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C C Malbon

Publications and source records attributed to C C Malbon.

At least 19 recordsLinked to original sources
PubMed2001

Lithium suppresses signaling and induces rapid sequestration of beta2-adrenergic receptors.

Lithium is a monovalent cation used therapeutically to treat a range of affective disorders (1), although the cellular mechanisms of lithium regulation that might contribute to its therapeutic effects at the level of neurotransmitter receptors are not known. Herein we report the ability of lithium to stimulate the internalization of beta2-adrenergic receptors. Lithium treatment of A431 human epidermoid carcinoma cells resulted in a rapid, prominent desensitization and internalization of beta2-adrenergic receptors. The ability of these receptors to generate a cyclic AMP response was strongly inhibited by lithium, at concentrations therapeutic in humans. Receptors for the serotonin (5HT1c) and for opiates (mu-opioid), in sharp contrast, resisted the effects of lithium on internalization. These data provide the first receptor-based mechanism to be described for lithium that could explain, in part, the therapeutic effects of lithium on affective disorders.

Adrenergic beta-2 Receptor Antagonists↗
PubMed2001

Wnt signaling and heterotrimeric G-proteins: strange bedfellows or a classic romance?

Wnts are secreted ligands with diverse roles in animal development. Wnts bind to cell surface membrane proteins termed Frizzleds. Molecular cloning of members of the Frizzled family revealed hydropathy plots with seven putative, transmembrane-spanning regions, conserved in Frizzleds characterized in mice, humans, flies, and worms. Understanding how Frizzled translates binding of their cognate Wnts into intracellular signals controlling aspects of development has been an elusive goal. Earlier observations gathered from a variety of model systems provided compelling, but indirect, support that the Frizzled receptors may be members of the superfamily of G-protein-coupled receptors that possess seven transmembrane-spanning domains. Search for a linkage between Frizzled and possible downstream heterotrimeric G-proteins has been advanced by the use of bacterial toxins, antisense DNA, and novel chimeric receptor constructs. New data establish that Frizzleds are indeed bona fide G-protein-coupled receptors. Frizzled-1 couples via G-proteins Go and Gq to the canonical beta-catenin-Lef-Tcf pathway. Frizzled-2 couples via Gq and Gt to downstream effectors including calcium mobilization. Frizzleds and G-proteins might once have been considered strange bedfellows, not likely partners in signaling. The new data, consistent with the properties known for virtually all members of the G-protein-coupled receptors, reveal a more classic romance of signaling elements controlling aspects of early development.

Animals↗
PubMed2001

Galpha(i2) enhances insulin signaling via suppression of protein-tyrosine phosphatase 1B.

Suppression of the expression of the heterotrimeric G-protein Galpha(i2) in vivo has been shown to provoke insulin resistance, whereas enhanced insulin signaling is observed when Galpha(i2) is overexpressed in vivo. The basis for Galpha(i2) regulation of insulin signaling was explored in transgenic mice with targeted expression of the GTPase-deficient, constitutively active Q205L Galpha(i2) in fat and skeletal muscle. Phosphorylation of insulin receptor and IRS-1 in response to insulin challenge in vivo was markedly amplified in fat and skeletal muscle expressing Q205L Galpha(i2). The expression and activity of the protein-tyrosine phosphatase 1B (PTP1B), but not protein-tyrosine phosphatases SHP-1, SHP-2, and LAR, were constitutively decreased in tissues expressing the Q205L Galpha(i2), providing a direct linkage between insulin signaling and Galpha(i2). The loss of PTP1B expression may explain, in part, the loss of PTP1B activity in the iQ205L transgenic mice. Activation of Galpha(i2) in mouse adipocytes with lysophosphatidic acid was shown to decrease PTP1B activity, whereas pertussis toxin inactivates Galpha(i2), blocks lysophosphatidic acid-stimulated inhibition of PTP1B activity, and blocks tonic suppression of PTP1B activity by Galpha(i2). Elevation of intracellular cAMP in fat cells is shown to increase PTP1B activity, whereas either depression of cAMP levels or direct activation of Galpha(i2) suppresses PTP1B. These data provide the first molecular basis for the interplay between Galpha(i2) and insulin signaling, i.e. activation of Galpha(i2) can suppress both the expression and activity of PTP1B in insulin-sensitive tissues.

