The frizzled receptors, which mediate development and display seven hydrophobic, membrane-spanning segments, are cell membrane-localized. We constructed a chimeric receptor with the ligand-binding and transmembrane segments from the beta2-adrenergic receptor (beta2AR) and the cytoplasmic domains from rat Frizzled-1 (Rfz1). Stimulation of mouse F9 clones expressing the chimera (beta2AR-Rfz1) with the beta-adrenergic agonist isoproterenol stimulated stabilization of beta-catenin, activation of a beta-catenin-sensitive promoter, and formation of primitive endoderm. The response was blocked by inactivation of pertussis toxin-sensitive, heterotrimeric guanine nucleotide-binding proteins (G proteins) and by depletion of Galphaq and Galphao. Thus, G proteins are elements of Wnt/Frizzled-1 signaling to the beta-catenin-lymphoid-enhancer factor (LEF)-T cell factor (Tcf) pathway.

A-kinase-anchoring protein 250 (AKAP250; gravin) acts as a scaffold that binds protein kinase A (PKA), protein kinase C and protein phosphatases, associating reversibly with the b 2 -adrenergic receptor. The receptor-binding domain of the scaffold and the regulation of the receptor±scaffold association was revealed through mutagenesis and biochemical analyses. The AKAP domain found in other members of this superfamily is essential for the scaffold±receptor interactions. Gravin constructs lacking the AKAP domain displayed no binding to the receptor. Metabolic labeling studies in vivo demonstrate agonist-stimulated phosphorylation of gravin and enhanced gravin±receptor association. Analysis of the AKAP domain revealed two canonical PKA sites phosphorylated in response to elevated cAMP, blocked by PKA inhibitor, and essential for scaffold±receptor association and for resensitization of the receptor. The AKAP appears to provide the catalytic PKA activity responsible for phosphorylation of the scaffold in response to agonist activation of the receptor as well as for the association of the scaffold with the receptor, a step critical to receptor resensitization. Keywords: b-adrenergic receptor/AKAP/gravin/protein kinase A/protein kinase C/scaffold IntroductionRecent advances in our understanding of the workings of cell signaling have highlighted the roles of molecules, often referred to as`scaffolds', in organizing the interactions of molecules in signaling pathways. In Saccharomyces cerevisiae, for example, STE5 is an essential component of the pheromone-mediated mitogen-activated protein kinase (MAPK) pathway. The STE5 scaffold recruits MAPK cascade members STE11, STE7 and FUS3 to generate speci®city for the pheromone pathway (Kim et al., 1998). In vertebrates, A-kinase-anchoring proteins (AKAPs) constitute a diverse family of such scaffold molecules, organizing not only protein kinases, but also protein phosphatases and other molecules, into multivalent signaling complexes (Pawson and Scott, 1997;Colledge and Scott, 1999;Bauman and Scott, 2002). AKAPs bind the RII subunits of the cAMP-dependent kinase (PKA), providing discrete localization of PKA within the cell.The muscle-speci®c AKAP, termed`mAKAP', coordinates multivalent signaling complexes in both the sarcoplasmic reticulum and perinuclear membrane of muscle cells . Another family of AKAPs are derived from a single gene, producing the splice products AKAP350, AKAP450, CG-NAP and Yotiao that are involved in coordinating multivaltent signaling complexes with N-methyl-D-aspartate (NMDA) receptors, protein phosphatases PP1 and calcineurin, as well as PKA Bauman and Scott, 2002). Gravin (also known as AKAP250 and AKAP12) is another prominent member of the AKAP family, a family that includes >50 members. As scaffold molecules, the AKAPs display binding sites for several well known protein kinases and phosphatases. Gravin, for example, binds the RII subunit of PKA, protein kinase C (PKC), protein phosphatase 2B (PP2B) (Nauert et al., 1997;Shih et al., 1999;Fan et al.,...

The Frizzled-2 receptor (Rfz2) from rat binds Wnt proteins and can signal by activating calcium release from intracellular stores. We show that wild-type Rfz2 and a chimeric receptor consisting of the extracellular and transmembrane portions of the beta2-adrenergic receptor with cytoplasmic domains of Rfz2 also signaled through modulation of cyclic guanosine 3',5'-monophosphate (cGMP). Activation of either receptor led to a decline in the intracellular concentration of cGMP, a process that was inhibited in cells treated with pertussis toxin, reduced by suppression of the expression of the heterotrimeric GTP-binding protein (G protein) transducin, and suppressed through inhibition of cGMP-specific phosphodiesterase (PDE) activity. Moreover, PDE inhibitors blocked Rfz2-induced calcium transients in zebrafish embryos. Thus, Frizzled-2 appears to couple to PDEs and calcium transients through G proteins.

