Wnt3a-stimulated LRP6 phosphorylation is dependent upon arginine methylation of G3BP2.

Wnt signaling is initiated upon binding of Wnt proteins to Frizzled proteins and their co-receptors LRP5 and 6. The signal is then propagated to several downstream effectors, mediated by the phosphoprotein scaffold, dishevelled. We report a novel role for arginine methylation in regulating Wnt3a-stimulated LRP6 phosphorylation. G3BP2, a dishevelled-associated protein, is methylated in response to Wnt3a. The Wnt3a-induced LRP6 phosphorylation is attenuated by G3BP2 knockdown, chemical inhibition of methyl transferase activity or expression of methylation-deficient mutants of G3BP2. Arginine methylation of G3BP2 appears to be a Wnt3a-sensitive 'switch' regulating LRP6 phosphorylation and canonical Wnt-ß-catenin signaling.

Arginine methylation of G3BP1 in response to Wnt3a regulates ß-catenin mRNA.

Wnt/ß-catenin signaling is essential for normal mammalian development. Wnt3a activates the Wnt/ß-catenin pathway through stabilization of ß-catenin; a process in which the phosphoprotein Dishevelled figures prominently. Protein arginine methylation in signaling complexes containing Dishevelled was investigated. Mass spectrometry of a prominent arginine-methylated, Dishevelled-associated protein identified the Ras GTPase activating protein-binding protein 1 G3BP1. Stimulation of totipotent mouse embryonic F9 cells with Wnt3a provoked increased methylation of G3BP1. We show that G3BP1 is a novel Ctnnb1 mRNA binding protein. Methylation of G3BP1 constitutes a molecular switch that regulates Ctnnb1 mRNA in response to Wnt3a. Thus, the protein arginine methylation that targets G3BP1 acts as a novel regulator of Ctnnb1 mRNA.

Dishevelled-KSRP complex regulates Wnt signaling through post-transcriptional stabilization of beta-catenin mRNA.

Canonical Wnt/beta-catenin signaling is crucial during embryonic development. Upon Wnt stimulation, Dishevelled proteins relay the signal from upstream Frizzled receptors to downstream effectors. By using affinity purification followed by ion-trap mass spectrometry we identified K-homology splicing regulator protein (KSRP) as a novel Dishevelled-interacting protein. We show that KSRP negatively regulates Wnt/beta-catenin signaling at the level of post-transcriptional CTNNB1 (beta-catenin) mRNA stability. Thus, Dishevelled-KSRP complex operates in Wnt regulation of beta-catenin, functioning post-transcriptionally upon CTNNB1 mRNA stability.

Wnt-dependent assembly of supermolecular Dishevelled-3-based complexes.

Dishevelled-3 (Dvl3) is a multivalent scaffold protein that is essential to Wnt signaling during development. Although Dvl-based punctae have been visualized by fluorescence microscopy; the physical nature and dynamic character of the such complexes are enigmatic. We use steric-exclusion chromatography, affinity pull-downs, proteomics and fluorescence correlation microscopy to characterize supermolecular Dvl3-based complexes of totipotent mouse F9 cells. The molecular mass of the complexes ranges from that of homodimeric Dvl3 to well-defined peaks harboring supermolecular complexes of 0.4 to 2.0 MDa. Addition of Wnt3a stimulates the formation of Dvl3-based complexes of greater molecular mass within 30 minutes. The presence of DKK1 and knockdown of Dishevelled proteins block formation of the 2 MDa Dvl3-based complexes and also block Wnt3a stimulation of the canonical pathway. Fluorescent correlation microscopy identified supermolecular Dvl3-based complexes with a molecular mass >30 MDa in live cells; these complexes were provoked to form structures with even greater molecular mass by Wnt3a. We establish for the first time the physical and functional nature of very large, supermolecular Dvl3-based complexes.

Wnt signalling: the case of the 'missing' G-protein.