Adipose Tissue↗
PubMed2001

Galpha i2 enhances in vivo activation of and insulin signaling to GLUT4.

Heterotrimeric G-proteins, including Galpha(i2), have been implicated in modulating glucose disposal and insulin signaling. This cross-talk between G-protein-coupled and tyrosine kinase-coupled signaling pathways is a focal point for the study of integration of cell signaling. Herein we study the role of Galpha(i2) in modulating glucose transport, focusing upon linkages to insulin signaling. Utilizing mice harboring a transgene that directs the expression of a constitutively activated, GTPase-deficient mutant of Galpha(i2) (Q205L) in adipose tissue, skeletal muscle, and liver, we demonstrate that Galpha(i2) regulates the translocation of the insulin-sensitive GLUT4 glucose transporter in skeletal muscle and adipose tissue. The expression of Q205L Galpha(i2) increased glucose transport and translocation of GLUT4 to the plasma membrane in vivo in the absence of insulin stimulation. Adipocytes from the Q205L Galpha(i2) mice displayed enhanced insulin-stimulated glucose transport and GLUT4 translocation to the plasma membrane to levels nearly twice that of those from littermate controls. Phosphatidylinositol 3-kinase and Akt activities were constitutively activated in tissues expressing the Q205L Galpha(i2). Studies of adipocytes from wild-type mice displayed short term activation of phosphatidylinositol 3-kinase, Akt, and GLUT4 translocation in response to activation of Galpha(i2) by lysophosphatidic acid, a response sensitive to pertussis toxin. These data provide an explanation for the marked glucose tolerance of the Q205L Galpha(i2) mice and demonstrate a linkage between Galpha(i2) and GLUT4 translocation.

Adipose Tissue↗
PubMed2001

Insulin stimulates tyrosine phosphorylation and inactivation of protein-tyrosine phosphatase 1B in vivo.

Protein-tyrosine phosphatase (PTP) 1B has been implicated in negative regulation of insulin action, although little is known of the ability of insulin to regulate PTP1B itself. The ability of insulin to regulate phosphorylation and activation of PTP1B was probed in vivo. Challenge with insulin in vivo provoked a transient, sharp increase in the phosphotyrosine content of PTP1B in fat and skeletal muscle that peaked within 15 min. Insulin stimulated a decline of 60--70% in PTP1B activity. In mouse adipocytes, the inhibition of PTP1B activity and increased tyrosine phosphorylation of the enzyme were blocked by the insulin receptor tyrosine kinase inhibitor AG1024. Phosphoserine content of PTP1B declined in response to insulin stimulation. Elevation of intracellular cyclic AMP provokes a sharp increase in PTP1B activity and leads to increased phosphorylation of serine residues and decreased tyrosine phosphorylation. Suppression of cyclic AMP levels or inhibition of protein kinase A leads to a sharp decline in PTP1B activity, a decrease in phosphoserine content, and an increase in PTP1B phosphotyrosine content. PTP1B appears to be a critical point for insulin and catecholamine counter-regulation.

Adipocytes↗
PubMed2001

G protein signaling from activated rat frizzled-1 to the beta-catenin-Lef-Tcf pathway.

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.

Amino Acid Sequence↗
PubMed2001

The scaffold protein gravin (cAMP-dependent protein kinase-anchoring protein 250) binds the beta 2-adrenergic receptor via the receptor cytoplasmic Arg-329 to Leu-413 domain and provides a mobile scaffold during desensitization.

