Independent ��-Arrestin2 and Gq/Protein Kinase C�� Pathways for ERK Stimulated by Angiotensin Type 1A Receptors in Vascular Smooth Muscle Cells Converge on Transactivation of the Epidermal Growth Factor Receptor

Recent studies in receptor-transfected cell lines have demonstrated that extracellular signal-regulated kinase (ERK) activation by angiotensin type 1A receptor and other G protein-coupled receptors can be mediated by both G protein-dependent and β-arrestin-dependent mechanisms. However, few studies have explored these mechanisms in primary cultured cells expressing endogenous levels of receptors. Accordingly, here we utilized the β-arrestin biased agonist for the angiotensin type 1A receptor, SII-angiotensin (SII), and RNA interference techniques to investigate angiotensin II (ANG)-activated β-arrestin-mediated mitogenic signaling pathways in rat vascular smooth muscle cells. Both ANG and SII induced DNA synthesis via the ERK activation cascade. Even though SII cannot induce calcium influx (G protein activation) after receptor stimulation, it does cause ERK activation, although less robustly than ANG. Activation by both ligands is diminished by depletion of β-arrestin2 by small interfering RNA, although the effect is more complete with SII. ERK activation at early time points but not later time points is strongly inhibited by those protein kinase C inhibitors that can block protein kinase Cζ. Moreover, ANG- and SII-mediated ERK activation require transactivation of the epidermal growth factor receptor via metalloprotease 2/9 and Src kinase. β-Arrestin2 facilitates ANG and SII stimulation of Src-mediated phosphorylation of Tyr-845 on the EGFR, a known site for Src phosphorylation. These studies delineate a convergent mechanism by which G protein-dependent and β-arrestin-dependent pathways can independently mediate ERK-dependent transactivation of the EGFR in vascular smooth muscle cells thus controlling cellular proliferative responses.G protein-coupled receptors, also known as seven transmembrane (7TM)² receptors, control virtually all known physiological processes in mammals (1). The various functions of these receptors are mediated and modulated by three families of proteins, which share the property that they interact virtually universally with the receptors in a strictly stimulus-dependent way (1). These three families of proteins are the heterotrimeric G proteins, the G protein-coupled receptor kinases (GRKs), and the β-arrestins. Activation of the receptors stimulates classical G protein-dependent signaling, often involving regulation of levels of second messengers such as cAMP and diacyglycerol. However, as has been known for many years, interaction of activated receptors with GRKs leading to their phosphorylation, and subsequent interaction with β-arrestins leads to desensitization of G protein signaling.In recent years, however, it has become increasingly clear that the β-arrestin-GRK system is in fact bifunctional (2). Thus, even as it desensitizes G protein signaling by the receptors, it also serves as a signal transduction system in its own right, activating a growing list of signaling pathways. These positive signaling functions are often mediated by the ability of β-arrestin to serve as an adaptor or scaffold molecule, bringing elements of diverse signaling pathways into proximity with one another and the receptors and thereby facilitating their activation. This new paradigm for understanding the previously unrecognized signaling properties of the β-arrestin-GRK system has been explored in a wide variety of transfected cultured cell systems.However, to date, relatively little investigation of these novel signaling pathways has been carried out in primary cell culture systems expressing endogenous levels of 7TM receptors. In seeking such a system in which to characterize and compare β-arrestin and G protein-mediated signaling pathways from a typical 7TM receptor, our attention was drawn to cultured rat vascular smooth muscle cells (VSMCs). Several features of rat VSMCs suggest this to be a relevant system for these purposes. Rat VSMCs express a variety of physiologically important 7TM receptors including the angiotensin II type 1A receptor (AT1R) (3). This receptor has been the focus of extensive study in transfected cell systems with respect to its β-arrestin-mediated signaling to a variety of pathways, most particularly extracellular signal-regulated kinase (ERK). Moreover, the AT1R mediates the physiologically important effects of angiotensin II (ANG) on vascular tone as well as on proliferation and chemotaxis (4, 5). Pathophysiologically, ANG stimulation of this receptor has been implicated in VSMC proliferation and chemotaxis, which are thought to play an important role in such important disease processes as atherosclerosis and restenosis after angioplasty (6, 7). Moreover, a ligand has been characterized [Sar¹,Ile⁴,Ile⁸](SII)-angiotensin (SII), a triply mutated angiotensin octapeptide that, in transfected cell systems, acts as a specific agonist for β-arrestin-mediated signaling, although not activating G protein-mediated signaling (8).Accordingly, in the studies described here, we set out to investigate the characteristics of activation of ERK in rat VSMCs that might be mediated through G protein as well as β-arrestin signaling. The results not only demonstrate the importance of β-arrestin-mediated signaling in ERK-mediated proliferative responses of these cells, but also shed new light on the molecular mechanisms and interrelationships between the β-arrestin and classical G protein-mediated activation of these pathways.     