Wnt signalling remains a hot topic for cell signalling sleuthhounds. The trail of signalling downstream of the seven-transmembrane segment Frizzleds, which bind Wnt ligands, is replete of clues [e.g. LPR5/6 (lipoprotein receptor-related protein 5/6), G-proteins or Dishevelled] and yet remains the 'final problem'. Although the heptahelical nature of Frizzleds places them well within a populous family of G-protein-coupled receptors, resistance to this theme has waxed and waned amid increasing demands for 'proof'. The Wnt Homepage (http://www.stanford.edu/group/nusselab/cgi-bin/wnt/) has acted as a dynamic real-time arbiter of the controversy, highlighted by the appearance and later the disappearance of the G-protein from its central diagramming and tabulations. A recent publication in this issue of the Biochemical Journal offers a solution to the 'final problem', demonstrating under native conditions that Frizzleds expressed in mammalian brain preparations act functionally to catalyse guanine-nucleotide exchange in response to stimulation with Wnt3a. Lensed from the fictional character of Sherlock Holmes, The Case of the Missing G-Protein is investigated.

Dishevelled-2 docks and activates Src in a Wnt-dependent manner.

Wnt3a activates the ;canonical' signaling pathway, stimulating the nuclear accumulation of beta-catenin and activation of Lef/Tcf-sensitive transcription of developmentally important genes. Using totipotent mouse F9 teratocarcinoma cells expressing frizzled-1 (Fz1), we investigated roles of tyrosine kinase activity in Wnt/beta-catenin signaling. Treatment with either genistein or Src family kinase inhibitor PP2 attenuates Wnt3a-stimulated Lef/Tcf transcription activation and primitive endoderm formation. siRNA-induced knockdown of Src likewise attenuates Lef/Tcf transcription and primitive endoderm formation in response to Wnt3a, implicating Src as a positive regulator of Wnt/beta-catenin signaling. We discovered that Src binds dishevelled-2 (Dvl2), a key phosphoprotein in Wnt signaling, at two positions: an SH3-binding domain and a C-terminal domain. The Y18F mutant of Dvl2 attenuates the Wnt3a-stimulated Lef/Tcf-sensitive transcriptional response. Wnt3a stimulates Src docking to Dvl2 and activation of this tyrosine kinase. Activated Src, in turn, enhances Wnt activation of the canonical pathway. We show that Dvl2 and beta-catenin are crucially important substrates for tyrosine phosphorylation in the canonical Wnt/beta-catenin pathway.

OSTM1 regulates beta-catenin/Lef1 interaction and is required for Wnt/beta-catenin signaling.

The Wnt/beta-catenin signaling pathway controls key aspects of embryonic development and adult tissue homeostasis, including the formation and maintenance of bone. Recently, mutations in the OSTM1 gene were found to be the cause of severe autosomal recessive osteopetrosis in both the mouse and humans. This disorder is characterized by increased bone mass resulting from a defect in osteoclast maturation. The possible role of OSTM1 in signaling of the Wnt/beta-catenin "canonical" pathway was investigated in totipotent mouse F9 embryonal teratocarcinoma cells. Overexpression of OSTM1 in F9 cells increased Wnt3a-responsive beta-catenin accumulation and Lef/Tcf-sensitive transcription. Similarly, knockdown of endogenous OSTM1 attenuated the ability of Wnt3a to stimulate the canonical signaling pathway. An OSTM1 mutant (detected in humans with osteopetrosis) was expressed in F9 cells and found to inhibit Wnt-stimulated beta-catenin stabilization, gene transcription, and primitive endoderm formation. Expression of this OSTM1 C-terminal deletion mutant attenuated Lef/Tcf-sensitive gene transcription, even when transcription was activated by expression of a constitutively-active form of beta-catenin. However, expression of this OSTM1 C-terminal deletion mutant was unable to alter Lef/Tcf-sensitive gene transcription when transcription was activated by expression of a beta-catenin/Lef chimeric protein. From the standpoint of protein-protein interactions, expression of wild-type OSTM1 stimulated whereas mutant OSTM1 inhibited, the Wnt-dependent association of beta-catenin and Lef1. On the foundation of these experiments, we propose that the human mutations in OSTM1 such as the C-terminal deletion mutant studied herein provoke dysregulation of the canonical Wnt/beta-catenin signaling pathway, providing a molecular basis for severe autosomal recessive osteopetrosis.

Insulin-like growth factor-I provokes functional antagonism and internalization of beta1-adrenergic receptors.