The cyclic AMP-dependent kinase-anchoring proteins (AKAPs) function as scaffolds for a wide-range of protein-protein interactions. The 250-kDa AKAP known as gravin plays a central role in organizing G-protein-coupled receptors to the protein kinases and phosphatases that regulate receptor function in desensitization, resensitization, and sequestration. Although gravin is critical for G-protein-linked receptor biology, the molecular features of the receptor necessary for interaction with this scaffold are not known. Herein, we map the regions of the beta(2)-adrenergic receptor that are required for binding to gravin. Intracellular loops 1, 2, and 3 appear not to participate in the binding of the receptor to the scaffold. In contrast, the C-terminal cytoplasmic region of the receptor (Arg-329 to Leu-413) competes readily for the binding of the beta(2)-adrenergic receptor by gravin, both using in vitro and in vivo assays. C-terminally truncated peptides with sequences ranging from Arg-329 to Leu-342 (13 aminoacyl residues), to Asn-352 (23 residues), to Tyr-366 (37 residues), to Asp-380 (51 residues), or to His-390 (61 residues), as well as N-terminally truncated peptides from Gln-391 to Leu-413 (23 residues) or Leu-381 to Leu-413 (33 residues) displayed no ability to block binding of receptor to gravin. The combination of Arg-329 to His-390 peptide and Gln-391 to Leu-413 peptide, however, reconstitutes a fragmented but full-length C-terminal region and also potently blocks the ability of gravin to bind the beta(2)-adrenergic receptor. The gravin-receptor interaction was examined in response to agonist by confocal microscopy. Remarkably, the association of the receptor with gravin was not disrupted during agonist-induced sequestration. The receptor-scaffold complex was maintained during agonist-induced sequestration. These data, in agreement with the biochemical data, reveal that gravin binds the receptor through the beta(2)-adrenergic receptor C-terminal cytoplasmic domain and that this interaction is maintained as the receptor is internalized. This is the first report of an AKAP scaffold protein translocating with its receptor, in this case a G-protein-coupled receptor.

A Kinase Anchor Proteins↗
PubMed2001

c-Src tyrosine kinase binds the beta 2-adrenergic receptor via phospho-Tyr-350, phosphorylates G-protein-linked receptor kinase 2, and mediates agonist-induced receptor desensitization.

The nonreceptor tyrosine kinase Src has been implicated in the switching of signaling of beta2-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 beta2-adrenergic receptors. Both the expression of dominant-negative Src and treatment with the 4-amine-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) inhibitor of Src kinase activity blocks agonist-induced desensitization. Agonist triggers tyrosine phosphorylation of the beta2-adrenergic receptor and recruitment and activation of Src. Because phosphorylation of the Tyr-350 residue of the beta2-adrenergic receptor creates a conditional, canonical SH2-binding site on the receptor, we examined the effect of the Y350F mutation on Src phosphorylation, Src recruitment, and desensitization. Mutant beta2-adrenergic receptors with a Tyr-to-Phe substitution at Tyr-350 do not display agonist-induced desensitization, Src recruitment, or Src activation. Downstream of binding to the receptor, Src phosphorylates and activates G-protein-linked receptor kinase 2 (GRK2), a response obligate for agonist-induced desensitization. Constitutively active Src increases GRK phosphorylation, whereas either expression of dominant-negative Src or treatment with the PP2 inhibitor abolishes tyrosine phosphorylation of GRK and desensitization. Thus, in addition to its role in signal switching to the mitogen-activated protein kinase pathway, Src recruitment to the beta2-adrenergic receptor and activation are obligate for normal agonist-induced desensitization.

Adrenergic beta-Agonists↗
PubMed2000

Insulin activation of mitogen-activated protein kinases Erk1,2 is amplified via beta-adrenergic receptor expression and requires the integrity of the Tyr350 of the receptor.

Insulin activates a complex set of intracellular responses, including the activation of mitogen-activated protein kinases Erk1,2. The counterregulatory actions of insulin on catecholamine action are well known and include phosphorylation of the beta(2)-adrenergic receptor on Tyr(350), Tyr(354), and Tyr(364) in the C-terminal cytoplasmic domain, as well as enhanced sequestration of the beta(2)-adrenergic receptor. Both beta-adrenergic agonists and insulin provoke sequestration of beta(2)-adrenergic receptors in a synergistic manner. In the current work, cross-talk between insulin action and beta(2)-adrenergic receptors revealed that insulin activation of Erk1,2 was amplified via beta(2)-adrenergic receptors. In Chinese hamster ovary cells, expression of beta(2)-adrenergic receptors enhanced 5-10-fold the activation of Erk1,2 by insulin and prolonged the activation, the greatest enhancement occurring at 5 min post-insulin. The potentiation of insulin signaling on Erk1,2 was proportional to the level of expression of beta(2)-adrenergic receptor. The potentiation of insulin signaling requires the integrity of Tyr(350) of the beta(2)-adrenergic receptor, a residue phosphorylated in response to insulin. beta(2)-adrenergic receptors with a Y350F mutation failed to potentiate insulin activation of Erk1,2. Expression of the C-terminal domain of the beta(2)-adrenergic receptor (Pro(323)-Leu(418)) in cells expressing the intact beta(2)-adrenergic receptor acts as a dominant negative, blocking the potentiation of insulin activation of Erk1,2 via the beta(2)-adrenergic receptor. Blockade of beta(2)-adrenergic receptor sequestration does not alter the ability of the beta(2)-adrenergic receptor to potentiate insulin action on Erk1,2. We propose a new paradigm in which a G-protein-linked receptor, such as the beta(2)-adrenergic receptor, itself acts as a receptor-based scaffold via its binding site for Src homology 2 domains, facilitating signaling of the mitogen-activated protein kinase pathway by insulin.

