The metabotropic glutamate 1 (mGlu1) receptor in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Here we show that the G protein-coupled receptor kinases (GRK) 2 and 4, which are expressed in these cells, regulate the mGlu1 receptor by at least in part different mechanisms. Using kinase-dead mutants in HEK293 cells, we found that GRK4, but not GRK2, needs the intact kinase activity to desensitize the mGlu1 receptor, whereas GRK2, but not GRK4, can interact with and regulate directly the activated Gαq. In cells transfected with GRK4 and exposed to agonist, ॆ-arrestin was first recruited to plasma membranes, where it was co-localized with the mGlu1 receptor, and then internalized in vesicles. The receptor was also internalized but in different vesicles. The expression of ॆ-arrestin V53D dominant negative mutant, which did not affect the mGlu1 receptor internalization, reduced by 70–807 the stimulation of mitogen-activated protein (MAP) kinase activation by the mGlu1 receptor. The agonist-stimulated differential sorting of the mGlu1 receptor and ॆ-arrestin as well as the activation of MAP kinases by mGlu1 agonist was confirmed in cultured cerebellar Purkinje cells. A major involvement of GRK4 and of ॆ-arrestin in agonist-dependent receptor internalization and MAP kinase activation, respectively, was documented in cerebellar Purkinje cells using an antisense treatment to knock down GRK4 and expressing ॆ-arrestin V53D dominant negative mutant by an adenovirus vector. We conclude that GRK2 and GRK4 regulate the mGlu1receptor by different mechanisms and that ॆ-arrestin is directly involved in glutamate-stimulated MAP kinase activation by acting as a signaling molecule. The metabotropic glutamate 1 (mGlu1) receptor in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Here we show that the G protein-coupled receptor kinases (GRK) 2 and 4, which are expressed in these cells, regulate the mGlu1 receptor by at least in part different mechanisms. Using kinase-dead mutants in HEK293 cells, we found that GRK4, but not GRK2, needs the intact kinase activity to desensitize the mGlu1 receptor, whereas GRK2, but not GRK4, can interact with and regulate directly the activated Gαq. In cells transfected with GRK4 and exposed to agonist, ॆ-arrestin was first recruited to plasma membranes, where it was co-localized with the mGlu1 receptor, and then internalized in vesicles. The receptor was also internalized but in different vesicles. The expression of ॆ-arrestin V53D dominant negative mutant, which did not affect the mGlu1 receptor internalization, reduced by 70–807 the stimulation of mitogen-activated protein (MAP) kinase activation by the mGlu1 receptor. The agonist-stimulated differential sorting of the mGlu1 receptor and ॆ-arrestin as well as the activation of MAP kinases by mGlu1 agonist was confirmed in cultured cerebellar Purkinje cells. A major involvement of GRK4 and of ॆ-arrestin in agonist-dependent receptor internalization and MAP kinase activation, respectively, was documented in cerebellar Purkinje cells using an antisense treatment to knock down GRK4 and expressing ॆ-arrestin V53D dominant negative mutant by an adenovirus vector. We conclude that GRK2 and GRK4 regulate the mGlu1receptor by different mechanisms and that ॆ-arrestin is directly involved in glutamate-stimulated MAP kinase activation by acting as a signaling molecule. metabotropic glutamate G protein-coupled receptor GTP-binding protein G protein-coupled receptor kinase the N-terminal domain of GRK inositol phosphate green fluorescent protein the α subunit of G protein extracellular signal-regulated kinase mitogen-activated protein mitogen-activated protein kinase regulators of G protein signaling glutathione S-transferase phosphate-buffered saline bovine serum albumin platelet-activating factor PAF receptor Metabotropic glutamate (mGlu)1 receptors, which are activated by the excitatory amino acid glutamate, are part of an original family of G protein-coupled receptors (GPCR) called the family 3 GPCRs (1De Blasi A. Conn P.J. Pin J.-P. Nicoletti F. Trends Pharmacol. Sci. 2001; 22: 114-120Google Scholar, 2Nakanishi S. Neuron. 1994; 29: 1031-1037Google Scholar, 3Conn P.J. Pin J.-P. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 205-237Google Scholar). These include all the mGlu receptor subtypes, Ca2+-sensing and GABAB receptors, and some putative olfactory, pheromone, and taste receptors. Eight subtypes of mGlu receptors have been identified, which are implicated in different aspects of physiology and pathology of the central nervous system. Group I mGlu receptors (mGlu1 and mGlu5), which stimulate polyphosphoinositide hydrolysis by coupling to Gq, are localized in the peripheral parts of postsynaptic dendrites and contribute to the regulation of synaptic plasticity. For example, mGlu1 receptor present in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Similar to many other GPCRs, the signal transduction of the mGlu1 receptor is strictly regulated by multiple mechanisms acting at different levels of signal propagation (1De Blasi A. Conn P.J. Pin J.-P. Nicoletti F. Trends Pharmacol. Sci. 2001; 22: 114-120Google Scholar). After prolonged or repeated stimulation, receptors are profoundly desensitized. Protein kinase C is clearly involved in this process, although a protein kinase C-independent component of mGlu1 receptor desensitization was also observed (4Catania M.V. Aronica E. Sortino M.A. Canonico P.L. Nicoletti F. J. Neurochem. 1991; 56: 1329-1335Google Scholar). The activated α subunit of the Gq(Gαq) can in turn be inhibited by RGS (forregulators of G protein signaling) proteins (5Saugstad J.A. Marino M.J. Folk J.A. Hepler J.R. Conn P.J. J. Neurosci. 1998; 18: 905-913Google Scholar). These RGS proteins work by interacting with Gα and by increasing the intrinsic GTPase activity of Gα, acting as GTPase-activating proteins (6Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Google Scholar, 7Hepler J.R. Trends Pharmacol. Sci. 1999; 20: 376-382Google Scholar). Recent studies from our and other laboratories have documented that G protein-coupled receptor kinases (GRKs) and arrestins are involved in the mechanism of agonist-stimulated mGlu1 receptor phosphorylation, desensitization, and internalization. Using transfected HEK293 cells, it was shown that the mGlu1 receptor is phosphorylated and desensitized by different GRK subtypes (8Sallese M. Salvatore L. D'Urbano E. Sala G Storto M. Launey T. Nicoletti F. Knopfel T. De Blasi A. FASEB J. 2000; 14: 2569-2580Google Scholar, 9Dale B.D. Bhattacharya M. Anborgh P.H. Murdoch B. Bhatia M. Nakanishi S. Ferguson S.S.G. J. Biol. Chem. 2000; 275: 38213-38220Google Scholar) in an agonist-dependent manner. In these cells agonist treatment induced the internalization of the mGlu1 receptor, and this mechanism was ॆ-arrestin- and dynamin-dependent (10Mundell S.J. Matharu A. Pula G. Roberts P.J. Kelly E. J. Neurochem. 2001; 78: 546-551Google Scholar, 11Dale L.B. Bhattacharya M. Seachrist J.L. Anborgh P.H. Ferguson S.S.G. Mol. Pharmacol. 