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 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.

Probing the stoichiometry of ß2-adrenergic receptor phosphorylation by targeted mass spectrometry.

BACKGROUND: Protein phosphorylation of G-protein-coupled receptors (GPCR) is central to the myriad of functions that these ubiquitous receptors perform in biology. Although readily addressable with the use of phospho-specific antibodies, analysis phosphorylation at the level of stoichiometry requires receptor isolation and advanced proteomics. We chose two key sites of potential phosphorylation of human beta2-adrenergic receptor (ß2AR residues S355 and S356) to ascertain the feasibility of applying targeted mass spectrometry to establishing the stoichiometry of the phosphorylation. METHOD: We stimulated HEK293 cells stably expressing Flag-tagged ß2AR-eGFP with 10 µM beta-adrenergic agonist (isoproterenol) and made use of proteomics and targeted mass spectrometry (MS) to quantify the molar ration of phosphorylation on S355 and S356 versus non-phosphorylated receptor in agonist-treated cells. RESULTS: Phosphorylation of either S355 or S356 residue occurred only for agonist-occupied ß2AR. The results demonstrated that pS356 is the dominant site of protein phosphorylation. The abundance of the p356 was 8.6-fold more than that of pS355. Calculation of the molar ratio of phosphorylated (pS355 plus pS356) versus non-phosphorylated receptor reveals that at high occupancy of the receptor only 12.4% of the ß2AR is phosphorylated at these sites. CONCLUSIONS: Application of advanced proteomics and use of the most sensitive targeted MS strategy makes possible the detection and quantification of phosphorylation of very low abundance peptide digests of ß2AR. Establishing the stoichiometry of two key sites of agonist-stimulated phosphorylation with ß2AR is an essential first-step to global analysis of the stoichiometry of GPCR phosphorylation.

Dvl3 translocates IPMK to the cell membrane in response to Wnt.

Wnt3a binds Frizzled-1 and the LRP5/6 co-receptors, ultimately activating Lef/Tcf-sensitive gene transcription in development. Inositol polyphosphate multikinase, IPMK, which possesses inositol phosphate kinase and lipid inositol kinase activities, is essential in Wnt3a regulation of its canonical pathway as well as physiologically in AMPK signaling. In the current report we show that translocation of IPMK to the cell membrane, where its substrates exist in high abundance, is obligate to its function in Wnt signaling. Translocation of IPMK to the cell membrane occurs within 5 min after Wnt3a stimulation. IPMK ducking onto Dishevelled-3 (Dvl3) requires a PDZ domain and the COOH-terminal prolyly-rich tail of Dvl3. Wnt3a-stimulates mobilization of Dvl3 to the cell membrane, translocating IPMK to the cell membrane also, to facilitate downstream signaling of Frizzled1. Deletion mutant of IPMK lacking the NH2-terminal variable region, IPMKΔN, fails to translocate to the cell membrane and to propagate canonical signaling. Targeting the IPMKΔN back to the cell membrane by addition of an isoprenylated CAAX box rescues its function in Wnt3a downstream signaling.

Assembly of Dishevelled 3-based supermolecular complexes via phosphorylation and Axin.

BACKGROUND: Dishevelled-3 (Dvl3) is a multivalent scaffold essential to cell signaling in development. Dsh/Dvls enable a myriad of protein-protein interactions in Wnt signaling. In the canonical Wnt/ß-catenin pathway specifically, Dvl3 polymerizes to form dynamic protein aggregates, so-called "signalsomes", which propagate signals from the Wnt receptor Frizzled to downstream elements. RESULTS: Very large Dvl3-based supermolecular complexes form in response to Wnt3a. These complexes are identified by steric-exclusion chromatography, affinity pull-downs, proteomics, and fluorescence correlation microscopy (fcs). In the current work, the roles of Dvl3 phosphorylation and of Axin in the assembly of Dvl3-based supermolecular complexes in response to Wnt3a are probed in totipotent mouse F9 teratocarcinoma cells. Point mutations of phosphorylation sites of Dvl3 which interfere with Lef/Tcf-sensitive transcriptional activation by Wnt3a are shown to interfere more proximally with the assembly of Dvl3-based supermolecular complexes. Axin, a Dvl-interacting protein, plays a central role in organizing the beta-catenin destruction complex. The assembly of Dvl3-based supermolecular complexes is blocked either by depletion of Axin or by mutation of Axin sites necessary for polymerization in response to Wnt3a. CONCLUSION: These data demonstrate that Wnt3a activation of the canonical pathway requires specific phosphorylation events as well as Axin to assemble very large, Dvl3-based supermolecular complexes; these complexes are a prerequisite to activation of Lef/Tcf-sensitive transcription.