Hormones that activate receptor tyrosine kinases have been shown to regulate G protein-coupled receptors, and herein we investigate the ability of IGF-I to regulate the beta(1)-adrenergic receptor. Treating Chinese hamster ovary cells in culture with IGF-I is shown to functionally antagonize the ability of expressed beta(1)-adrenergic receptors to accumulate intracellular cAMP in response to stimulation by the beta-adrenergic agonist Iso. The attenuation of beta(1)-adrenergic action was accompanied by internalization of beta(1)-adrenergic receptors in response to IGF-I. Inhibiting either phosphatidylinositol 3-kinase or the serine/threonine protein kinase Akt blocks the ability of IGF-I to antagonize and to internalize beta(1)-adrenergic receptors. Mutation of one potential Akt substrate site Ser412Ala, but not another Ser312Ala, of the beta(1)-adrenergic receptor abolishes the ability of IGF-I to functionally antagonize and to sequester the beta(1)-adrenergic receptor. We also tested the ability of IGF-I to regulate beta(1)-adrenergic receptors and their signaling in adult canine cardiac myocytes. IGF-I attenuates the ability of beta(1)-adrenergic receptors to accumulate intracellular cAMP in response to Iso and promotes internalization of beta(1)-adrenergic receptors in these cardiac myocytes.

A-kinase anchoring proteins: trafficking in G-protein-coupled receptors and the proteins that regulate receptor biology.

Scaffold proteins, such as members of the A-kinase anchoring protein (AKAP) family, constitute molecular 'tool boxes' that assist in modulating the amplitude, integration and transduction of information along signaling pathways. As AKAPs are multivalent and often display trafficking in response to the activation of a signaling pathway, each represents a scaffold with multiple, high-value targets for new drug discovery. Recent efforts at the molecular description of a subset of AKAPs that dynamically interact with members of the superfamily of G-protein-coupled receptors and ion channels provide an ideal starting point for drug discovery and development, one that has already produced a lead-like compound. Each of the docking sites for receptors/channels, protein kinases, phosphoprotein phosphatases and adaptor molecules may prove to be a suitable candidate for molecular description and for the identification of small molecules that can interfere with and/or modulate the activity of the overall signaling pathway, providing benefits to health or in treating disease states.

Structure-function analysis of Frizzleds.

Frizzleds, cell surface receptors that mediate the actions of Wnt ligands on early development, are heptahelical (based upon hydropathy analysis) and couple to heterotrimeric G proteins. The primary structure of all ten mammalian Frizzleds display many landmarks observed in virtually all G protein-coupled receptors, including an exofacial N-terminus that is N-glycosylated, the presence of seven hydrophobic transmembrane segments predicted to form alpha-helixes, and three intracellular loops as well as a cytoplasmic, C-terminal tail that harbor suspected sites for protein phosphorylation. Prediction of the G proteins to which Frizzleds mediate signaling based upon a bioinformatic analysis of the primary sequence of the intracellular domains are in good agreement with functional screens in Drosophila, zebrafish, and mouse models of development, e.g., predicting Frizzled-1 to interact with members of the Gi/Go protein family. Likewise various Wnt signaling pathways are sensitive to treatment with pertussis toxin and knock-down of specific G protein alpha-subunits. Homology among the sequences encoding the cytoplasmic domains of human Frizzleds is high and the various Frizzleds can be segregated into subsets predicted to share some common downstream signaling elements. Among different species, homologies can reveal conservation of signaling to cognate G protein partners. Additionally, cytoplasmic domains of the prototypic beta2-adrenergic receptor can be substituted with those from either Frizzled-1 or Frizzled-2 to create chimeric receptors that are activated by beta-adrenergic agonists, yet signal with high fidelity to the Wnt/beta-catenin and Wnt/Ca2+, cyclic GMP pathways, respectively, regulating key aspects of early development. The nature of Frizzled-based signaling complexes, their temporal assembly, and spatial distribution via scaffold protein remains to be elucidated, as does whether or not these Wnt receptors display agonist-induced desensitization, internalization, and re-cycling to the cell membrane.

G-protein-coupled receptors and tyrosine kinases: crossroads in cell signaling and regulation.