2001; 60: 1243-1253Google Scholar). The mGlu1 receptor is also internalized tonically (i.e. in an agonist-independent manner) by a mechanism that is ॆ-arrestin- and dynamin-independent and likely involves a clathrin-mediated endocytic pathway (11Dale L.B. Bhattacharya M. Seachrist J.L. Anborgh P.H. Ferguson S.S.G. Mol. Pharmacol. 2001; 60: 1243-1253Google Scholar). Based on these results and on studies with different receptor types, it was suggested that multiple endocytic pathways may contribute to the internalization of the same GPCR (11Dale L.B. Bhattacharya M. Seachrist J.L. Anborgh P.H. Ferguson S.S.G. Mol. Pharmacol. 2001; 60: 1243-1253Google Scholar). We have recently shown that GRK4, one GRK subtype originally identified in testis and sperm cells, may play a major role in the regulation of the mGlu1 receptor. In HEK293 cells transfected with GRK4 and in cultured cerebellar Purkinje cells, which naturally express high levels of GRK4, we demonstrated that this kinase is important for the desensitization and for rapid internalization of the mGlu1receptor (8Sallese M. Salvatore L. D'Urbano E. Sala G Storto M. Launey T. Nicoletti F. Knopfel T. De Blasi A. FASEB J. 2000; 14: 2569-2580Google Scholar). The present study shows that the stimulation of the mGlu1receptor induces the rapid redistribution of ॆ-arrestin, which is first recruited to plasma membranes and then internalized in intracellular vesicles. Our results support the possibility that ॆ-arrestin acts as a signaling protein that mediates the mGlu1 receptor-mediated activation of MAP kinases (MAPK). Polyclonal anti-mGlu1 antibody was from Upstate Biotechnology; polyclonal anti-RGS4, anti-ERK1, and anti-Gαq were from Santa Cruz Biotechnology (Santa Cruz, CA); monoclonal anti-phospho-ERK1/2 was from Cell Signaling Technology; monoclonal anti-GRK2/3 was from Upstate Biotechnology; polyclonal anti-GRK4 was from Santa Cruz; monoclonal anti-FLAG M5 was from Eastman Kodak Co.; Alexa-594 protein labeling kit, Alexa-488 anti-mouse, and anti-rabbit IgGs were from Molecular Probes (Eugene, OR); monoclonal anti-ॆ-arrestin antibody (F4C1) was kindly provided by Dr. L. A. Donoso; Cy3-conjugated anti-rabbit IgG was from Sigma. To generate a fusion protein between GST and the N-terminal domain of GRK2 (GST-GRK2-Nter) and of GRK4 (GST-GRK4-Nter), we used a PCR-based method as previously described (12Sallese M. Mariggiò S. D'Urbano E. Iacovelli L. De Blasi A. Mol. Pharmacol. 2000; 57: 826-831Google Scholar). The GRK4-(K216M,K217M) was prepared as previously described (8Sallese M. Salvatore L. D'Urbano E. Sala G Storto M. Launey T. Nicoletti F. Knopfel T. De Blasi A. FASEB J. 2000; 14: 2569-2580Google Scholar). The following plasmids were generous gifts: GRK2-(K220R) from C. Scorer (Glaxo Wellcome, Stevenage, UK), Gαq from A. Gilman (University of Texas, Dallas, TX), Gαq(Q209L) from N. Dhanasekaran (Temple University, Philadelphia, PA), ॆ-arrestin V53D from Federico Mayor (Universidad Autonoma de Madrid, Madrid, Spain), human mGlu1 receptor in pcDNA 3 from M. Corsi (Glaxo Wellcome, Verona, Italy), the human EAAC1 from J. P. Pin (CNRS, Montpellier, France) and M. A. Hediger (Harvard Medical School, Boston, MA), and PAF receptor (PAFr) in pCDM8-FLAG plasmid from C. Gerard (Harvard Medical School, Boston, MA). Cerebellar neurons were prepared from Wistar rats as previously described (8Sallese M. Salvatore L. D'Urbano E. Sala G Storto M. Launey T. Nicoletti F. Knopfel T. De Blasi A. FASEB J. 2000; 14: 2569-2580Google Scholar, 13Furuya S. Makino A. Hirabayashi Y. Brain Res. Protoc. 1998; 3: 192-198Google Scholar), with minor modifications to obtain a Purkinje cell-rich culture. Seven-day-old pups were sacrificed by cervical dislocation and the cerebella excised and minced with a scalpel. The cerebellar cells were disgregated with 0.0257 trypsin and 0.017 DNase I in Krebs Ringer plus 0.037 MgSO4, 0.37 BSA for 15 min at 37 °C. The cells were washed with the same buffer containing 40 ॖg/ml trypsin inhibitor and 0.017 DNase I and dissociated by repeated passage through a fine-tipped pipette. The cell suspension was centrifuged at 400 × g for 2 min, and cells resuspended carefully in 2 ml of the same buffer. After 30–45 min, the Purkinje cells are enriched from granules by gravity. The upper part of the suspension (granules) was removed very carefully, and the sediment (Purkinje cells) was rinsed with culture medium. Recovered cells were plated at a density of 20–25 × 104 cells/cm2 onto poly-l-lysine-coated chamber slides in serum-free defined medium: Eagle's medium supplemented with 1 mg/ml BSA, 10 ॖg/ml insulin, 0.1 nm l-thyroxin, 0.1 mg/ml transferrin, 1 ॖg/ml aprotinin, 30 nm selenium, 100 ॖg/ml streptomycin, and 100 units/ml penicillin. The cultures were maintained in a humidified atmosphere of 57 CO2 in air at 37 °C. The cultures, which consisted of ∼2–37 Purkinje cells (assessed by calbindin immunostaining), were used after 15–20 daysin vitro. HEK293 cells were transfected as described (8Sallese M. Salvatore L. D'Urbano E. Sala G Storto M. Launey T. Nicoletti F. Knopfel T. De Blasi A. FASEB J. 2000; 14: 2569-2580Google Scholar). One day after transfection, the cells were washed in PBS and incubated for 18 h with Dulbecco's modified Eagle's medium/Glutamax-1 (Invitrogen), then washed and incubated overnight with minimal essential medium/Glutamax-1 containing 3 ॖCi/well myo-[3H]inositol (Amersham Biosciences). On the third day, IP production was measured as described (8Sallese M. Salvatore L. D'Urbano E. Sala G Storto M. Launey T. Nicoletti F. Knopfel T. De Blasi A. FASEB J. 2000; 14: 2569-2580Google Scholar). Briefly, cells were washed twice and incubated for 1–2 h at 37 °C in 1 ml of HEPES-buffered saline (146 mmNaCl, 4.2 mm KCl, 0.5 mm MgCl2, 0.17 glucose, 20 mm HEPES, pH 7.4), washed again with HEPES-buffered saline, and pre-incubated for 15 min in the same buffer containing 10 mm LiCl, 1.8 units/ml glutamic pyruvic transaminase, 2 mm sodium pyruvate. The stimulus was carried out for 30 min with 100 ॖm quisqualate, unless otherwise indicated. The reaction was stopped by replacing the incubation medium with 1 ml of ice-cold perchloric acid (57). Inositol phosphates were separated by an ion exchange chromatography column of Dowex AG1-X8 (formiate form) (200–400-mesh, 350-ॖl bed volume). Usually 1 × 106 cells were co-transfected with 1 ॖg of mGlu1 plasmid along with 5 ॖg of GRK cDNA, or empty vector. For mGlu1a 5 ॖg of the plasmid encoding the glutamate transporter EAAC1 (8Sallese M. Salvatore L. D'Urbano E. Sala G Storto M. Launey T. Nicoletti F. Knopfel T. De Blasi A. FASEB J. 2000; 14: 2569-2580Google Scholar) was included. For antisense oligonucleotide treatment, the experiments were performed as previously described (8Sallese M. Salvatore L. D'Urbano E. Sala G Storto M. Launey T. Nicoletti F. Knopfel T. De Blasi A. FASEB J. 2000; 14: 2569-2580Google Scholar). Cerebellar neurons were prepared as described (8Sallese M. Salvatore L. D'Urbano E. Sala G Storto M. Launey T. Nicoletti F. Knopfel T. De Blasi A. FASEB J. 2000; 14: 2569-2580Google Scholar) and maintained in culture for 2–3 weeks, at which time two-end phosphorothioate oligonucleotides at 1 ॖm final concentration were added for 4–5 days. The sequence of the GRK4 antisense oligonucleotide and of the scrambled oligonucleotide (used as control) are as described in Ref. 8Sallese M. Salvatore L. D'Urbano E. Sala G Storto M. Launey T. Nicoletti F. Knopfel T. De Blasi A. FASEB J. 2000; 14: 2569-2580Google Scholar. Recombinant adenovirus was prepared according to Ref. 14He T.