"Shaping" of cell signaling via AKAP-tethered PDE4D: Probing with AKAR2-AKAP5 biosensor.

BACKGROUND: PKA, a key regulator of cell signaling, phosphorylates a diverse and important array of target molecules and is spatially docked to members of the A-kinase Anchoring Protein (AKAP) family. AKAR2 is a biosensor which yields a FRET signal in vivo, when phosphorylated by PKA. AKAP5, a prominent member of the AKAP family, docks several signaling molecules including PKA, PDE4D, as well as GPCRs, and is obligate for the propagation of the activation of the mitogen-activated protein kinase cascade from GPCRs to ERK1,2. RESULTS: Using an AKAR2-AKAP5 fusion "biosensor", we investigated the spatial-temporal activation of AKAP5 undergoing phosphorylation by PKA in response to ß-adrenergic stimulation. The pattern of PKA activation reported by AKAR2-AKAP5 is a more rapid and spatially distinct from those "sensed" by AKAR2-AKAP12. Spatial-temporal restriction of activated PKA by AKAP5 was found to "shape" the signaling response. Phosphatase PDE4D tethered to AKAP5 also later reverses within 60 s elevated intracellular cyclic AMP levels stimulated by ß-adrenergic agonist. AKAP12, however, fails to attenuate the rise in cyclic AMP over this time. Fusion of the AKAP5 PDE4D-binding-domain to AKAP12 was found to accelerate a reversal of accumulation of intracellular cyclic AMP. CONCLUSION: AKAPs, which are scaffolds with tethered enzymes, can "shape" the temporal and spatial aspects of cell signaling.

AKAP12 and AKAP5 form higher-order hetero-oligomers.

BACKGROUND: The family of A-kinase-anchoring proteins, AKAPs, constitutes a group of molecular scaffolds that act to catalyze dynamic interactions of protein kinase A, protein kinase C, tyrosine kinases, G-protein-coupled receptors and ion channels. AKAP5 (MW ~47 kDa) and AKAP12 (MW ~191 kDa) homo-oligomerize, but whether or not such AKAPs can hetero-oligomerize into supermolecular scaffolds of increased complexity is unknown. RESULTS: Affinity chromatography using immobilized AKAPs as "bait" demonstrates unequivocally that AKAP5 and AKAP12 do form minimally hetero-dimers. Steric-exclusion chromatography of AKAP5 and AKAP12 mixtures revealed the existence of very large, supermolecular complexes containing both AKAPs. Docking of AKAP5 to AKAP12 was increased 4-fold by beta-adrenergic agonist stimulation. Overexpression of AKAP12 was found to potentiate AKAP5-mediated Erk1/2 activation in response to stimulation with beta-adrenergic agonist. CONCLUSION: AKAP5 and AKAP12 are capable of forming hetero-oligomeric supermolecular complexes that influence AKAP locale and function.

AKAP5 and AKAP12 Form Homo-oligomers.

BACKGROUND: A-kinase-anchoring proteins, AKAPs, constitute a family of scaffolds that play an essential role in catalyzing the spatial-temporal, dynamic interactions of protein kinase A, protein kinase C, tyrosine kinases, G-protein-coupled receptors and ion channels. We studied AKAP5 (AKAP79; MW ~47 kDa) and AKAP12 (gravin, SSECKS; MW ~191 kDa) to probe if these AKAP scaffolds oligomerize. RESULTS: In gel analysis and sodium-dodecyl sulfate denaturation, AKAP12 behaved with a MW of a homo-dimer. Only in the presence of the chaotropic agent 8 M urea did gel analysis reveal a monomeric form of AKAP12. By separation by steric-exclusion chromatography, AKAP12 migrates with MW of ~840 kDa, suggestive of higher-order complexes such as a tetramer. Interestingly, the N-(1-840) and C-(840-1782) terminal regions of AKAP12 themselves retained the ability to form dimers, suggesting that the structural basis for the dimerization is not restricted to a single "domain" found within the molecule. In either sodium dodecyl sulfate or urea, AKAP5 displayed a relative mobility of a monomer, but by co-immunoprecipitation in native state was shown to oligomerize. When subjected to steric-exclusion chromatography, AKAP5 forms higher-order complexes with MW ~220 kDa, suggestive of tetrameric assemblies. CONCLUSION: Both AKAP5 and AKAP12 display the capacity to form supermolecular homo-oligomeric structures that likely influence the localization and function of these molecular scaffolds.