G-protein-coupled receptors and protein tyrosine kinases represent two prominent pathways for cellular signaling. As our knowledge of cell signaling pathways mediated by the superfamily of G-protein-coupled receptors and the smaller family of receptor tyrosine kinases expands, so does our appreciation of how these two major signaling platforms share information and modulate each other, otherwise termed "cross-talk". Cross-talk between G-protein-coupled receptors and tyrosine kinases can occur at several levels, including the receptor-to-receptor level, and at crucial downstream points (e.g. phosphatidylinositol-3-kinase, Akt/protein kinase B and the mitogen-activated protein kinase cascade). Regulation of G-protein-coupled receptors by non-receptor tyrosine kinases, such as Src family members, also operates in signaling. A broader understanding of how G-protein-coupled receptors and tyrosine kinases cross-talk reveals new insights into signaling modalities in both health and disease.

pp60Src mediates insulin-stimulated sequestration of the beta(2)-adrenergic receptor: insulin stimulates pp60Src phosphorylation and activation.

Insulin stimulates a rapid phosphorylation and sequestration of the beta(2)-adrenergic receptor. Analysis of the signaling downstream of the insulin receptor with enzyme inhibitors revealed roles for both phosphatidylinositol 3-kinase and pp60Src. Inhibition of Src with PP2, like the inhibition of phosphatidylinositol 3-kinase with LY294002 [2-(4-morpholynyl)-8-phenyl-4H-1-benzopyran-4-one], blocked the activation of Src as well as insulin-stimulated sequestration of the beta(2)-adrenergic receptor. Depletion of Src with antisense morpholinos also suppressed insulin-stimulated receptor sequestration. Src is shown to be phosphorylated/activated in response to insulin in human epidermoid carcinoma A431 cells as well as in mouse 3T3-L1 adipocytes and their derivative 3T3-F422A cells, well-known models of insulin signaling. Inhibition of Src with PP2 blocks the ability of insulin to sequester beta(2)-adrenergic receptors and the translocation of the GLUT4 glucose transporters. Insulin stimulates Src to associate with the beta(2)-adrenergic receptor/AKAP250/protein kinase A/protein kinase C signaling complex. We report a novel positioning of Src, mediating signals from insulin to phosphatidylinositol 3-kinase and to beta(2)-adrenergic receptor trafficking.

G-Protein-coupled receptor-associated A-kinase anchoring proteins: AKAP79 and AKAP250 (gravin).

A-kinase anchoring proteins (AKAPs) define an expanding group of scaffold proteins that display a signature binding site for the RI/RII subunit of protein kinase A. AKAPs are multivalent and a subset of these scaffold proteins also display the ability to associate with the prototypic member of G-protein-coupled receptors, the beta(2)-adrenergic receptor. Both AKAP79 (also known as AKAP5) and AKAP250 (also known as gravin or AKAP12) have been shown to associate with the beta(2)-adrenergic receptor, but each directs downstream signaling events in decidedly different manners. The primary structures, common and unique protein motifs are of interest. Both proteins display largely natively unfolded primary sequences that provide a necklace on which short, structured regions of sequence are found. Membrane association appears to involve both interactions with the lipid bilayer via docking to a G-protein-coupled receptor as well as interactions of short positively charged domains with the inner leaflet of the cell membrane. Gravin, unlike AKAP79, displays a canonical site at its N-terminus that is subject to N-myristoylation. AKAP79 appears to function in switching signaling pathways of the receptor from adenylylcyclase to activation of the mitogen-activated protein kinase cascade. Gravin, in contrast, is essential for the resensitization and recycling of the receptors following agonist-induced activation, desensitization, and internalization. Each AKAP provides a template that enables space-time continuum features to G-protein-coupled signaling pathways as well as a paradigm for explaining apparent compartmentalization of cell signaling.

Beta-catenin, cancer, and G proteins: not just for frizzleds anymore.