Galpha-mediated inhibition of developmental signal response

BACKGROUND: Seven-transmembrane receptor (7-TMR)-G protein networks are molecular sensors of extracellular signals in all eukarya. These pathways cycle through activated (sensitized) and inhibited (desensitized) states, and, while many of the molecular components for signal activation have been described, inhibitory mechanisms are not well characterized. In Dictyostelium, 7-TM cAMP receptors direct chemotaxis and development but also regulate the periodic synthesis of their own ligand, the chemoattractant/morphogen cAMP. We now demonstrate through loss-of-function/gain-of-function studies that the novel heterotrimeric Galpha9 protein subunit regulates an inhibitory pathway during early Dictyostelium development for the cAMP signal response.RESULTS: galpha9 null cells form more cAMP signaling centers, are more resistant to compounds that inhibit cAMP signaling, and complete aggregation sooner and at lower cell densities than wild-type cells. These phentoypes are consistent with the loss of an inhibitory signaling pathway during development of galpha9 null cells. Cells expressing constitutively activated Galpha9 are defective in cAMP signaling center formation and development at low cell density and display an increased sensitivity to cAMP signal inhibition that is characteristic of enhanced suppression of the cAMP signal response. Finally, we demonstrate that galpha9 null cells, which have been codeveloped with a majority of wild-type cells, primarily establish cAMP signaling centers and are able to non-autonomously direct wild-type cells to adopt a galpha9 null-like phenotype.CONCLUSIONS: We suggest that Galpha9 functions in an inhibitory-feedback pathway that regulates cAMP signaling center formation and propagation. Galpha9 may be part of the mechanism that regulates lateral signal inhibition or that modulates receptor desensitization.     

Insect G protein-coupled receptors and signal transduction

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins (7-TM) that transduce extracellular signals into cellular physiological responses through the activation of heterotrimeric guanine nucleotide binding proteins (alpha beta gamma subunits). Their general properties are remarkably well conserved during evolution. Despite this general resemblance, a large variety of different signals are mediated via this category of receptors. Several GPCR-(sub)families have an ancient origin that is situated before the divergence of Protostomian and Deuterostomian animals. Nevertheless, an enormous diversification has occurred since then. The availability of novel sequence information is growing very rapidly as a result of molecular cloning experiments and of metazoan genome (Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens) and EST (expressed sequence tags) sequencing projects. The Drosophila Genome Sequencing Project will certainly have an important impact on insect signal transduction and receptor research. In parallel, convenient expression systems and functional assay procedures will be needed to investigate insect receptor properties and to monitor the effects of natural and artificial ligands. The study of the evolutionary aspects of G protein-coupled receptors and of their signaling pathways will probably reveal insect-specific features. More insight into these features may result in novel methods and practical applications. Arch.     