Probing the physical nature and composition of signalsomes.

BACKGROUND: Recent advances in our understanding of cell signaling have revealed assemblies of signaling components often viewed in fluorescence microscopy as very large, irregular "punctae". These punctae are often dynamic in nature, appearing to act as mobile scaffolds that function in integrating protein-protein interactions from large arrays of signaling components. The visualization of these punctae, termed "signalsomes" when applied to protein assemblies involved in cell signaling provokes the question, what is the physical nature of these structures made visible in live cells through the expression of fluorescently-tagged fusion molecules? RESULTS: Steric-exclusion chromatography on wide-bore matrices, fluorescence correlation spectroscopy, and advanced proteomics permits the analysis of several important physical properties of signalsomes. Wnt canonical signaling is essential to normal cell development and dysregulation can lead to cancers in humans. Punctae/signalsomes have been reported based upon the study of fluorescently-tagged mammalian Dishevelleds. Dishevelleds are phosphoprotein scaffolds that demonstrate dynamic character and mobility in cells stimulated with Wnt3a. Recent studies have successfully isolated Dvl3-based signalsomes from mouse totipotent embryonic teratocarcinoma F9 cells in culture and sized by application of steric exclusion chromatography (SEC), displaying large discrete Mr (0.5 and 2 MDa). Activation of the Wnt canonical ß-catenin/LEF-Tcf-sensitive transcriptional response leads to an upfield shift of >5 MDa of the Dvl3-based signalsome. Fluorescence correlation spectroscopy (fcs) is a single molecule analysis performed in live cells that experimentally measures the diffusion coefficient and permits calculation of MW of the signalsome (0.2 and 30 MDa species in vivo), which also reveal an upfield shift in MW in response to Wnt3a. Proteomics provides for molecular dissection of the composition of the signalsome isolated from untreated and Wnt3a-treated cells. CONCLUSION: Dvl3-based punctae/signalsomes made visible by fluorescent microscopy now can be interrogated by advanced physical means, defining such properties as signalsome Mr/MW, molecular composition, and intracellular locale.

Dishevelled-3 C-terminal His single amino acid repeats are obligate for Wnt5a activation of non-canonical signaling.

BACKGROUND: The Wnt non-canonical pathway (Wnt5a > Frizzled-2 > cyclic GMP phosphodiesterase/Ca2+-mobilization pathway regulates the activation of NF-AT) is mediated by three mammalian Dishevelleds (Dvl1, Dvl2, and Dvl3) and the role of the C-terminal region unique to Dvl3 was interrogated. RESULTS: Dvl1, Dvl2, and Dvl3 are expressed at varying levels in mouse totipotent F9 embryonal teratocarcinoma cells. The expression of each endogenous Dvl isoform, as defined by knock-down with siRNA, was obligate for Wnt5a to activate NF-AT-sensitive transcription. Elements upstream of effectors, e.g., cGMP phosphodiesterase and Ca2+-mobilization, were blocked by knock-down of any one of the Dvls; thus, with respect to Wnt5a activation of NF-AT Dvls are not redundant. Among the three Dvl isoforms, the C-terminal sequence of Dvl3 is the most divergent. Deletion of region of Dvl3 abolishes Wnt5a-stimulated signaling. Alanine (Ala)-substitution of histidine (His) single amino acid repeats at 637,638 and/or 647,648 in Dvl3, like C-terminal deletion, abolishes Wnt 5a signal propagation. Phenylalanine (Phe)-substitution of the same His-repeats in Dvl3 mimics Wnt5a stimulated NF-AT-sensitive transcription. CONCLUSIONS: The C-terminal third of Dvl3 and His single amino acid repeats 637,638 and 647,648 (which are unique to and conserved in Dvl3) are essential for Wnt5a activation of the non-canonical pathway, but not the Wnt3a activation of the canonical pathway.

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.

AKAR2-AKAP12 fusion protein "biosenses" dynamic phosphorylation and localization of a GPCR-based scaffold.