-C. Zhou S. Da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Google Scholar with minor modifications. The ॆ-arrestin V53D cDNA was subcloned into the multiple cloning site of the shuttle plasmid (pAd-CMV-TRK) by standard cloning procedures. The purified shuttle plasmid was digested with the restriction enzyme PmeI to obtain the 舠rescue fragment.舡 The fragment was then purified on agarose gel, and 2 ॖg of purified rescue fragment was used for homologous recombination. The adenoviral plasmid pAdEasy-1 (14He T.-C. Zhou S. Da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Google Scholar) was then mixed with the rescue fragment, and the DNA mixture was transformed into the BJ5183 bacterial strain and incubated overnight. Colonies were screened by digesting the DNA withBglII and performing a Southern blot to confirm the presence of the cDNA insert. The DNA with the proper orientation was transformed into the DH5a bacterial strain. The recombinant construct, purified using Qiagen Maxi preparation kit, was digested overnight withPacI and transfected into HEK293 cells using LipofectAMINE (Invitrogen). Adenoviral plaques were purified twice by infecting HEK293 cells in agar. Virus was purified by CsCl gradient centrifugation, dialyzed, and titrated by plaque assay. The recombinant adenovirus obtained expresses both green fluorescent protein (GFP) and ॆ-arrestin V53D under independent cytomegalovirus promoters. A virus expressing only GFP was used as a control. For infection cerebellar Purkinje cells were incubated with the recombinant adenovirus at a multiplicity of infection of 50 plaque-forming units/cell for 3 h at 37 °C in medium without serum. The virus-containing medium was then removed, and cells were incubated in standard medium plus serum. At 48 h after infection, >957 of the cultured cerebellar Purkinje cells were infected, as assessed by the expression of GFP. The expression of ॆ-arrestin V53D was confirmed by immunocytochemistry in cerebellar Purkinje cells or by immunoblot in a U87MG glioblastoma cell line. These experiments were performed as described previously (12Sallese M. Mariggiò S. D'Urbano E. Iacovelli L. De Blasi A. Mol. Pharmacol. 2000; 57: 826-831Google Scholar). Cytosolic proteins (150 ॖg) from HEK293 cells transfected with the Gαq subunit were mixed with 40 ॖl of slurry containing GST-GRK-Nter fusion proteins bound to glutathione agarose beads in a final volume of 400 ॖl of binding buffer (20 mm Tris-HCl, pH 7.5, 1 mm dithiothreitol, 100 mm NaCl, 0.17 Lubrol, 10 ॖm GDP, 3 mm MgCl2), in the presence or absence of 47 mm MgCl2, 30 ॖmAlCl3, and 20 mm NaF. After 1 h at 4 °C the beads were washed three times with 1 ml of ice-cold binding buffer and the resins containing the eventual bound proteins were analyzed by immunoblotting, using anti-Gαq antibody (Santa Cruz Biotechnology). One fraction of starting material (30–40 ॖg, ∼257 of the total cytosolic proteins used for binding) was also included in the gel (indicated as S in Fig. 1). Immunoprecipitation was done as follows. After treatments, cells were rapidly washed in ice-cold PBS and solubilized in Triton X-100 lysis buffer (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 17 Triton-X 100, 1 mm EDTA, 107 glycerol, 1 mm phenylmethylsulfonyl fluoride, 10 ॖg/ml aprotinin, 1 mm sodium orthovanadate, 50 mm sodium fluoride, and 10 mm ॆ-glycerophosphate) for 15 min. The lysates were clarified by centrifugation (10,000 × g for 10 min). Protein concentration of supernatants was determined, and 600 ॖg of total proteins were incubated with 5 ॖg of anti-Gαq antibodies for 2h at 4 °C followed by addition of 50 ॖl of di-protein A-Sepharose pre-equilibrated in HNTG buffer (20 mm Hepes, 150 mm NaCl, 0,17 Triton X-100, 57 glycerol), and an additional 1-h incubation at 4 °C. Immunoprecipitates were washed four times in HNTG buffer, and the pellets were boiled in Laemmli buffer for 5 min before electrophoresis. Immunoprecipitates and starting materials were subjected to 107 SDS-polyacrylamide gel under reducing condition. After electrophoresis, proteins were transferred to polyvinylidene difluoride membrane and immunoblotted using anti-GRK2 (Upstate Biotechnology) and anti-GRK4 (Santa Cruz) antibodies. After treatments, cells were rapidly washed in ice-cold PBS and solubilized in Triton X-100 lysis buffer (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 17 Triton X-100, 1 mm EDTA, 107 glycerol, 1 mmphenylmethylsulfonyl fluoride, 10 ॖg/ml aprotinin, 1 mmsodium orthovanadate, 50 mm sodium fluoride, and 10 mm ॆ-glycerophosphate) for 15 min. The lysates were clarified by centrifugation (10,000 × g for 10 min), and 80–100 ॖg of proteins were separated by SDS-PAGE electrophoresis, blotted onto nitrocellulose, and probed using a commercial anti-phosphospecific antibody against phosphorylated ERK1/2. Monoclonal anti-phospho-ERK1/2 antibody was used at 1:2000 dilution. The membranes were stripped according to instructions from the manufacturer and reprobed with polyclonal anti-ERK1/2 antibody at 1:5000 dilution (Santa Cruz Biotechnology). Other Western blot analyses were performed as described (12Sallese M. Mariggiò S. D'Urbano E. Iacovelli L. De Blasi A. Mol. Pharmacol. 2000; 57: 826-831Google Scholar). The immunoreactive bands were visualized either by enhanced chemiluminescence using horseradish peroxidase-linked secondary antibody. HEK293 cells transfected as above and 20-day-old Purkinje cell primary cultures were fixed with 47 paraformaldehyde in PBS for 15 min at room temperature. The autofluorescence was quenched by incubation for 30 min in 50 mm NH4Cl, 50 mm glycine in PBS, and nonspecific interactions were blocked by treatment with blocking solution (0.057 saponin, 0.57 BSA in PBS) for 30 min at room temperature. Cells were incubated (8Sallese M. Salvatore L. D'Urbano E. Sala G Storto M. Launey T. Nicoletti F. Knopfel T. De Blasi A. FASEB J. 2000; 14: 2569-2580Google Scholar) with anti-mGlu1a (0.3 ॖg/ml, overnight at 4 °C), anti-ॆ-arrestin (F4C1) (12.5 ॖg/ml, 1 h, room temperature), anti-phospho-ERK1/2 (4 ॖg/ml, 1 h, room temperature), anti-ERK1 (2 ॖg/ml, 1 h, room temperature), and anti-RGS4 (2 ॖg/ml, overnight at 4 °C) antibodies in blocking solution. The chamber slides were then incubated with blocking solution containing Alexa-488 anti-rabbit (1:400, Molecular Probes), Cy3-conjugated