Adrenergic beta-Agonists↗
PubMed2000

MEKK4 mediates differentiation in response to retinoic acid via activation of c-Jun N-terminal kinase in rat embryonal carcinoma P19 cells.

Differentiation of P19 embryonal carcinoma cells in response to the morphogen retinoic acid is regulated by Galpha(12/13) and is associated with activation of c-Jun N-terminal kinase. The role of MEKK1 and MEKK4 upstream of the c-Jun N-terminal kinase was investigated in P19 cells. P19 clones stably expressing constitutively active and dominant negative mutants of MEKK1 and MEKK4 were created and characterized. Expression of the constitutively active form of either MEKK1 or MEKK4 mimicked the action of retinoic acid, inducing these embryonal carcinoma cells to primitive endoderm. Expression of the dominant negative form of MEKK1 had no influence on the ability of retinoic acid to induce either JNK activation or primitive endoderm formation in P19 stem cells. Expression of the dominant negative form of MEKK4, in contrast, effectively blocks both morphogen-induced activation of JNK and cellular differentiation. These data identify MEKK4 as upstream of c-Jun N-terminal kinase in the pathway mediating differentiation of P19 stem cells to primitive endoderm.

Animals↗
PubMed2000

Gravin-mediated formation of signaling complexes in beta 2-adrenergic receptor desensitization and resensitization.

Agonist-induced desensitization and resensitization of G-protein-linked receptors involve the interaction of receptors with protein kinases, phosphatases, beta-arrestin, and clathrin organized by at least one scaffold protein. The dynamic composition of the signaling complexes and the role of the scaffold protein AKAP250 (gravin) in agonist-induced attenuation and recovery of beta-adrenergic receptors were explored by co-immunoprecipitation of target elements, antisense suppression, and confocal microscopy. Gravin associated with unstimulated receptor, and the association was increased significantly after agonist stimulation for up to 60 min. Agonist stimulation also induced a robust association of the receptor-gravin complex with protein kinases A and C, G-protein-linked receptor kinase-2, beta-arrestin, and clathrin. Confocal microscopy of the green fluorescence protein-tagged beta(2)-adrenergic receptor showed that the receptor underwent sequestration after agonist stimulation. Suppression of gravin expression via antisense oligodeoxynucleotides disrupted agonist-induced association of the receptor with G-protein-linked receptor kinase-2, beta-arrestin, and clathrin as well as receptor recovery from desensitization. Gravin deficiency also inhibited agonist-induced sequestration. These data reveal that gravin-mediated formation of signaling complexes with protein kinases/phosphatases, beta-arrestin, and clathrin is essential in agonist-induced internalization and resensitization of G-protein-linked receptors.

A Kinase Anchor Proteins↗
PubMed2000

Ca(2+)/calmodulin-dependent protein kinase II is stimulated by Wnt and Frizzled homologs and promotes ventral cell fates in Xenopus.

Wnt ligands working through Frizzled receptors have a differential ability to stimulate release of intracellular calcium (Ca(2+)) and activation of protein kinase C (PKC). Since targets of this Ca(2+) release could play a role in Wnt signaling, we first tested the hypothesis that Ca(2+)/calmodulin-dependent protein kinase II (CamKII) is activated by some Wnt and Frizzled homologs. We report that Wnt and Frizzled homologs that activate Ca(2+) release and PKC also activate CamKII activity in Xenopus embryos, while Wnt and Frizzled homologs that activate beta-catenin function do not. This activation occurs within 10 min after receptor activation in a pertussis toxin-sensitive manner, concomitant with autophosphorylation of endogenous CamKII. Based on data that Wnt-5A and Wnt-11 are present maternally in Xenopus eggs, and activate CamKII, we then tested the hypothesis that CamKII participates in axis formation in the early embryo. Measurements of endogenous CamKII activity from dorsal and ventral regions of embryos revealed elevated activity on the prospective ventral side, which was suppressed by a dominant negative Xwnt-11. If this spatial bias in CamKII activity were involved in promoting ventral cell fate one might predict that elevating CamKII activity on the dorsal side would inhibit dorsal cell fates, while reducing CamKII activity on the ventral side would promote dorsal cell fates. Results obtained by expression of CamKII mutants were consistent with this prediction, revealing that CamKII contributes to a ventral cell fate.