Extracellular ATP as a signaling molecule for epithelial cells

The charge of this invited review is to present a convincing case for the fact that cells release their ATP for physiological reasons. Many of our "purinergic" colleagues as well as ourselves have experienced resistance to this concept, because it is teleologically counter-intuitive. This review serves to integrate the three main tenets of extracellular ATP signaling: ATP release from cells, ATP receptors on cells, and ATP receptor-driven signaling within cells to affect cell or tissue physiology. First principles will be discussed in the Introduction concerning extracellular ATP signaling. All possible cellular mechanisms of ATP release will then be presented. Use of nucleotide and nucleoside scavengers as well as broad-specificity purinergic receptor antagonists will be presented as a method of detecting endogenous ATP release affecting a biological endpoint. Innovative methods of detecting released ATP by adapting luciferase detection reagents or by using "biosensors" will be presented.Because our laboratory has been primarily interested in epithelial cell physiology and pathophysiology for several years, the role of extracellular ATP in regulation of epithelial cell function will be the focus of this review. For ATP release to be physiologically relevant, receptors for ATP are required at the cell surface. The families of P2Y G protein-coupled receptors and ATP-gated P2X receptor channels will be introduced. Particular attention will be paid to P2X receptor channels that mediate the fast actions of extracellular ATP signaling, much like neurotransmitter-gated channels versus metabotropic heptahelical neurotransmitter receptors that couple to G proteins. Finally, fascinating biological paradigms in which extracellular ATP signaling has been implicated will be highlighted. It is the goal of this review to convert and attract new scientists into the exploding field of extracellular nucleotide signaling and to convince the reader that extracellular ATP is indeed a signaling molecule.     

The role of beta-arrestins in the formyl peptide receptor-like 1 internalization and signaling

The N-formyl peptide receptor-like 1 (FPRL1) is a G protein-coupled receptor (GPCR) that transmits intracellular signals in response to a variety of agonists, many of them being clearly implicated in human pathology. beta-arrestins are adaptor proteins that uncouple GPCRs from G protein and regulate receptor internalization. They can also function as signal transducers through the scaffolding of signaling molecules, such as components of the extracellular signal-regulated kinase (ERK) cascade. We investigated the role of beta-arrestins in ligand-induced FPRL1 internalization and signaling. In HEK293 cells expressing FPRL1, fluorescence microscopy revealed that agonist-stimulated FPRL1 remained co-localized with beta-arrestins during endocytosis. Internalization of FPRL1, expressed in a mouse embryonic fibroblast (MEF) cell line lacking endogenous beta-arrestins, was highly compromised. This distinguishes FPRL1 from the prototypical formyl peptide receptor FPR that is efficiently internalized in the absence of beta-arrestins. In both HEK293 and MEF cells, FPRL1-mediated ERK1/2 activation was a rapid and transient event. The kinetics and extent of ERK1/2 activation were not significantly modified by beta-arrestin overexpression. The pattern of FPRL1-mediated ERK1/2 activation was similar whether cells express or not beta-arrestins. Furthermore, treatment of the FPRL1 expressing cells with pertussis toxin inhibited ERK1/2 activation in MEF and in HEK293 cells. These results led us to conclude that activation of ERK1/2 mediated by FPRL1 occurs primarily through G protein signaling. Since beta-arrestin-mediated signaling has been observed essentially for receptors coupled to G proteins other than G(i), this may be a characteristic of G(i) protein-coupled chemoattractant receptors.     

G protein-coupled chemokine receptors induce both survival and apoptotic signaling pathways

Chemokine receptors are essential for triggering chemotaxis to immune cells; however, a number of them can also mediate death when engaged by nonchemokine ligands. When the chemokine receptor CXCR4 is engaged by stromal cell-derived factor (SDF1)alpha, it triggers cells to chemotax, and in some cell types such as neurons, causes cell death. To elucidate this dual and opposing receptor function, we have investigated whether CXCR4 activation by its chemokine SDF1alpha could lead to the simultaneous activation of both anti- and proapoptotic signaling pathways; the balance ultimately influencing cell survival. CXCR4 activation in CD4 T cells by SDF1alpha led to the activation of the prosurvival second messengers, Akt and extracellular signal-regulated protein kinase. Selective inhibition of each signal demonstrated that extracellular signal-regulated protein kinase is essential for mediating SDF1alpha-triggered chemotaxis but does not confer an antiapoptotic state. In contrast, Akt activation through CXCR4 by SDF1alpha interactions is necessary to confer resistance to apoptosis. The proapoptotic signaling pathway triggered by SDF1alpha-CXCR4 interaction involves the G(ialpha) protein-independent activation of the proapoptotic MAPK (p38). Furthermore, other chemokines and chemokine receptors also signal chemotaxis and proapoptotic effects via similar pathways. Thus, G(ialpha) protein-coupled chemokine receptors can function as death prone receptors and the balance between the above signaling pathways will ultimately mandate the fate of the activated cell.     