anti-rabbit (1:200, Sigma), or Alexa-488 anti-mouse IgG (1:400, Molecular Probes) for 1 h at room temperature. For PAFr localization, an anti-FLAG M5 monoclonal antibody directed at MDYKDDDDKEF amino acid sequence at the N-terminal Met-FLAG fusion protein of PAFr was used; this antibody was conjugated to the fluorochrome Alexa-594 and diluted at 1 ॖg/ml in blocking buffer (1 h, room temperature). Each incubation step was carried out in the dark and followed by careful washes with PBS (six times/3 min each). After immunostaining the coverslips were mounted on slides with Mowiol 4–88 and analyzed by a Zeiss LSM 510 laser scanning microscope equipped with an Axiovert 100 m-BP and by the Confocal Imaging System Ultraview (PerkinElmer Life Sciences). The internalization of the mGlu1 receptor and of PAF receptors was quantified as previously described (8Sallese M. Salvatore L. D'Urbano E. Sala G Storto M. Launey T. Nicoletti F. Knopfel T. De Blasi A. FASEB J. 2000; 14: 2569-2580Google Scholar). Co-localization was quantified as previously reported (8Sallese M. Salvatore L. D'Urbano E. Sala G Storto M. Launey T. Nicoletti F. Knopfel T. De Blasi A. FASEB J. 2000; 14: 2569-2580Google Scholar) or using the 舠co-localization and correlation舡 option of the Ultraview software. GRK2 and GRK4 are the two GRK subtypes expressed in cerebellar Purkinje cells (8Sallese M. Salvatore L. D'Urbano E. Sala G Storto M. Launey T. Nicoletti F. Knopfel T. De Blasi A. FASEB J. 2000; 14: 2569-2580Google Scholar). As these cells represent one relevant site of the mGlu1 receptor expression and function, we investigated the role of GRK2 and GRK4 in the regulation of this receptor. Previous studies have shown that the agonist-dependent phosphorylation of the mGlu1areceptor expressed in HEK293 cells is significantly enhanced when GRK2 (9Dale B.D. Bhattacharya M. Anborgh P.H. Murdoch B. Bhatia M. Nakanishi S. Ferguson S.S.G. J. Biol. Chem. 2000; 275: 38213-38220Google Scholar) or GRK4 (8Sallese M. Salvatore L. D'Urbano E. Sala G Storto M. Launey T. Nicoletti F. Knopfel T. De Blasi A. FASEB J. 2000; 14: 2569-2580Google Scholar) are co-transfected. To assess the role of GRK-dependent receptor phosphorylation in the homologous desensitization of the mGlu1 receptor-stimulated IP production, we used GRK2 and GRK4 kinase-dead mutants in which the kinase activity was disrupted by site-directed mutagenesis of key amino acids located in the catalytic domain. Both mutants, which are named GRK2-(K220R) and GRK4-(K216M,K217M) for GRK2 and GRK4, respectively, lost their ability to phosphorylate receptor substrates (8Sallese M. Salvatore L. D'Urbano E. Sala G Storto M. Launey T. Nicoletti F. Knopfel T. De Blasi A. FASEB J. 2000; 14: 2569-2580Google Scholar, 12Sallese M. Mariggiò S. D'Urbano E. Iacovelli L. De Blasi A. Mol. Pharmacol. 2000; 57: 826-831Google Scholar). We determined the agonist-stimulated IP production in HEK293 cells transfected with the mGlu1 receptor and the effect of co-expression of different GRK mutants (Fig.1A). According to our previous results (8Sallese M. Salvatore L. D'Urbano E. Sala G Storto M. Launey T. Nicoletti F. Knopfel T. De Blasi A. FASEB J. 2000; 14: 2569-2580Google Scholar), the co-transfection of either GRK2 or GRK4 resulted in a 35–407 reduction of the agonist-stimulated response. By contrast, the effect of the two kinase-dead mutants was substantially different; the GRK4-(K216M,K217M) mutant was ineffective, whereas the GRK2-(K220R) desensitized the mGlu1 receptor-stimulated signaling to the same extent as the GRK2 wild type. These results indicated that the phosphorylation of the mGlu1 receptor was necessary for GRK4-mediated receptor desensitization, whereas GRK2 utilized, at least in part, a phosphorylation-independent mechanism for receptor regulation. The likely mechanism by which GRK2 could regulate mGlu1receptor signaling in a phosphorylation-independent manner is provided by the RGS-like domain present in the N terminus of GRK2, because we and others have previously shown that this is a functionally active domain able to regulate the GPCR-stimulated Gq signaling by direct binding and inhibition of the activated Gαq (12Sallese M. Mariggiò S. D'Urbano E. Iacovelli L. De Blasi A. Mol. Pharmacol. 2000; 57: 826-831Google Scholar,15Carman C.V. Parent J.L. Day P.W. Pronin A.N. Stenweis P.M. Wedegaertner P.B. Gilman A.G. Benovic J.L. Kozasa T. J. Biol. Chem. 1999; 274: 34483-34492Google Scholar). According to this hypothesis, our results suggest that, unlike GRK2, the GRK4 N terminus, which also contains an RGS homology domain, should be unable to interact with Gαq and to regulate its signaling cascade at the G protein level. To test this possibility we prepared a GST-GRK4-Nter fusion protein and we measured the binding of this domain to Gαq, using the GST-GRK2-Nter as a positive control. For binding experiments, the cytosolic proteins from HEK293 cells transfected with Gαq were incubated with agarose-conjugated GST-GRK-Nter fusion proteins. Unbound proteins were removed by extensive washing, and Gαq bound to GST-GRK-Nter proteins was revealed by immunoblot. According to previous findings (12Sallese M. Mariggiò S. D'Urbano E. Iacovelli L. De Blasi A. Mol. Pharmacol. 2000; 57: 826-831Google Scholar), when the incubation was done in the presence of AlF4− (i.e. Gαq was in the active state), a substantial fraction of Gαq was bound to GST-GRK2-Nter, whereas in the absence of the AlF4− (with Gαq in the inactive state), the Gαqbound to GST-GRK2-Nter was undetectable (Fig. 1B). When similar experiments were done using GST-GRK4-Nter, the amount of Gαq interacting with this domain was significantly lower even when Gαq was in the active state (Fig.1B). The amount of the Gαq bound to GRK4-Nter (in the presence of AlF4− ) was estimated to be ∼3–57 of the starting material, whereas that bound to GRK2-N-ter was estimated at ∼257 of the starting material. To assess whether GRK2, but not GRK4, interacts with the activated Gαq in cells, we investigated the interaction of Gαq and GRKs by co-immunoprecipitation in transfected HEK293 cells. We used both the wild type Gαq or the constitutively active mutant Gαq(Q209L), which can bind in vitro to the GRK2-Nter even in the absence of AlF4− (12Sallese M. Mariggiò S. D'Urbano E. Iacovelli L. De Blasi A. Mol. Pharmacol. 2000; 57: 826-831Google Scholar). Gαq was immunoprecipitated from cells transfected with GRK2 or GRK4 plus Gαq or Gαq(Q209L), and the presence of GRK subtypes in the immunoprecipitates was assessed by immunoblot (Fig. 1C). GRK2 was co-immunoprecipitated in an agonist-dependent manner from cells expressing Gαq, showing that GRK2 and Gαq interact in intact cells and that this binding depends on the active state of Gαq. In cells expressing Gαq(Q209L), GRK2 was co-immunoprecipitated even in the absence of agonist, although we consistently found that the amount of GRK2 co-immunoprecipitated with Gαq(Q209L) was enhanced by quisqualate treatment. This indicates that the activation of the mGlu1 receptor by agonist may favor the interaction between GRK2 and Gαq in intact cells, perhaps by modulating the active state of GRK2, which, in turn, could govern the interaction with Gαq. This hypothesis is consistent with our previous finding showing that the presence of an agonist-stimulated receptor increased the ability of GRK2-Nter to inhibit Gαq-stimulated IP production (Fig. 4 of Ref. 12Sallese M. Mariggiò S. D'Urbano E. Iacovelli L. De Blasi A. Mol. Pharmacol. 