Animals↗
PubMed2000

The full-length, cytoplasmic C-terminus of the beta 2-adrenergic receptor expressed in E. coli acts as a substrate for phosphorylation by protein kinase A, insulin receptor tyrosine kinase, GRK2, but not protein kinase C and suppresses desensitization when expressed in vivo.

The ability of the cytoplasmic, full-length C-terminus of the beta 2-adrenergic receptor (BAC1) expressed in Escherichia coli to act as a functional domain and substrate for protein phosphorylation was tested. BAC1 was expressed at high-levels, purified, and examined in solution as a substrate for protein phosphorylation. The mobility of BAC1 on SDS-PAGE mimics that of the native receptor itself, displaying decreased mobility upon chemical reduction of disulfide bonds. Importantly, the C-terminal, cytoplasmic domain of the receptor expressed in E. coli was determined to be a substrate for phosphorylation by several candidate protein kinases known to regulate G-protein-linked receptors. Mapping was performed by proteolytic degradation and matrix-assisted laser desorption ionization, time-of-flight mass spectrometry. Purified BAC1 is phosphorylated readily by protein kinase A, the phosphorylation occurring within the predicted motif RRSSSK. The kinetic properties of the phosphorylation by protein kinase A displayed cooperative character. The activated insulin receptor tyrosine kinase, which phosphorylates the beta-adrenergic receptor in vivo, phosphorylates BAC1. The Y364 residue of BAC1 was predominantly phosphorylated by the insulin receptor kinase. GRK2 catalyzed modest phosphorylation of BAC1. Phosphorylation of the human analog of BAC1 in which Cys341 and Cys378 were mutated to minimize disulfide bonding constraints, displayed robust phosphorylation following thermal activation, suggesting under standard conditions that the population of BAC1 molecules capable of assuming the "activated" conformer required by GRKs is low. BAC1 was not a substrate for protein kinase C, suggesting that the canonical site in the second cytoplasmic loop of the intact receptor is preferred. The functional nature of BAC1 was tested additionally by expression of BAC1 protein in human epidermoid carcinoma A431 cells. BAC1 was found to act as a dominant-negative, blocking agonist-induced desensitization of the beta-adrenergic receptor when expressed in mammalian cells. Thus, the C-terminal, cytoplasmic tail of this G-protein-linked receptor expressed in E. coli acts as a functional domain, displaying fidelity with regard to protein kinase action in vivo and acting as a dominant-negative with respect to agonist-induced desensitization.

Amino Acid Sequence↗
PubMed1999

Activation of a frizzled-2/beta-adrenergic receptor chimera promotes Wnt signaling and differentiation of mouse F9 teratocarcinoma cells via Galphao and Galphat.

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 beta-adrenergic agonist by using the ligand-binding domains of the beta(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 beta-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 Galphao, Galphat2, and Gbeta2. Our results demonstrate the requirement of two pertussis toxin-sensitive G proteins, Galphao and Galphat, for signaling by the Frizzled-2 receptor.

Amino Acid Sequence↗
PubMed1999

Analysis of the AU-rich elements in the 3'-untranslated region of beta 2-adrenergic receptor mRNA by mutagenesis and identification of the homologous AU-rich region from different species.