When trafficking and signaling mix: How subcellular location shapes G protein‐coupled receptor activation of heterotrimeric G proteins

G protein‐coupled receptors (GPCRs) physically connect extracellular information with intracellular signal propagation. Membrane trafficking plays a supportive role by “bookending” signaling events: movement through the secretory pathway delivers GPCRs to the cell surface where receptors can sample the extracellular environment, while endocytosis and endolysosomal membrane trafficking provide a versatile system to titrate cellular signaling potential and maintain homeostatic control. Recent evidence suggests that, in addition to these important effects, GPCR trafficking actively shapes the cellular signaling response by altering the location and timing of specific receptor‐mediated signaling reactions. Here, we review key experimental evidence underlying this expanding view, focused on GPCR signaling mediated through activation of heterotrimeric G proteins located in the cytoplasm. We then discuss lingering and emerging questions regarding the interface between GPCR signaling and trafficking.      

HERG K+ channels activation during beta(1) integrin-mediated adhesion to fibronectin induces an up-regulation of alpha(v)beta(3) integrin in the preosteoclastic leukemia cell line FLG 29.1

Integrin receptors have been demonstrated to mediate either "inside-to-out" and "outside-to-in" signals, and by this way are capable of regulating many cellular functions, such as cell growth and differentiation, cell migration, and activation. Among the various integrin-centered signaling pathways discovered so far, we demonstrated that the modulation of the electrical potential of the plasma membrane (V(REST)) is an early integrin-mediated signal, which is related to neurite emission in neuroblastoma cells. This modulation is sustained by the activation of HERG K(+) channels, encoded by the ether-à-go-go-related gene (herg). The involvement of integrin-mediated signaling is being discovered in the hemopoietic system: in particular, osteoclasts are generated as well as induced to differentiate by interaction of osteoclast progenitors with the stromal cells, through the involvement of integrin receptors. We studied the effects of cell interaction with the extracellular matrix protein fibronectin (FN) in a human leukemic preosteoclastic cell line (FLG 29.1 cells), which has been demonstrated to express HERG currents. We report here that FLG 29.1 cells indeed adhere to purified FN through integrin receptors, and that this adhesion induces an osteoclast phenotype in these cells, as evidenced by the appearance of tartrate-resistant acid phosphatase, as well as by the increased expression of CD51/alpha(v)beta(3) integrin and calcitonin receptor. An early activation of HERG current (I(HERG)), without any increase in herg RNA or modifications of HERG protein was also observed in FN-adhering cells. This activation is apparently sustained by the beta(1) integrin subunit activation, through the involvement of a pertussis-toxin sensitive G(i) protein, and appears to be a determinant signal for the up-regulation of alpha(v)beta(3) integrin, as well as for the increased expression of calcitonin receptor.     

Ras-dependent ERK activation by the human G(s)-coupled serotonin receptors 5-HT4(b) and 5-HT7(a)

Receptor tyrosine kinases activate mitogen-activated protein (MAP) kinases through Ras, Raf-1, and MEK. Receptor tyrosine kinases can be transactivated by G protein-coupled receptors coupling to G(i) and G(q). The human G protein-coupled serotonin receptors 5-HT(4(b)) and 5-HT(7(a)) couple to G(s) and elevate intracellular cAMP. Certain G(s)-coupled receptors have been shown to activate MAP kinases through a protein kinase A- and Rap1-dependent pathway. We report the activation of the extracellular signal-regulated kinases (ERKs) 1 and 2 (p44 and p42 MAP kinase) through the human serotonin receptors 5-HT(4(b)) and 5-HT(7(a)) in COS-7 and human embryonic kidney HEK293 cells. In transfected HEK293 cells, 5-HT-induced activation of ERK1/2 is sensitive to H89, which indicates a role for protein kinase A. The observed activation of ERK1/2 does not require transactivation of epidermal growth factor receptors. Furthermore, 5-HT induced activation of both Ras and Rap1. Whereas the presence of Rap1GAP1 did not influence the 5-HT-mediated activation of ERK1/2, the activation of ERK1/2 was abolished in the presence of dominant negative Ras (RasN17). ERK1/2 activation was reduced in the presence of "dominant negative" Raf1 (RafS621A) and slightly reduced by dominant negative B-Raf, indicating the involvement of one or more Raf isoforms. These findings suggest that activation of ERK1/2 through the human G(s)-coupled serotonin receptors 5-HT(4(b)) and 5-HT(7(a)) in HEK293 cells is dependent on Ras, but independent of Rap1.     