2000; 57: 826-831Google Scholar). By contrast, GRK4 was never co-immunoprecipitated with Gαq, indicating that this kinase does not interact with Gαq. The levels of expression of GRKs (Fig.1C), Gαq, and Gαq(Q209L) (data not shown) were comparable in different samples. In our experimental conditions ॆ-arrestin was not co-immunoprecipitated with GRK2 or with GRK4 in either the presence or absence of agonist stimulation (not shown). GRK4 is also primarily involved in mGlu1 receptor internalization (Fig. 2). In HEK293 cells transiently expressing the mGlu1 receptor, exposure to quisqualate for 5 min did not induce a significant level of receptor internalization and the co-expression of GRK2 resulted in a 2–3-fold increase of receptor internalization. In cells transfected with GRK2, the maximal internalization was observed after 20–30 min of agonist treatment (Fig. 2). The expression of GRK4 drastically enhanced the internalization of the mGlu1 receptor induced by the quisqualate, and this effect was rapid with a maximal peak at 5 min of agonist stimulation and was reversible within 30 min (Fig. 2). Previous work from our laboratory documented a substantial co-localization of the mGlu1 receptor and GRK4 both under basal conditions and after internalization. We in
WNT factors represent key mediators of many processes in animal development and homeostasis and act through a receptor complex comprised of members of the Frizzled and low density lipoprotein-related receptors (LRP). In mammals, 19 genes encoding Wingless and Int-related factor (WNTs), 10 encoding Frizzled, and 2 encoding LRP proteins have been identified, but little is known of the identities of individual Frizzled-LRP combinations mediating the effects of specific WNT factors. Additionally, several secreted modulators of WNT signaling have been identified, including at least three members of the Dickkopf family. WNT7A is a WNT family member expressed in the vertebrate central nervous system capable of modulating aspects of neuronal plasticity. Gene knock-out models in the mouse have revealed that WNT7A plays a role in cerebellar maturation, although its function in the development of distal limb structures and of the reproductive tract have been more intensely studied. To identify a receptor complex for this WNT family member, we have analyzed the response of the rat pheochromocytoma cell line PC12 to WNT7A. We find that PC12 cells are capable of responding to WNT7A as measured by increased β-catenin stability and activation of a T-cell factor-based luciferase reporter construct and that these cells express three members of the Frizzled family (Frizzled-2, -5, and -7) and LRP6. Our functional analysis indicates that WNT7A can specifically act via a Frizzled-5·LRP6 receptor complex in PC12 cells and that this activity can be antagonized by Dickkopf-1 and Dickkopf-3. WNT factors represent key mediators of many processes in animal development and homeostasis and act through a receptor complex comprised of members of the Frizzled and low density lipoprotein-related receptors (LRP). In mammals, 19 genes encoding Wingless and Int-related factor (WNTs), 10 encoding Frizzled, and 2 encoding LRP proteins have been identified, but little is known of the identities of individual Frizzled-LRP combinations mediating the effects of specific WNT factors. Additionally, several secreted modulators of WNT signaling have been identified, including at least three members of the Dickkopf family. WNT7A is a WNT family member expressed in the vertebrate central nervous system capable of modulating aspects of neuronal plasticity. Gene knock-out models in the mouse have revealed that WNT7A plays a role in cerebellar maturation, although its function in the development of distal limb structures and of the reproductive tract have been more intensely studied. To identify a receptor complex for this WNT family member, we have analyzed the response of the rat pheochromocytoma cell line PC12 to WNT7A. We find that PC12 cells are capable of responding to WNT7A as measured by increased β-catenin stability and activation of a T-cell factor-based luciferase reporter construct and that these cells express three members of the Frizzled family (Frizzled-2, -5, and -7) and LRP6. Our functional analysis indicates that WNT7A can specifically act via a Frizzled-5·LRP6 receptor complex in PC12 cells and that this activity can be antagonized by Dickkopf-1 and Dickkopf-3. Members of the WNT gene family encode structurally related secreted glycoprotein factors, modulating a vast array of processes during vertebrate and invertebrate embryonic development as well as several aspects of tissue homeostasis in the adult (1Dale T.C. Biochem. J. 1998; 329: 209-223Crossref PubMed Scopus (439) Google Scholar, 2Miller, J. R. (2002) Genome Biol., 3, Reviews 3001Google Scholar, 3Seidensticker M.J. Behrens J. Biochim. Biophys. Acta. 2000; 1495: 168-182Crossref PubMed Scopus (239) Google Scholar, 4Wodarz A. Nusse R. Annu. Rev. Cell Dev. Biol. 1998; 14: 59-88Crossref PubMed Scopus (1743) Google Scholar). In embryos, signaling by WNT factors controls the organization of the body plan during the early stages of development as well as organogenesis at later developmental stages. Postnatally, WNT signaling is involved in normal biological events such as tissue maturation and homeostasis and in several neoplastic pathologies (2Miller, J. R. (2002) Genome Biol., 3, Reviews 3001Google Scholar, 5Behrens J. Ann. N. Y. Acad. Sci. 2000; 910: 21-33Crossref PubMed Scopus (215) Google Scholar, 6Roose J. Clevers H. Biochim. Biophys. Acta. 1999; 1424: M23-M37PubMed Google Scholar, 7van Noort M. Clevers H. Dev. Biol. 2002; 244: 1-8Crossref PubMed Scopus (174) Google Scholar, 8Smalley M.J. Dale T.C. Cancer Metastasis Rev. 1999; 18: 215-230Crossref PubMed Scopus (187) Google Scholar). For example, in the mammalian central nervous system (CNS) 1The abbreviations used are: CNS, central nervous system; LRP, low density lipoprotein; ERK, extracellular signal-regulated kinase; FZD, Frizzled; WNT, Wingless and Int-related; TCF, T-cell factor; FRP, Frizzled-related protein; DKK, Dickkopf. WNT signal transduction is involved in neural induction and patterning in early embryogenesis (2Miller, J. R. (2002) Genome Biol., 3, Reviews 3001Google Scholar, 4Wodarz A. Nusse R. Annu. Rev. Cell Dev. Biol. 1998; 14: 59-88Crossref PubMed Scopus (1743) Google Scholar) as well as in organogenesis and neuronal homeostasis at later stages (9Patapoutian A. Reichardt L.F. Curr. Opin. Neurobiol. 