The frizzled gene family of putative Wnt receptors encodes proteins that have a seven-transmembrane-spanning motif characteristic of G protein-linked receptors, though no loss-of-function studies have demonstrated a requirement for G proteins for Frizzled signaling. We engineered a Frizzled-2 chimera responsive to ␤-adrenergic agonist by using the ligand-binding domains of the ␤2-adrenergic receptor. The expectation was that the chimera would be sensitive both to drug-mediated activation and blockade, thereby circumventing the problem of purifying soluble and active Wnt ligand to activate Frizzled. Expression of the chimera in zebrafish embryos demonstrated isoproterenol (ISO)-stimulated, propranolol-sensitive calcium transients, thereby confirming the ␤-adrenergic nature of Wnt signaling by the chimeric receptor. Because F9 embryonic teratocarcinoma cells form primitive endoderm after stable transfection of Frizzled-2 chimera and stimulation with ISO, they were subject to depletion of G protein subunits. ISO stimulation of endoderm formation of F9 stem cells expressing the chimeric receptor was blocked by pertussis toxin and by oligodeoxynucleotide antisense to G␣o, G␣t2, and G␤2. Our results demonstrate the requirement of two pertussis toxin-sensitive G proteins, G␣o and G␣t, for signaling by the Frizzled-2 receptor.

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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.

Fully-differentiated mouse 3T3-L1 fibroblasts accumulate large amounts of lipid at 7-10 days after induction by insulin or by dexamethasone and a methyl xanthine. G proteins mediate transmembrane signalling from a diverse group of cell-surface receptors to effector units that include phospholipase C, adenylyl cyclase and ion channels. They are also targets of regulation themselves. 3T3-L1 fibroblasts display marked changes in levels of G protein when induced to differentiate to adipocytes. Here we show that cholera toxin, which ADP-ribosylates and activates the G protein subunit Gs alpha, blocks the induction of differentiation, whereas increasing intracellular cyclic AMP directly with the dibutyryl analogue or indirectly with pertussis toxin or forskolin does not affect differentiation. Oligodeoxynucleotides antisense to the sequence encoding Gs alpha accelerate differentiation markedly. The time course of adipogenesis declined from 7-10 days in controls to roughly 3 days in cultures treated with antisense-Gs alpha oligodeoxynucleotides, whereas oligodeoxynucleotides, antisense to Gi alpha 1, Gi alpha 3, and sense and missense to Gs alpha, had no such effect. Antisense-Gs alpha alone induced differentiation by day 7, indicating that Gs alpha activity modulates differentiation in 3T3-L1 cells, acting in a new role which is independent of increased intracellular cAMP.

The nonreceptor tyrosine kinase Src has been implicated in the switching of signaling of ␤2-adrenergic receptors from adenylylcyclase coupling to the mitogen-activated protein kinase pathway. In the current work, we demonstrate that Src plays an active role in the agonist-induced desensitization of ␤2-adrenergic The nonreceptor tyrosine kinase family of Src functions in a wide spectrum of cell signaling (1). The recruitment and activation of Src kinases is obligate for the mediation of Ras activation by G-protein-linked receptors (GPLRs) 1 (2, 3). GPLRs display four well known responses to the stimulation by agonists: namely, activation, desensitization, internalization, and eventual resensitization (4, 5). The activation of adenylylcyclase in response to agonist stimulation of ␤2-adrenergic receptors results in elevation of intracellular cyclic AMP levels, activation of protein kinase A, and later phosphorylation of the receptor by protein kinase A and G-protein-linked receptor kinases (GRKs) (6). Phosphorylation leads to desensitization and receptor sequestration, dependent upon the binding of ␤-arrestin to the phosphorylated receptor as well as to clathrin (7,8). Internalization occurs via clathrin-mediated processes (9), and resensitization/dephosphorylation follows by the action of the protein phosphatase 2A (10) or 2B (11). These activities are organized by the protein kinase A-anchoring protein AKAP250 or gravin (12). Gravin acts as a scaffold for interactions among protein kinase A, protein kinase C, PP2B, and the ␤2-adrenergic receptor (13).Src has been shown to participate in the formation of ␤2-adrenergic receptor-Src complexes, in association with ␤-arrestin, to switch the signaling from adenylylcyclase to activation of Ras and its downstream effector, the mitogen-activated protein kinase (3). Src has been reported to phosphorylate and activate GRKs (14), observations confined to systems in which the elements are overexpressed at high levels or examined in reconstituted systems, leaving unanswered the question whether or not Src interacts directly with the ␤2-adrenergic receptor and functions in desensitization. The proposed model for Src action defines a temporal sequence in which GRK2 phosphorylates the ␤2-adrenergic receptor, and an Src-arrestin complex then targets to the receptor, facilitating internalization and signaling to Ras (3). In the current work we reveal that Src targets a conditional SH2-binding site of the ␤2-adrenergic receptor, is recruited to the phospho-Tyr-350 receptor, is activated, and then phosphorylates GRK2. This temporal sequence is shown to be obligate for agonist-induced desensitization. EXPERIMENTAL PROCEDURESCell Culture-Human epidermoid carcinoma cells (A431) and Chinese hamster ovary (CHO) cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT), penicillin (60 g/ml), and streptomycin (100 g/ml) and grown in a humidified atmosphere of 5% CO 2 and 95% air at 37°C. Cells were stably transfected with mamma...