BACKGROUND: The cAMP-dependent protein kinase A (PKA) plays a pivotal role in virtually all cells, there being a multitude of important target molecules that are substrates for PKA in cell signaling. The spatial-temporal dynamics of PKA activation in living cells has been made accessible by the development of clever biosensors that yield a FRET signal in response to the phosphorylation by PKA. AKAR2 is genetically encoded fluorescent probe that acts as a biosensor for PKA activation. AKAP12 is a scaffold that docks PKA, G-protein-coupled receptors, cell membrane negatively-charged phospholipids, and catalyzes receptor resensitization and recycling. In the current work, the AKAR2 biosensor was fused to the N-terminus of AKAP12 to evaluate its ability to function and report on dynamic phosphorylation of the AKAP12 scaffold. RESULTS: AKAR2-AKAP12 can be expressed in mammalian cells, is fully functional, and reveals the spatial-temporal activation of AKAP12 undergoing phosphorylation by PKA in response to beta-adrenergic activation in human epidermoid carcinoma A431 cells. CONCLUSION: The dynamic phosphorylation of AKAP12 "biosensed" by AKAR2-AKAP12 reveals the scaffold in association with the cell membrane, undergoing rapid phosphorylation by PKA. The perinuclear, cytoplasmic accumulation of phosphorylated scaffold reflects the phosphorylated, PKA-activated form of AKAP12, which catalyzes the resensitization and recycling of desensitized, internalized G-protein-coupled receptors.

Wnt and Frizzled RNA expression in human mesenchymal and embryonic (H7) stem cells.

BACKGROUND: Wnt signals are important for embryonic stem cells renewal, growth and differentiation. Although 19 Wnt, 10 Frizzled genes have been identified in mammals, their expression patterns in stem cells were largely unknown. RESULTS: We conducted RNA expression profiling for the Wnt ligands, their cellular receptors "Frizzleds" and co-receptors LRP5/6 in human embryonic stem cells (H7), human bone marrow mesenchymal cells, as well as mouse totipotent F9 teratocarcinoma embryonal cells. Except failing to express Wnt2 gene, totipotent F9 cells expressed RNA for all other 18 Wnt genes as well as all 10 members of Frizzled gene family. H7 cells expressed RNA for each of the 19 Wnt genes. In contrast, human mesenchymal cells did not display detectable RNA expression of Wnt1, Wnt8a, Wnt8b, Wnt9b, Wnt10a, and Wnt11. Analysis of Frizzled RNAs in H7 and human mesechymal cells revealed expression of 9 members of the receptor gene family, except Frizzled8. Expression of the Frizzled co-receptor LRP5 and LRP6 genes were detected in all three cell lines. Human H7 and mouse F9 cells express nearly a full complement of both Wnts and Frizzleds genes. The human mesenchymal cells, in contrast, have lost the expression of six Wnt ligands, i.e. Wnt1, 8a, 8b, 9b, 10a and 11. CONCLUSION: Puripotent human H7 and mouse F9 embryonal cells express the genes for most of the Wnts and Frizzleds. In contrast, multipotent human mesenchymal cells are deficient in expression of Frizzled-8 and of 6 Wnt genes.

Differential mediation of the Wnt canonical pathway by mammalian Dishevelleds-1, -2, and -3.