2000; 10: 392-399Crossref PubMed Scopus (273) Google Scholar). In the adult, WNTs play a role in the control of neuronal plasticity and are implicated in CNS neoplasias such as medulloblastoma (10Dahmen R.P. Koch A. Denkhaus D. Tonn J.C. Sorensen N. Berthold F. Behrens J. Birchmeier W. Wiestler O.D. Pietsch T. Cancer Res. 2001; 61: 7039-7043PubMed Google Scholar, 11Gan D.D. Reiss K. Carrill T. Del Valle L. Croul S. Giordano A. Fishman P. Khalili K. Oncogene. 2001; 20: 4864-4870Crossref PubMed Scopus (61) Google Scholar, 12Hall A.C. Lucas F.R. Salinas P.C. Cell. 2000; 100: 525-535Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar, 13Koch A. Waha A. Tonn J.C. Sorensen N. Berthold F. Wolter M. Reifenberger J. Hartmann W. Friedl W. Reifenberger G. Wiestler O.D. Pietsch T. Int. J. Cancer. 2001; 93: 445-449Crossref PubMed Scopus (150) Google Scholar, 14Morin P.J. BioEssays. 1999; 21: 1021-1030Crossref PubMed Scopus (817) Google Scholar). The analysis of the signaling events mediated by WNTs has uncovered at least three signal transduction pathways, each involved in the mediation of specific biological responses (2Miller, J. R. (2002) Genome Biol., 3, Reviews 3001Google Scholar, 15Miller J.R. Hocking A.M. Brown J.D. Moon R.T. Oncogene. 1999; 18: 7860-7872Crossref PubMed Scopus (607) Google Scholar). The most studied and best understood signaling cascade elicited by WNTs involves an interaction with a receptor complex comprising members of the Frizzled (FZD) class of 7-transmembrane receptors and a member of the low density lipoprotein receptor (LRP) family of single-pass membrane proteins (16Tamai K. Semenov M. Kato Y. Spokony R. Liu C. Katsuyama Y. Hess F. Saint-Jeannet J.P. He X. Nature. 2000; 407: 530-535Crossref PubMed Scopus (1103) Google Scholar). WNT interaction with its receptor results in an increase in the stability of β-catenin, whose accumulation results in translocation to the nucleus where it can interact with members of the TCF class of transcription factors and therefore modulate gene expression. The stability of β-catenin is controlled by WNT through the modulation of a large cytoplasmic protein complex comprised of the proteins AXIN, APC, GBP/FRAT, and GSK3β, the latter controlling directly the level of β-catenin phosphorylation and its consequent degradation by the proteasome pathway (2Miller, J. R. (2002) Genome Biol., 3, Reviews 3001Google Scholar, 17Woodgett, J. R. (2001) Science's STKE http://stke.org/cgi/content/full/OC_sigtrans;2001/100/rel2.Google Scholar). WNT action can also be modulated at the extracellular level by several classes of secreted factors, including members of the Dickkopf, Cerberus, and FRP protein families (2Miller, J. R. (2002) Genome Biol., 3, Reviews 3001Google Scholar, 18Nusse R. Nature. 2001; 411: 255-256Crossref PubMed Scopus (61) Google Scholar). The vast array of processes controlled by WNTs is reflected in the numerous mammalian WNT and Frizzled classes, numbering 19 and 10 members, respectively, in man (2Miller, J. R. (2002) Genome Biol., 3, Reviews 3001Google Scholar). During development, many of these genes are expressed in a temporally and spatially regulated fashion, often in overlapping domains, whereas others are more widely expressed (19Dickinson M.E. McMahon A.P. Curr. Opin. Genet. Dev. 1992; 2: 562-566Crossref PubMed Scopus (54) Google Scholar, 20Gavin B.J. McMahon J.A. McMahon A.P. Genes Dev. 1990; 4: 2319-2332Crossref PubMed Scopus (406) Google Scholar, 21Sala C.F. Formenti E. Terstappen G.C. Caricasole A. Biochem. Biophys. Res. Commun. 2000; 273: 27-34Crossref PubMed Scopus (32) Google Scholar). These observations suggest a degree of specificity of action of the individual ligands and of relative affinities in the ligand-receptor interactions. Given the wider expression patterns of LRP genes and the tissue-specificity of expression displayed by many of the more numerous FZD genes, the specificity of the cellular response to individual WNT ligands is likely to depend largely on the individual FZD proteins expressed on the cell membrane. Despite the fact that a number of studies have investigated the association between individual Wnts and Frizzled proteins (22He X. Saint-Jeannet J.P. Wang Y. Nathans J. Dawid I. Varmus H. Science. 1997; 275: 1652-1654Crossref PubMed Scopus (419) Google Scholar, 23Holmen S.L. Salic A. Zylstra C.R. Kirschner M.W. Williams B.O. J. Biol. Chem. 2002; 277: 34727-34735Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 24Hsieh J.C. Rattner A. Smallwood P.M. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3546-3551Crossref PubMed Scopus (292) Google Scholar, 25Karasawa T. Yokokura H. Kitajewski J. Lombroso P.J. J. Biol. Chem. 2002; 277: 37479-37486Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 26Slusarski D.C. Corces V.G. Moon R.T. Nature. 1997; 390: 410-413Crossref PubMed Scopus (551) Google Scholar, 27Wu W. Glinka A. Delius H. Niehrs C. Curr. Biol. 2000; 10: 1611-1614Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 28Yang-Snyder J. Miller J.R. Brown J.D. Lai C.J. Moon R.T. Curr. Biol. 1996; 6: 1302-1306Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar) and of the wealth of information on the biological responses elicited by WNTs in different models systems, relatively little information is available on the identity of the FZD and LRP components of the receptor complex transducing the signal of individual WNT factors. Much interest is presently focusing on WNT signaling in the CNS because many of the effects mediated by WNTs in neuronal tissue can be phenocopied by LiCl, a GSK3 inhibitor widely employed in the treatment of mood disorders that displays well characterized neuroprotective and neuromodulatory activities (29Manji H.K. Moore G.J. Chen G. Biol. Psychiatry. 1999; 46: 929-940Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 30Manji H.K. Moore G.J. Chen G. J. Clin. Psychiatry. 2000; 61: 82-96PubMed Google Scholar). In particular, several studies have characterized the modulation of neuronal plasticity by WNT7A and LiCl in primary neuronal cultures, identifying WNT7A as a modulator of axonal remodeling and synaptic differentiation. Interestingly, recent evidence suggests that modulation of GSK3 activity in growth cones contributes to the signaling mechanisms by which semaphorins modulate neuronal plasticity, thus further implicating components of the WNT pathway in this process (31Eickholt B.J. Walsh F.S. Doherty P. J. Cell Biol. 2002; 157: 211-217Crossref PubMed Scopus (216) Google Scholar). No information is available regarding the receptor complex involved in the mediation of WNT7A effects in neuronal cells in mammals. We have investigated WNT7A responses in a cell line widely employed as a neuronal model, and well known to respond to WNT factors, to characterize a receptor complex capable of transducing WNT7A signals and to analyze the modulation of WNT7A signaling by members of the Dickkopf family. Our data indicate that a FZD5·LRP6 receptor complex can transduce a WNT7A signal in PC12 cells and that DKK1 and DKK3 can antagonize this signal. Cell Culture and RNA Isolation—PC12 cells were cultured in Dulbecco's modified Eagle's medium supplemented with glutamine 2 mm and 10% fetal bovine serum and maintained at 37 °C in a 10% CO2 humidified atmosphere. Total RNA was extracted from subconfluent monolayers as described (32Auffray C. Rougeon F. Eur. J. Biochem. 