The 35000-Mr beta-adrenergic receptor mRNA binding protein (beta ARB) is induced by beta-adrenergic agonists and binds to G-protein-linked receptor mRNAs that exhibit agonist-induced destabilization. Recently, we identified a 20-nucleotide, AU-rich region in the 3'-untranslated region of the hamster beta 2-adrenergic receptor mRNA consisting of an AUUUUA hexamer flanked by U-rich regions, which constitutes the binding domain for beta ARB. U to G substitution in the hexamer region attenuates the binding of beta ARB, whereas U to G substitution of hexamer and flanking U-rich domains abolishes binding of beta ARB and stabilizes beta 2-adrenergic receptor mRNA levels in transfectant clones challenged with either isoproterenol or cyclic AMP. In the study presented here, we mutated the 20-nucleotide ARE region to establish the minimal AU-rich sequence required for beta ARB binding. U to G substitutions of flanking poly(U) regions and of the hexamer established the nature of the binding properties. Using various mutants, we demonstrated also that binding of beta ARB correlates with the extent of destabilization of beta 2-adrenergic receptor mRNA in response to agonist stimulation. High-affinity binding of hamster, rat, mouse, porcine, and human ARE sequences to beta ARB was revealed by SDS-polyacrylamide gel electrophoresis following UV-catalyzed cross-linking and by gel mobility shift assays. Further, beta ARB was shown to bind more avidly to the 20-nucleotide ARE region than to well-established mRNA destablization sequences of tandem repeats of five pentamers. Thus, for beta 2-adrenergic receptor, mRNA destabilization likely occurs via conserved AU-rich elements present in the 3'-untranslated regions of receptor mRNAs.

3' Untranslated Regions↗
PubMed1999

Activation of rat frizzled-1 promotes Wnt signaling and differentiation of mouse F9 teratocarcinoma cells via pathways that require Galpha(q) and Galpha(o) function.

The frizzled gene family of putative Wnt receptors encodes proteins that have a seven transmembrane-spanning motif characteristic of G-protein-linked receptors, although no loss-of-function studies have demonstrated a requirement for G-proteins for Wnt signaling by the gene product of frizzled-1. Medium conditioned by mouse F9 teratocarcinoma stem cells stably transfected to express either Xenopus Wnt-5a or Wnt-8 was used to test primitive endoderm formation of F9 stem cells. F9 stem cells expressing the rat Frizzled-1 receptors demonstrated endoderm formation in response to conditioned medium containing Wnt-8 but not to medium containing Wnt-5a. Primitive endoderm formation stimulated by Wnt-8 acting on the rat Frizzled-1 receptor was blocked by treatment with pertussis toxin by depletion of either Galpha(o) or Galpha(q) via antisense oligodeoxynucleotides, as well as by inhibitors of protein kinase C (bisindoylmaleimide) and of mitogen-activated protein kinase kinase (PD98059). Our results demonstrate the requirement for G-protein subunits Galpha(o) (a pertussis toxin substrate) and Galpha(q) for signaling by Frizzled-1, and an obligate role for the protein kinase C (likely mediated through stimulation of Galpha(q)) and mitogen-activated protein kinase network at the level of mitogen-activated protein kinase kinase.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1999

G(s)alpha repression of adipogenesis via Syk.

G(s)alpha regulates the differentiation of 3T3-L1 mouse embryonic fibroblasts to adipocytes, a process termed adipogenesis. Inducers of adipogenesis lead to a loss of G(s)alpha and derepress differentiation to adipocytes. The broad spectrum tyrosine kinase inhibitor genistein is shown to block induction of adipogenesis, suggesting an early role of tyrosine phosphorylation in adipogenesis. Staining of phosphotyrosine identified prominent staining of a approximately 70-kDa protein, hypothesized to be the tyrosine kinase Syk. Reverse transcription and polymerase chain reaction amplification established the expression of Syk mRNA in these embryonic fibroblasts. Immunoprecipitations with Syk-specific antibodies demonstrated the presence of Syk in fibroblasts and a rapid increase in the amount of phospho-Syk, peaking at 24 h post induction. Clones constitutively expressing G(s)alpha, which can no longer be induced to differentiate, no longer display increased phospho-Syk levels in response to inducers. The linkage between G(s)alpha and Syk was probed by immunoprecipitations revealing association of Syk with G(s)alpha in the absence of induction. Upon induction of adipogenesis, G(s)alpha levels decline and phospho-Syk levels as well as Syk kinase activity increase. Expression of wild-type Syk both potentiates the ability of inducers to act as well as induces adipogenesis itself. Expression of the kinase-deficient Syk had no such effects on adipogenesis. These data provide a new insight into the control of adipogenesis, suggesting that G(s)alpha represses adipogenesis via Syk. Treatment with the inducers promotes a decline in G(s)alpha, increases in levels of phospho-Syk, and adipogenesis.

3T3 Cells↗