In the Drosophila, a single copy of the phosphoprotein Dishevelled (Dsh) is found. In the genomes of higher organism (including mammals), three genes encoding isoforms of Dishevelled (Dvl1, Dvl2, and Dvl3) are present. In the fly, Dsh functions in the Wnt-sensitive stabilization of intracellular beta-catenin and activation of the Lef/Tcf-sensitive transcriptional response known as the Wnt "canonical" pathway. In the current work we explore the expression of Dishevelleds in mammalian cells and provide an estimate of the relative cellular abundance of each Dvl. In mouse F9 cells, all three Dvls are expressed. Dvl2 constitutes more than 95% of the total pool, the sum of Dvl1 and Dvl3 constituting the remainder. Similarly, Dvl2 constitutes more than 80% of the Dvl1-3 pool in mouse P19 and human HEK 293 cells. siRNA-induced knock-down of individual Dvls was performed using Wnt3a-sensitive canonical pathway in F9 cells as the read-out. Activation of the canonical signaling pathway by Wnt3a was dependent upon the presence of Dvl1, Dvl2, and Dvl3, but to a variable extent. Wnt3a-sensitive canonical transcription was suppressible, by knock-down of Dvl1, Dvl2, or Dvl3. Conversely, the overexpression of any one of the three Dvls individually was found to be capable of promoting Lef/Tcf-sensitive transcriptional activation, in the absence of Wnt3a, i.e., overexpression of Dvl1, Dvl2, or Dvl3 is Wnt3a-mimetic. Graded suppression of individual Dvl isoforms by siRNA was employed to test if the three Dvls could be distinguished from one another with regard to mediation of the canonical pathway. Canonical signaling was most sensitive to changes in the abundance of either Dvl3 or Dvl1. Changes in expression of Dvl2, the most abundant of the three isoforms, resulted in the least effect on canonical signaling. Dvl-based complexes were isolated by pull-downs from whole-cell extracts with isoform-specific antibodies and found to include all three Dvl isoforms. Rescue experiments were conducted in which depletion of either Dvl3 or Dvl1 suppresses Wnt3a activation of the canonical pathway and the ability of a Dvl isoform to rescue the response evaluated. Rescue of Wnt3a-stimulated transcriptional activation in these siRNA-treated cells occurred only by the expression of the very same Dvl isoform depleted by the siRNA. Thus, Dvls appear to function cooperatively as well as uniquely with respect to mediation of Wnt3a-stimulated canonical signaling. The least abundant (Dvl1, 3) plays the most obvious role, whereas the most abundant (Dvl2) plays the least obvious role, suggesting that individual Dvl isoforms in mammals may operate as a network with some features in common and others rather unique.

Mitogen-activated protein kinase p38 regulates the Wnt/cyclic GMP/Ca2+ non-canonical pathway.

The non-canonical Wnt/cyclic GMP/Ca(2+)/NF-AT pathway operates via Frizzled-2, a member of the superfamily of G protein-coupled receptors. In scanning for signaling events downstream of the Frizzled-2/G alpha t2/PDE6 triad activated in response to Wnt5a, we observed a strong activation of the mitogen-activated protein kinase p38 in mouse F9 teratocarcinoma embryonal cells. The activation of p38 is essential for NF-AT transcriptional activation mediated via Frizzled2. Wnt5a-stimulated p38 activation was rapid, sensitive to pertussis toxin, to siRNA against either G alpha t2 or p38 alpha, and to the p38 inhibitor SB203580. Real-time analysis of intracellular cyclic GMP using the Cygnet2 biosensor revealed p38 to act at the level of cyclic GMP, upstream of the mobilization of intracellular Ca(2+). Fluorescence resonance energy transfer (FRET) imaging reveals the changes in cyclic GMP in response to Wnt5a predominate about the cell membrane, and likewise sensitive to either siRNA targeting p38 or to treatment with SB203580. Dishevelled is not required for Wnt5a activation of p38; siRNAs targeting Dishevelleds and expression of the Dishevelled antagonist Dapper-1 do not suppress the p38 response to Wnt5a stimulation. These novel results are the first to detail a Dishevelled-independent Wnt response, demonstrating a critical role of the mitogen-activated protein kinase p38 in regulating the Wnt non-canonical pathway.

Inositol pentakisphosphate mediates Wnt/beta-catenin signaling.

Wnt3a stimulates lymphoid enhancer factor/T-cell factor protein-sensitive transcription, i.e. the canonical pathway, in mouse F9 embryonal tetratocarcinoma cells expressing rat Frizzled-1. We explored the potential roles for inositol polyphosphates as mediators of Wnt signaling in the canonical path-way. Wnt3a triggers G-protein-linked phosphatidylinositol signaling, transiently generating inositol polyphosphates, especially inositol pentakisphosphate (IP(5)) accumulation. Knock-down of Galpha(q) abolishes, whereas expression of the Q209L constitutively active mutant of Galpha(q) mimics, the effects of Wnt3a on IP(5) generation and downstream signaling. Phospholipase Cbeta-1 and Cbeta-3 mediate the G protein signal to the level of phosphatidylinositol signaling. Knock-down and inhibitor studies of the enzymes responsible for generating IP(5) reveal inositol 1,4,5-trisphosphate 3-kinase and inositol polyphosphate multikinase as key mediators in the production of IP(5). Wnt3a stimulation of the canonical pathway requires accumulation of IP(5), which acts to inhibit the activity of glycogen synthase kinase-3beta, whereas stimulating casein kinase 2. Blockade of Wnt3a stimulation of IP(5) generation blocks beta-catenin accumulation, activation of lymphoid enhancer factor/T-cell factor protein-sensitive transcription, and promotion of primitive endoderm formation in response to Wnt3a. Phosphatidylinositol signaling mediates Wnt3a action in the canonical pathway, acting to generate inositol pentakisphosphate, a key second messenger of Wnt3a.