1980; 107: 303-314Crossref PubMed Scopus (2085) Google Scholar). Western Blotting—Western blot analysis was performed as previously described (33Iacovelli L. Bruno V. Salvatore L. Melchiorri D. Gradini R. Caricasole A. Barletta E. De Blasi A. Nicoletti F. J. Neurochem. 2002; 82: 216-223Crossref PubMed Scopus (102) Google Scholar). Briefly, 24 h post-transfection PC12 cells were lysed in Triton X lysis buffer (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100, 1 mm EDTA, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm sodium orthovanadate, 50 mm sodium fluoride, and 10 mm β-glycerophosphate) for 15 min. The cell lysates (PC12 cells cultured under standard conditions or in the presence of 10 mm LiCl for 24 h) were clarified by centrifugation (10,000 × g for 10 min). Protein cell lysates (80 μg) were separated by SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes, and probed using polyclonal anti-β-catenin antibody (sc-1496; Santa Cruz Biotechnology) at 1 μg/ml dilution. Membranes were also probed with polyclonal anti-ERK1 antibody (sc-93; Santa Cruz Biotechnology) at 0.5 μg/ml to control equal protein loading. The immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Biosciences), using horseradish peroxidase-linked secondary antibodies. For the analysis of FZD expression constructs, cells were plated at a density of 2 × 106 cells/10-mm Petri dish the day before transfection. Cells were transfected using 8 ml of LipofectAMINE 2000 (Invitrogen) and 10 μg of cDNA. 72 h post-transfection, cells were rapidly rinsed in ice-cold phosphate buffered-saline and lysed for 15 min in Triton X-100 lysis buffer (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100, 1 mm EDTA, 10% glycerol) added with protease and phosphatase inhibitors. The cell lysates were clarified by centrifugation (10,000 × g for 10 min). Protein cell lysates were separated by SDS-PAGE (8% gel for LRP5 and LRP6 and 10% gel for FZD2, FZD5, and FZD7) and blotted onto nitrocellulose. Membranes were probed using monoclonal anti-LRP5/6 (cat. 3801–106, 1:100 dilution; BioVision) or polyclonal anti-pan Fzd (cat. sc-9169, 1:100 dilution; Santa Cruz Biotechnology). The immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Biosciences) using horseradish peroxidase secondary antibodies. cDNA Synthesis and RT-PCR Studies—Synthesis of cDNA and RT-PCR analysis were carried out essentially as described (21Sala C.F. Formenti E. Terstappen G.C. Caricasole A. Biochem. Biophys. Res. Commun. 2000; 273: 27-34Crossref PubMed Scopus (32) Google Scholar, 34Caricasole A. Sala C. Roncarati R. Formenti E. Terstappen G.C. Biochim. Biophys. Acta. 2000; 1517: 63-72Crossref PubMed Scopus (27) Google Scholar, 35Caricasole A. Ferraro T. Rimland J.M. Terstappen G.C. Gene. 2002; 288: 147-157Crossref PubMed Scopus (48) Google Scholar). Briefly, human polyadenylated RNA samples from CNS and peripheral tissues were purchased from Clontech (Palo Alto, CA). cDNA was synthesized from 1 μl of polyadenylated RNA using Superscript II reverse transcriptase (Invitrogen) and oligo(dT) and random hexamer oligonucleotides (250 ng each) in a final volume of 20 μl, according to the manufacturer's instructions. Following first strand cDNA synthesis, the reaction volume was increased to 100 μl, and 1 μl was used for each PCR reaction. Assuming a 50% efficiency in the reverse transcription reaction, ∼5 ng of cDNA were employed in each PCR and TaqMan reaction. For the analysis of the expression of gene expression by RT-PCR, reaction conditions were 3 min at 94 °C, 30 s at 94 °C, then 30 s at 55 °C and 30 s at 72 °C for 35 cycles. For β-actin amplification, PCR conditions were the same except that primers were forward, 5′-TGAACCCTAAGGCCAACCGTG-3′, reverse, 5′-GCTCATAGCTCTTCTCCAGGG-3′. For the analysis of expression of FZD genes in PC12 cell cDNA, PCR conditions were the same as above except that the primers used were those reported previously (21Sala C.F. Formenti E. Terstappen G.C. Caricasole A. Biochem. Biophys. Res. Commun. 2000; 273: 27-34Crossref PubMed Scopus (32) Google Scholar). RT-PCR primers for LRP-5 and LRP-6 were as follows: LRP-5 (forward, 5′-GCAAGAAGCTGTACTGGACG-3′, and reverse, 5′-TGTTGCAGGCATGGATGGAG-3′), and LRP-6 (forward and reverse, 5′-GCATTTGGCTGCCTCTGTTG-3′). PCR Amplification Employing a Proofreading Thermostable DNA Polymerase—PCR amplification was carried out employing the Gene-Amp XL PCR kit, using human adult brain cDNA as a template, as described previously (21Sala C.F. Formenti E. Terstappen G.C. Caricasole A. Biochem. Biophys. Res. Commun. 2000; 273: 27-34Crossref PubMed Scopus (32) Google Scholar, 34Caricasole A. Sala C. Roncarati R. Formenti E. Terstappen G.C. Biochim. Biophys. Acta. 2000; 1517: 63-72Crossref PubMed Scopus (27) Google Scholar, 35Caricasole A. Ferraro T. Rimland J.M. Terstappen G.C. Gene. 2002; 288: 147-157Crossref PubMed Scopus (48) Google Scholar). Reaction conditions were according to the manufacturer's protocol, with a final Mg(OAc)2 concentration of 0.8 mm. Primers sequence were as follow: forward, 5′-GGGCGGGCTATGTTGATTGC-3′, reverse, 5′-ACAAGCTCAGCATCCTGCCA-3′ for WNT-7A; forward, 5′-GAGGAGAAGCGCAGTCAATCA-3′, reverse, 5′-GGTTCCGGTTGCAATTCTTGG-3′, for WNT5A; forward, 5′-GGCCCCGCAGCGCCCT-3′, reverse, 5′-CGTCCCTCACACGGTGGTCT-3′, for FZD2; forward, 5′-GGCGATGGCTCGGCCTGAC-3′, reverse, 5′-CCTCCTACACGTGCGACAG-3′, for FZD5; forward, 5′-GGCTGAGAGCACCGCTGCACT-3′, reverse, 5′-CTACCGTGCCTCTCCTCTTGC-3′, for FZD7. PCR products were analyzed by electrophoresis on a 1% agarose gel poured and run in 1× TAE buffer. Products were cloned using the TOPO TA cloning system (Invitrogen). Plasmid DNA was recovered and subjected to automated DNA sequencing by standard protocols using an ABI377 machine (PE Biosystems, Branchburg, NJ). Plasmids—Construction of plasmids was as follows. For the WNT7A expression construct, the WNT7A cDNA was amplified from human adult brain cDNA by using primers carrying EcoRV and XbaI restriction sites at the flanking ends. The amplified cDNA was sequenced and subcloned into the EcoRV and XbaI restriction enzyme sites of the eucaryotic expression pCIN4 vector (35Caricasole A. Ferraro T. Rimland J.M. Terstappen G.C. Gene. 2002; 288: 147-157Crossref PubMed Scopus (48) Google Scholar, 36Rees S. Coote J. Stables J. Goodson S. Harris S. Lee M.G. Biotechniques. 