT1 (mucosa-associated lymphoid tissue) protein, a critical mediator of T cell receptor-stimulated activation of NF-kappaB. The c-IAP2.MALT1 fusion protein constitutively activates the NF-kappaB pathway, and this is considered critical to malignant B cell transformation and lymphoma progression. The BIR domains of c-IAP1 and c-IAP2 interact with tumor necrosis factor receptor-associated factors 1 and 2 (TRAF1 and TRAF2). Here we investigated the importance of TRAF1 and TRAF2 for c-IAP2.MALT1-stimulated NF-kappaB activation. We identified a novel epitope within the BIR1 domains of c-IAP1 and c-IAP2 that is crucial for their physical interaction with TRAF1 and TRAF2. The c-IAP2.MALT1 fusion protein associates with TRAF1 and TRAF2 using the same binding site. We explored the functional relevance of this interaction and established that binding to TRAF1 and TRAF2 is not required for c-IAP2.MALT1-stimulated NF-kappaB activation. Furthermore, gene ablation of TRAF2 or combined down-regulation of TRAF1 and TRAF2 did not affect c-IAP2.MALT1-stimulated signaling. However, TRAF1/2-binding mutants of c-IAP2.MALT1 still oligomerize and activate NF-kappaB, suggesting that oligomerization might be important for signaling of the fusion protein. Therefore, the t(11, 18)(q21;q21) translocation creating the c-IAP2.MALT1 fusion protein activates NF-kappaB and contributes to human malignancy in the absence of signaling adaptors that might otherwise regulate its activity.     

Cell signalling diversity of the Gqalpha family of heterotrimeric G proteins

Many receptors for neurotransmitters and hormones rely upon members of the Gqalpha family of heterotrimeric G proteins to exert their actions on target cells. Galpha subunits of the Gq class of G proteins (Gqalpha, G11alpha, G14alpha and G15/16alpha) directly link receptors to activation of PLC-beta isoforms which, in turn, stimulate inositol lipid (i.e. calcium/PKC) signalling. Although Gqalpha family members share a capacity to activate PLC-beta, they also differ markedly in their biochemical properties and tissue distribution which predicts functional diversity. Nevertheless, established models suggest that Gqalpha family members are functionally redundant and that their cellular responses are a result of PLC-beta activation and downstream calcium/PKC signalling. Growing evidence, however, indicates that Gqalpha, G11alpha, G14alpha and G15/16alpha are functionally diverse and that many of their cellular actions are independent of inositol lipid signalling. Recent findings show that Gqalpha family members differ with regard to their linked receptors and downstream binding partners. Reported binding partners distinct from PLC-beta include novel candidate effector proteins, various regulatory proteins, and a growing list of scaffolding/adaptor proteins. Downstream of these signalling proteins, Gqalpha family members exhibit unexpected differences in the signalling pathways and the gene expression profiles they regulate. Finally, genetic studies using whole animal models demonstrate the importance of certain Gqalpha family members in cardiac, lung, brain and platelet functions among other physiological processes. Taken together, these findings demonstrate that Gqalpha, G11alpha, G14alpha and G15/16alpha regulate both overlapping and distinct signalling pathways, indicating that they are more functionally diverse than previously thought.