Lysophosphatidic acid regulates trafficking of beta2-adrenergic receptors: the Galpha13/p115RhoGEF/JNK pathway stimulates receptor internalization.

Lysophosphatidic acid is an important lipid ligand regulating many aspects of cell function, including proliferation and migration. Operating via heterotrimeric G proteins to downstream effectors, lysophosphatidic acid was shown to regulate the function and trafficking of the G protein-coupled beta(2)-adrenergic receptor. C3 exotoxin, expression of dominant negative RhoA, and inhibition of c-Jun N-terminal kinase blocked the ability of lysophosphatidic acid to sequester the beta(2)-adrenergic receptor, whereas expression of constitutively active Galpha(13), p115RhoGEF, or RhoA mimicked lysophosphatidic acid (LPA) action, stimulating the internalization of the Galpha(s)-coupled beta(2)-adrenergic receptor. This study revealed a novel cross-talk exerted from the LPA/Galpha(13)/p115RhoGEF/RhoA pathway to the beta(2)-adrenergic receptor/Galpha(s)/adenylyl cyclase pathway, attenuating the ability of beta-adrenergic agonists to act following stimulation of cells by LPA as may occur during beta-adrenergic therapy of an inflammatory response.

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.

Src docks to A-kinase anchoring protein gravin, regulating beta2-adrenergic receptor resensitization and recycling.

Gravin (AKAP12) is a membrane-associated scaffold that provides docking for protein kinases, phosphatases, and adaptor molecules obligate for resensitization and recycling of beta(2)-adrenergic receptors. Gravin binds to the cell membrane in a Ca(2+)-sensitive manner and to receptors through well characterized protein-protein interactions. Although the interaction of serine/threonine, cyclic AMP-dependent protein kinase with protein kinase A-anchoring proteins is well described and involves a kinase regulatory subunit binding domain in the C terminus of these proteins, far less is known about tyrosine kinase docking to members of this family of scaffolds. The non-receptor tyrosine kinase Src regulates resensitization of beta(2)-adrenergic receptors and docks to gravin. Gravin displays nine proline-rich domains distributed throughout the molecule. One class I ligand for Src homology domain 3 docking, found in the N terminus ((10)RXPXXP(15)) of gravin, is shown to bind Src. Binding of Src to gravin activates the intrinsic tyrosine kinase of Src. Mutagenesis/deletion of the class I ligand (P15A,P16A) on the N terminus of gravin abolishes both the docking of Src to gravin as well as the receptor resensitization and recycling catalyzed by gravin. The Src-binding peptide-(1-51) of gravin behaves as a dominant-negative for AKAP gravin regulation of receptor resensitization/recycling. The tyrosine kinase Src plays an essential role in the AKAP gravin-mediated receptor resensitization and recycling, an essential aspect of receptor biology.

Suppression of cyclic GMP-dependent protein kinase is essential to the Wnt/cGMP/Ca2+ pathway.

Novel downstream effectors sensing changes in intracellular concentrations of Ca2+ and cyclic GMP in response to activation of the Wnt/Frizzled-2 pathway were sought. Activation of Frizzled-2 suppressed protein kinase G activity while activating NF-AT-dependent transcription. Each of these responses was abolished by pertussis toxin and by knock-down of the expression of either Galphat2 or Galphao. Activation of NF-AT-dependent transcription in response to Wnt5a stimulation was suppressed by activation of protein kinase G and by buffering intracellular Ca2+. Elevation of intracellular cyclic GMP either by inhibition of cyclic GMP phosphodiesterase or by addition of 8-bromocyclic GMP was shown to activate protein kinase G, to block Ca2+ mobilization, as well as to markedly attenuate activation of NF-AT-dependent transcription in response to Wnt5a stimulation. Chemical inhibition of protein kinase G by Rp-8-pCPT-cGMP, conversely, was shown to provoke increased NF-AT gene transcription and Ca2+ mobilization in the absence of Wnt stimulation. Protein kinase G is shown to be a critical downstream effector of the noncanonical Wnt-Frizzled-2/cGMP/Ca2+ pathway.