The lipid metabolite lysophosphatidic acid (LPA) mediates an impressive set of responses that includes morphogenesis, cell proliferation, cell survival, cell adhesion, and cell migration. LPA exerts its downstream signaling by binding to the LPA(1), LPA(2), and LPA(3) (formerly Edg-2, -4, and -7) family of seven-transmembrane, segmented, heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors. LPA actions of therapeutic interest include effects on wound healing, atherogenesis, thrombogenesis, and, of course, cancer. LPA has been implicated in the progression of human breast, ovarian, prostate, head and neck, and colon malignancies. In view of these earlier observations, a recent report that LPA stimulates the proliferation of colon cancer-derived cell lines was greeted with great anticipation for its possible contribution to the unraveling of details of cancer signaling downstream of LPA. LPA was shown to stimulate nuclear accumulation of beta-catenin in a manner that depended on activation of Galpha(q) by LPA(2,3'), activation of phospholipase Cbeta, activation of a conventional protein kinase C, and phosphorylation and inhibition of glycogen synthase kinase 3-beta. The phosphorylation of beta-catenin by this kinase marks the protein for intracellular degradation; LPA suppresses this degradation and stimulates beta-catenin accumulation. Beta-catenin is a pivotal molecule in the control of cell cycle progression and gene expression, activating both processes in combination with lymphoid-enhancing factor/T cell-factor-sensitive transcription and inhibiting both processes in combination with FOXO transcription factors. The ability of LPA to increase the cytoplasmic and nuclear accumulation of beta-catenin provides a new dimension of knowledge linking lipid mediators to the dysregulation of beta-catenin signaling in cancer.

Frizzled-4 C-terminus Distal to KTXXXW Motif is Essential for Normal Dishevelled Recruitment and Norrin-stimulated Activation of Lef/Tcf-dependent Transcriptional Activation.

The carboxy (C)-termini of G protein coupled receptors (GPCR) dictate essential functions. The KTXXXW motif C-terminus of Frizzleds (FZD) has been implicated in recruitment of Dishevelled (DVL). Through study of FZD4 and its associated ligand Norrin, we report that a minimum of three residues distal to the KTXXXW motif in the C-terminal tail of Frizzled-4 are essential for DVL recruitment and robust Lef/Tcf-dependent transcriptional activation in response to Norrin.

The 15-amino acid motif of the C terminus of the beta2-adrenergic receptor is sufficient to confer insulin-stimulated counterregulation to the beta1-adrenergic receptor.

Insulin counterregulates catecholamine action in part by inducing the sequestration of beta2-adrenergic receptors. Although similar to agonist-induced sequestration, insulin-induced internalization of beta2-adrenergic receptors operates through a distinct and better-understood cellular pathway. The effects of insulin treatment on the function and trafficking of both beta1- and beta2-adrenergic receptors were tested. The beta2-adrenergic receptors were counterregulated and internalized in response to insulin. The beta1-adrenergic receptors, in sharp contrast, are shown to be resistant to the ability of insulin to counterregulate function and induce receptor internalization. Using chimeric receptors composed of beta1-/beta2-adrenergic receptors in tandem with mutagenesis, we explored the role of the C-terminal cytoplasmic tail of the beta2-adrenergic receptors for insulin-induced counterregulation. Substitution of the C-terminal cytoplasmic tail of the beta2-adrenergic receptor on the beta1-adrenergic receptor enabled the chimeric G protein-coupled receptor to be functionally and spatially regulated by insulin. Truncation of the beta2-adrenergic receptor C-terminal cytoplasmic tail to a 15-amino acid motif harboring a potential Src homology 2-binding domain at Y350 and an Akt phosphorylation site at S345,346 was sufficient to enable receptor regulation by insulin.

Insulin signalling: putting the 'G-' in protein-protein interactions.

Cell signalling via receptor tyrosine kinases, such as the insulin receptor, and via heterotrimeric G-proteins, such as Galpha(i), Galpha(s) and Galpha(q) family members, constitute two of most avidly studied paradigms in cell biology. That elements of these two populous signalling pathways must cross-talk to achieve proper signalling in the regulation of cell proliferation, differentiation and metabolism has been anticipated, but the evolution of our thinking and the analysis of such cross-talk have lagged behind the ever-expanding troupe of players and the recognition of multivalency as the rule, rather than the exception, in signalling biology. New insights have been provided by Kreuzer et al. in this issue of the Biochemical Journal, in which insulin is shown to provoke recruitment of Galpha(i)-proteins to insulin-receptor-based complexes that can regulate the gain of insulin-receptor-catalysed autophosphorylation, a proximal point in the insulin-sensitive cascade of signalling. Understanding the convergence and cross-talk of signals from the receptor tyrosine kinases and G-protein-coupled receptor pathways in physical, spatial and temporal contexts will remain a major challenge of cell biology.