Theme and variations on kinetics of GPCR activation/deactivation

G protein-coupled receptors (GPCRs) initiate intracellular signaling pathways in response to physiologically and medically important extracellular ligands such as peptide and large glycoprotein hormones, neurotransmitters, sensory stimuli (odorant and taste molecules, light), calcium, l-amino acids, and are the target of many clinical drugs. The conversion of these extracellular stimuli into intracellular signals involves sequential and reversible reactions that initially take place at the plasma membrane. These reactions are mediated not only by dynamic interactions between ligands, receptors and heterotrimeric G proteins, but also by conformational changes associated with the activation/deactivation process of each protein. This review discusses the kinetic characteristics and rate-limiting reactions engaged in signal propagation that are involved in systems as diverse as neurotransmitter and hormonal signaling, and that have been recorded in live cells by Förster resonance energy transfer (FRET) approaches.     

Fibronectin, integrins, and growth control

Cell proliferation is controlled not only by soluble mitogens but also by components of the extracellular matrix (ECM) such as fibronectin, to which cells adhere via the integrin family of transmembrane receptors. Input from both growth factor receptors and integrins is required to stimulate progression through the G1 phase of the cell cycle, via induction of G1 cyclins and suppression of inhibitors of the G1 cyclin-dependent kinases. Extensive crosstalk takes place between integrin and growth factor receptor signaling pathways, and mitogenic signaling is weak and transient in the absence of integrin-mediated cell adhesion. In normal untransformed cells, all of the important mitogenic signal transduction cascades, namely those downstream of the Ras and Rho family small GTPases and the phosphoinositide 3-OH kinase-PKB/Akt pathway, are regulated by integrin-mediated cell adhesion. As a result, these cells are anchorage-dependent for growth. In contrast, constitutive activity of each of these pathways has been reported in cancer cells, which not only reduces their mitogen dependence but also allows these cells to grow in an anchorage-independent fashion.     

Expression and biophysical analysis of two double-transmembrane domain-containing fragments from a yeast G protein-coupled receptor

Fragments of G protein-coupled receptors (GPCRs) are widely used as models to investigate these polytopic integral-membrane, signal-transducing molecules, but have proven difficult to prepare in quantities necessary for NMR analyses. We report on the biosynthesis of two double transmembrane (TM) containing fragments of Ste2p, the alpha-factor GPCR from the yeast Saccharomyces cerevisiae. Ste2p(G31-T110) [TM1-TM2] and Ste2p(R231-S339) [TM6-TM7-CT40] were expressed as TrpDeltaLE fusion proteins in Escherichia coli and released by CNBr cleavage. Expression yields were optimized using different strains and induction parameters, and by performing CNBr cleavage directly on inclusion bodies. Nonlabeled and uniformly labeled [15N]-TM1-TM2 and TM6-TM7-CT40, as well as uniformly labeled [15N,13C]-TM1-TM2 and TM1-TM2 selectively labeled with [15N-Ala], [15N-Phe], [15N-Leu], [15N-Ile], and [15N-Val] were prepared. Yields of target peptides with >95% homogeneity varied from 3 mg/L of fermentation ([15N]-TM6-TM7-CT40) to 20 mg/L (selectively labeled TM1-TM2). The high level biosynthesis and the efficient CNBr processing and purification yields allowed the initiation of a comprehensive biophysical analysis of TM1-TM2 and TM6-TM7-CT40. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that TM1-TM2 was monomeric in this micellar environment, whereas TM6-TM7-CT40 migrated as a dimer. CD analysis indicated that TM1-TM2 was highly helical in SDS and 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)], but had a tendency to aggregate in dodecylphosphocholine micelles. Similar results were found with TM6-TM7-CT40. Conditions for NMR measurements were optimized, and both TM1-TM2 and TM6-TM7-CT40 exhibited more than 90% of the expected crosspeaks in the [15N,1H]-HSQC spectrum. These findings set the stage for the determination of the 3D structure of these large domains of a GPCR in micelles using high-resolution NMR.     

