Activation of a frizzled-2/beta-adrenergic receptor chimera promotes Wnt signaling and differentiation of mouse F9 teratocarcinoma cells via Galphao and Galphat - PubMed (original) (raw)

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

X Liu et al. Proc Natl Acad Sci U S A. 1999.

Abstract

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.

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Figures

Figure 1

Figure 1

Conditioned medium from cells expressing Xwnt-5a, but not Xwnt-8, stimulates differentiation of mouse F9 teratocarcinoma cells expressing Rfz-2 to PE, as established by the expression of the PE marker, tPA. (A) F9 clones stably transfected with the vector expressing Rfz-2 receptor, Xwnt-5a, Xwnt-8, or empty vector (EV) were selected on the basis of expression of target mRNA, as measured by reverse transcription–PCR. The molecular markers (MK) indicate relative size in bp of the amplified product. Boldface tick marks identify expected product. (B) Conditioned medium was collected from F9 clones stably transfected with Xwnt-5a, Xwnt-8, or the empty vector and used to supplement 1:9 (ratio of conditioned medium of target Rfz-2-expressing clones to that of the Wnt-expressing clones) the medium of the F9 cells stably expressing either Rfz-2 or the empty vector (EV; Left). Nil denotes the clones to which no conditioned medium was added. The ratio of conditioned medium of the target Rfz-2-expressing clones to that of the Wnt-expressing clones was varied from 9:1 to 1:9, and the PE formation was followed by tPA activity (Right). The tPA activity is calculated as described (47). The results are presented as the mean values ± SEM of at least four separate experiments. *, P < 0.05 for difference from the mean. (C) Coculture of Rfz-2 receptor-expressing F9 clones with clones expressing green fluorescent protein (GFP) and either Xwnt-5a or Xwnt-8. Coverslips on which Rfz-2-expressing cells had been cultured were transferred to Petri dishes seeded with clones expressing a single Wnt ligand (phase contrast, PC). Epifluorescence (EF) analysis reveals the GFP/Wnt-producing clones; the Rfz-2-bearing clones are negative. Indirect immunofluorescence (IIF) analysis of coculture partners reveals positive TROMA staining (indicative of PE formation) only for cocultures of Rfz-2 cells with Xwnt-5a- but not Xwnt-8-producing clones.

Figure 2

Figure 2

Expression of a chimera of the exofacial and transmembrane domains of the β2AR with the cytoplasmic domains of Rfz-2 gene products enables ISO-stimulated differentiation of F9 cells to PE. (A) Schematic representation of the Rfz-2/β2AR construct. (B) Reverse transcription–PCR and immunoblotting of Rfz-2/β2AR stably expressed in F9 cells. The RNA of F9 clones harboring either the empty expression vector (EV) or the vector expressing the Rfz-2/β2AR chimera was reverse-transcribed and amplified. The molecular markers (MK) indicate the relative size in bp of the amplified products. Note the identification of the 55-kDa chimeric receptor species in the immunoblots of clones 1 and 2 when stained with anti-β2AR antibodies. (C) The β-adrenergic agonist ISO (10 μM) stimulates whereas the β-adrenergic antagonist propranolol (10 μM) blocks differentiation to PE in F9 clones expressing the Rfz-2/β2AR chimera. Clones were treated with ligand for 4 days and then fixed and stained with TROMA-1 antibody to reveal PE formation. Indirect immunofluorescence (IIF) and phase-contrast (PC) images are displayed. (D) Dose response for ISO-stimulated differentiation to PE in F9 clones expressing the Rfz-2/β2AR chimera. Formation of PE was revealed by positive staining with the TROMA-1 antibody, shown by IIF. Based on multiple dose responses, the _K_d for ISO-induced PE formation appears to be ≈2 μM in the F9 clones stably expressing the chimeric receptor.

Figure 3

Figure 3

Expression of the Rfz-2/β2AR chimera in zebrafish embryos results in appearance of ISO-stimulated calcium transients. The composites of fura-2-dextran fluorescence ratio–image analysis represent the frequency of intracellular Ca2+ transients in embryos injected with RNA encoding Rfz-2/β2AR and then treated with drugs for 20 min before imaging. (a) A representative embryo perfused with a solution of β-adrenergic agonist (ISO, 100 μM) to stimulate the receptor. (b) A sibling representative embryo perfused with a solution of β-adrenergic antagonist (propranolol, 100 μM). (c and d) A representative embryo was treated with β-adrenergic antagonist (propranolol, 100 μM) for 20 min, imaged for 20 min (c), and then switched to agonist (ISO, 100 μM) for 20 min and imaged (d). (e) As a control, one-half of a zebrafish embryo was injected with Rfz-2/β2AR RNA in Texas Red dextran, and the embryo was challenged with ISO. Note that only the half of the embryo microinjected with Rfz-2/β2AR RNA (determined by Texas Red fluorescence; not shown) displays a calcium transient in response to challenge with ISO. Data were collected during the time of mesoderm induction between the 32- and 64-cell stages and the 1,000-cell stage. The pseudocolor ratio images represented in this set are a compilation of a 20-min time course in which the color bar represents the number of transients: red denotes high numbers, violet represents lower numbers, and the peaks represent more active regions.

Figure 4

Figure 4

Analysis of downstream signaling of Rfz-2/β2AR chimera by using pertussis toxin treatment as well as treatment with ODN antisense to specific G protein subunits. Cells stably transfected with Rfz-2/β2AR chimera were treated either with ODN antisense to the specific G protein subunits indicated 48 hr in advance or with pertussis toxin 12 hr in advance of challenge with ISO (10 μM). For the control situation, only the clones expressing the Rfz-2/β2AR chimera and then treated with ISO differentiated to PE within 4 days. For pertussis toxin, the cells were intoxicated (10 ng toxin/ml) for 4 hr before the challenge with ISO. The PE phenotype was established by positive staining of the cells by TROMA antibody. The data are compiled from results of three separate trials for each ODN. The extent of subunit suppression in response to the treatment with specific ODNs was quantified from the immunoblots and ranged between 85% (Gαs) and 95% (Gαi2).

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