AKAPs (A-kinase anchoring proteins) and molecules that compose their G-protein-coupled receptor signalling complexes.

Cell signalling mediated via GPCRs (G-protein-coupled receptors) is a major paradigm in biology, involving the assembly of receptors, G-proteins, effectors and downstream elements into complexes that approach in design 'solid-state' signalling devices. Scaffold molecules, such as the AKAPs (A-kinase anchoring proteins), were discovered more than a decade ago and represent dynamic platforms, enabling multivalent signalling. AKAP79 and AKAP250 were the first to be shown to bind to membrane-embedded GPCRs, orchestrating the interactions of various protein kinases (including tyrosine kinases), protein phosphatases (e.g. calcineurin) and cytoskeletal elements with at least one member of the superfamily of GPCRs, the prototypical beta2-adrenergic receptor. In this review, the multivalent interactions of AKAP250 with the cell membrane, receptor, cytoskeleton and constituent components are detailed, providing a working model for AKAP-based GPCR signalling complexes. Dynamic regulation of the AKAP-receptor complex is mediated by ordered protein phosphorylation.

The Intracellular Loop 2 F328S Frizzled-4 Mutation Implicated in Familial Exudative Vitreoretinopathy Impairs Dishevelled Recruitment.

Familial exudative vitreoretinopathy (FEVR) is a disease state characterized by aberrant retinal angiogenesis. Norrin-induced activation of Frizzled-4 (Fz4) has a major role in regulating beta-catenin levels in the eye that, in turn, modulate the blood retina barrier (BRB). Here we gain insight on the basis of the pathology of a FEVR implicated F328S Fz4 mutant by study. The receptor exhibits a substantially reduced ability to activate Lef/Tcf-dependent transcription. This impaired activation correlates with a decreased ability to stabilize and recruit Dishevelled-2 (Dvl2) to the cell surface. Aromaticity at position 328 of the intracellular loop 2 (iloop2) is revealed similarly as a prerequisite for Dvl2 recruitment to the Fz4. This aromaticity at 328 enables normal Norrin-induced canonical activation. The corresponding position in iloop2 of other Frizzleds likely functions in Dvl recruitment.

Frizzleds: new members of the superfamily of G-protein-coupled receptors.

The superfamily of membrane receptors that signal via heterotrimeric G-proteins includes more than 1500 members, classified into five basic groups, representing about 5-10% of the human genome. These G-protein-coupled receptors operate through a comparatively smaller group of heterotrimeric G-protein family of approximately 20 members, each displaying an alpha subunit that binds and hydrolyzes GTP in combination with the beta-/gamma-subunit complex that is largely non-dissociable in vivo. Frizzleds represent the cell membrane receptors for a family of secreted glycoprotein ligands termed "Wnts" that play essential roles in development, including cell fate, adhesion, polarity, migration, and proliferation. Based upon a compelling set of experimental observations about the structure and downstream signaling of Wnt-Frizzled signaling, one can only conclude that Frizzleds are true members of the GPCR family and require heterotrimeric G-proteins to propagate signals from the Wnts to well-known effectors, including beta-catenin stabilization, mobilization of intracellular Ca2+, and activation of cyclic GMP phosphodiesterase. Careful study of primary structure of Frizzleds reveal heptihelical, 7-transmembrane segments, characteristic of GPCRs. Chimeric forms of Frizzleds, making use of the cytoplasmic domains of Frizzleds, substituted into the exofacial and transmembrane segments of the prototypic GPCR beta2-adrenergic receptor are functional and display the well-known GTP-shift in receptor affinity. Suppression of specific G-protein subunits suppress the ability of chimeric as well as authentic Frizzled-1 and Frizzled-2 to signal to their canonical pathways upon activation. The involvement of beta-arrestin, an important regulator of GPCR signaling, in Frizzled signaling is, therefore, not unexpected. Recognition of the GPCR character of Frizzled enables a more broad understanding of these receptors and of their mechanisms of downstream signaling.