Biased agonism of the calcium-sensing receptor

After the discovery of molecules modulating G protein-coupled receptors (GPCRs) that are able to selectively affect one signaling pathway over others for a specific GPCR, thereby "biasing" the signaling, it has become obvious that the original model of GPCRs existing in either an "on" or "off" conformation is too simple. The current explanation for this biased agonism is that GPCRs can adopt multiple active conformations stabilized by different molecules, and that each conformation affects intracellular signaling in a different way. In the present study we sought to investigate biased agonism of the calcium-sensing receptor (CaSR), by looking at 12 well-known orthosteric CaSR agonists in 3 different CaSR signaling pathways: G(q/11) protein, G(i/o) protein, and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Here we show that apart from G(q/11) and G(i/o) signaling, ERK1/2 is activated through recruitment of β-arrestins. Next, by measuring activity of all three signaling pathways we found that barium, spermine, neomycin, and tobramycin act as biased agonist in terms of efficacy and/or potency. Finally, polyamines and aminoglycosides in general were biased in their potencies toward ERK1/2 signaling. In conclusion, the results of this study indicate that several active conformations of CaSR, stabilized by different molecules, exist, which affect intracellular signaling distinctly.     

The novel tyrosine kinase ZAK1 activates GSK3 to direct cell fate specification

Inhibition of GSK3 by 7-TM Wnt/wg receptor signaling is critical for specifying embryonic cell fate patterns. In Dictyostelium, the 7-TM cAMP receptors regulate GSK3 by parallel, antagonistic pathways to establish a developmental body plan. We describe here a novel tyrosine kinase, ZAK1, downstream of 7-TM cAMP receptor signaling that is required for GSK3 activation during development. zak1-nulls have reduced GSK3 activity and are defective in GSK3-regulated developmental pathways. Moreover, recombinant ZAK1 phosphorylates and activates GSK3 in vitro. We propose that ZAK1 is a positive regulator of GSK3 activity required for cell pattern formation in Dictyostelium and speculate that similar mechanisms exist to antagonize Wnt/wg signaling for metazoan cell fate specification.     

G protein-coupled receptor-induced Akt activity in cellular proliferation and apoptosis

Akt (also known as protein kinase B) plays an integral role in many intracellular signaling pathways activated by a diverse array of extracellular signals that target several different classes of membrane-bound receptors. Akt plays a particularly prominent part in signaling networks that result in the modulation of cellular proliferation, apoptosis and survival. Thus, the overexpression of Akt subtypes has been measured in a number of cancer types, and dominant-negative forms of Akt can trigger apoptosis and reduce the survival of cancer cells. G protein-coupled receptors act as cell-surface detectors for a diverse spectrum of biological signals and are able to activate or inhibit Akt via several direct and indirect means. In this review, we shall document how G protein-coupled receptors are able to control Akt activity and examine the resulting biochemical and physiological changes, with particular emphasis on cellular proliferation, apoptosis and survival.     

Role of the PAR1 receptor 8th helix in signaling: the 7-8-1 receptor activation mechanism

The protease-activated receptors are tethered ligand G protein-coupled receptors that are activated by proteolytic cleavage of the extracellular domain of the receptor. The archetypic protease-activated receptor PAR1 strongly activates G(q) signaling pathways, but very little is known regarding the mechanism of signal transference between receptor and internally located G protein. The recent x-ray structure of rhodopsin revealed the presence of a highly conserved amphipathic 8th helix that is likely to be physically interposed between receptor and G protein. We found that the analogous 8th helix region of PAR1 was critical for activation of G(q)-dependent signaling. Engineering an 8th helix alpha-aneurysm with a downwards-directed alanine residue markedly interfered with signal transference to G(q). The 8th helix-anchoring cysteine palmitoylation sites were important for the affinity of ligand-dependent G protein coupling but did not affect the maximal signal. A network of H-bond and ionic interactions was found to connect the N-terminal portion of the 8th helix to the nearby NPXXY motif on transmembrane helix 7 and also to the adjacent intracellular loop-1. Disruption of these pairwise interactions caused additive defects in coupling to G protein, indicating that the transmembrane 7-8th helix-i1 loop may move in a coordinated manner to transfer the signal from PAR1 to G protein. This "7-8-1" interaction network was found to be prevalent in G protein-coupled receptors involved in endothelial signaling and angiogenesis.     

Coupling of endothelin receptors to the ERK/MAP kinase pathway. Roles of palmitoylation and G(alpha)q.

Endothelins are potent mitogens that stimulate extracellular signal-regulated kinases (ERK/MAP kinases) through their cognate G-protein-coupled receptors, ET(A) and ET(B). To address the role of post-translational ET receptor modifications such as acylation on ERK activation and to identify relevant downstream effectors coupling the ET receptor to the ERK signaling cascades we have constructed a panel of palmitoylation-deficient ET receptor mutants with differential G(alpha) protein binding capacity. Endothelin-1 stimulation of wild-type ET(A) or ET(B) induced a fivefold to sixfold increase in ERK in COS-7 and CHO cells whereas full-length nonpalmitoylated ET(A) and ET(B) mutants failed to stimulate ERK. A truncated ET(B) lacking the C-terminal tail domain including putative phosphorylation and arrestin binding site(s) but retaining the critical palmitoylation site(s) was still able to fully stimulate ERK activation. Using mutated ET receptors with selective G-protein-coupling we found that endothelin-induced stimulation of G(alpha)q, but not of G(alpha)i or G(alpha)s, is essential for endothelin-mediated ERK activation. Inhibition of protein kinases A and C or epidermal growth factor receptor kinase failed to prevent ET(A)- and ET(B)-mediated ERK activation whereas blockage of phospholipase C-beta completely abrogated endothelin-promoted ERK activation through ET(A) and ET(B) in recombinant COS-7 and native C6 cells. Complex formation of Ca2+ or inhibition of Src family tyrosine kinases prevented ET-1-induced ERK-2 activation in C6-cells. Our results indicate that endothelin-promoted ERK/MAPK activation criticially depends on palmitoylation but not on phosphorylation of ET receptors, and that the G(alpha)q/phospholipase C-beta/Ca2+/Src signaling cascade is necessary for efficient coupling of ET receptors to the ERK/MAPK pathway.     

Cross Talk between the TM4SF5/Focal Adhesion Kinase and the Interleukin-6/STAT3 Pathways Promotes Immune Escape of Human Liver Cancer Cells

TM4SF5 overexpressed in hepatocellular carcinoma activates focal adhesion kinase (FAK) during tumor cell migration. However, it remains unknown how TM4SF5 in hepatocellular carcinoma cells compromises with immune actions initiated by extracellular cytokines. Normal and cancerous hepatocytes with or without TM4SF5 expression were analyzed for the effects of cytokine signaling activity on TM4SF5/FAK signaling and metastatic potential. We found that interleukin-6 (IL-6) was differentially expressed in hepatocytes depending on cancerous malignancy and TM4SF5 expression. IL-6 treatment activated FAK and STAT3 and enhanced focal adhesion (FA) formation in TM4SF5-null cells, but it decreased TM4SF5-dependent FAK activity and FA formation in SNU761-TM4SF5 cells. STAT3 suppression abolished the IL-6-mediated effects in normal Chang cells, but it did not recover the TM4SF5-dependent FAK activity that was inhibited by IL-6 treatment in cancerous SNU761-TM4SF5 cells. In addition, modulation of FAK activity did not change the IL-6-mediated STAT3 activity in either the Chang or SNU761 cell system. TM4SF5 expression in SNU761 cells caused invasive extracellular matrix degradation negatively depending on IL-6/IL-6 receptor (IL-6R) signaling. Thus, it is likely that hepatic cancer cells adopt TM4SF5-dependent FAK activation and metastatic potential by lowering IL-6 expression and avoiding its immunological action through the IL-6-STAT3 pathway.