1996; 20: 102-110Crossref PubMed Scopus (263) Google Scholar). For the WNT5a expression plasmid, the WNT5A cDNA was amplified from human adult brain cDNA by using primers carrying SmaI and XbaI restriction sites at the flanking ends. The amplified cDNA was sequenced and subcloned into the EcoRV and XbaI restriction enzyme sites of pCIN4 vector. To prepare FZD2, FZD5, and FZD7 expression constructs, FZD2, FZD5, and FZD7 cDNAs were amplified from human adult brain. The amplified cDNAs were sequenced and subcloned into the EcoRI restriction enzyme site of pCIN4 vector in the sense (pCIN4/FZD2+, pCIN4/FZD5+, and pCIN4/FZD7+) and antisense direction (pCIN4/FZD2–, pCIN4/FZD5–, and pCIN4/FZD7–). The FZD cDNAs were also subcloned in the pCIN4/WNT7A plasmid to obtain plasmids expressing an FZD receptor and WNT7A ligand spaced by an IRES sequence (pCIN4/FZD2/WNT7A, pCIN4/FZD5/WNT7A, and pCIN4/FZD7/WNT7A). The construction details of expression plasmids for human LRP-5, LRP-6, and mouse DKK-1, DKK-2, and DKK-3 expression plasmids were kind gifts from Drs. C. Niehrs and M. Semenov, and were reported previously (16Tamai K. Semenov M. Kato Y. Spokony R. Liu C. Katsuyama Y. Hess F. Saint-Jeannet J.P. He X. Nature. 2000; 407: 530-535Crossref PubMed Scopus (1103) Google Scholar, 27Wu W. Glinka A. Delius H. Niehrs C. Curr. Biol. 2000; 10: 1611-1614Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 37Mao B. Wu W. Li Y. Hoppe D. Stannek P. Glinka A. Niehrs C. Nature. 2001; 411: 321-325Crossref PubMed Scopus (908) Google Scholar). Transient Transfection Assays for Reporter Studies—Transfection and reporter assays were carried out essentially as described (35Caricasole A. Ferraro T. Rimland J.M. Terstappen G.C. Gene. 2002; 288: 147-157Crossref PubMed Scopus (48) Google Scholar). Transient transfections of PC12 cells were carried out in triplicate employing LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. PC12 cells (∼6 × 105/well) were plated 1 day before transfection in collagen-coated 96-well culture plates. A total of 0.32 μg of DNA was transfected into each well, including luciferase reporter plasmid (100 ng), expression construct (100 ng of each expression construct, for up to 2 different plasmids), Renilla luciferase cytomegalovirus (CMV)-driven internal reporter (20 ng; Promega), and carrier plasmid DNA (pGEM4Z, to 320 ng; Promega) as appropriate. For transfection sets involving more than two expression plasmids (i.e. those depicted in Fig. 6, B and C), a total of 0.64 μg of DNA was transfected, including luciferase reporter plasmid (100 ng), expression construct (100 ng of each expression construct), Renilla luciferase CMV-driven internal reporter (20 ng; Promega), and carrier plasmid DNA (pGEM4Z, to 640 ng; Promega) as appropriate. The luciferase reporter plasmid is the p4TCF, comprising four copies of a TCF-responsive element upstream of a TATA element luciferase coding sequence transcriptional unit (38Bettini E. Magnani E. Terstappen G.C. Neurosci. Lett. 2002; 317: 50-52Crossref PubMed Scopus (14) Google Scholar). Luciferase activity was measured using the Promega dual luciferase assay reagent and read using a Berthold LUMAT LB3907 tube luminometer. Readings were from triplicate transfections and were automatically normalized relative to the internal standard (Renilla luciferase). For experiments involving LiCl, cells transfected with the reporter were in the presence of 10 mm LiCl for 24 h after transfection. WNT7A Signals via the Canonical Pathway in PC12 Cells— PC12 cells are widely employed as a neuronal cell model, are known to activate the canonical WNT pathway in response to WNT1 (39Bournat J.C. Brown A.M. Soler A.P. J. Neurosci. Res. 2000; 61: 21-32Crossref PubMed Scopus (95) Google Scholar, 40Bradley R.S. Cowin P. Brown A.M. J. Cell Biol. 1993; 123: 1857-1865Crossref PubMed Scopus (232) Google Scholar, 41Giarre M. Semenov M.V. Brown A.M. Ann. N. Y. Acad. Sci. 1998; 857: 43-55Crossref PubMed Scopus (51) Google Scholar, 42Issack P.S. Ziff E.B. Cell Growth Differ. 1998; 9: 827-836PubMed Google Scholar, 43Porfiri E. Rubinfeld B. Albert I. Hovanes K. Waterman M. Polakis P. Oncogene. 1997; 15: 2833-2839Crossref PubMed Scopus (133) Google Scholar, 44Shackleford G.M. Willert K. Wang J. Varmus H.E. Neuron. 1993; 11: 865-875Abstract Full Text PDF PubMed Scopus (49) Google Scholar), and do not express WNT7A (45Erdreich-Epstein A. Shackleford G.M. Growth Factors. 1998; 15: 149-158Crossref PubMed Scopus (9) Google Scholar). We therefore selected this cell model to assay the capacity of WNT7A to activate the canonical WNT pathway. This was performed by transiently transfecting a WNT7A expression construct (35Caricasole A. Ferraro T. Rimland J.M. Terstappen G.C. Gene. 2002; 288: 147-157Crossref PubMed Scopus (48) Google Scholar) in PC12 cells followed by an analysis of the intracellular accumulation of β-catenin and the activation of a co-transfected TCF-luciferase construct, using culture in the presence of 10 mm LiCl as a positive control. As illustrated in Fig. 1, expression of WNT7A in PC12 cells can induce the accumulation of β-catenin (Fig. 1A) to a degree comparable with that achieved by LiCl. Functional activation of the canonical WNT pathway by WNT7A in this cell model is demonstrated by the activation of a TCF-luciferase construct, an effect that is dependent on the amount of transfected WNT7A expression construct (Fig. 2A). The effect is dependent on the integrity of the encoded WNT7A protein, because an expression construct encoding a truncated, non-functional WNT7A cDNA (WNT7Apx) does not result in reporter gene activation (Fig. 2B). This latter mutation represents a human allele serendipitously isolated during the amplification of full-length WNT7A cDNA (35Caricasole A. Ferraro T. Rimland J.M. Terstappen G.C. Gene. 2002; 288: 147-157Crossref PubMed Scopus (48) Google Scholar) analogous to that associated with the postaxial hemimelia mutation in the mouse, which was predicted to encode a non-functional protein (46Parr B.A. Avery E.J. Cygan J.A. McMahon A.P. Dev. Biol. 1998; 202: 228-234Crossref PubMed Scopus (43) Google Scholar). The data presented here demonstrate that the px WNT7A allele does indeed encode a non-functional product.Fig. 2PC12 cells respond specifically in a TCF reporter assay to WNT7A in transient transfection assays. A, effects of culture in the presence of 10 mm LiCl or of transfection with expression plasmids encoding WNT7A, a truncated non-functional WNT7A allele (WNT7Apx) or WNT5A on TCF-mediated transcriptional activity, employing the TCF-luciferase reporter. B, the TCF reporter response to WNT7A is dependent on the dose of transfected WNT7A expression plasmid. Increasing amounts of WNT7A expression plasmid (70–440 ng) result in increasing TCF-mediated transcriptional activity, whereas transfection of the WNT7Apx expression plasmid does not result in comparable reporter activation. The total amount of DNA transfected was maintained equivalent in all samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Interestingly, although WNT7A can activate the canonical pathway in PC12 cells, it is a rather inefficient inducer of a Ca2+-dependent Nuclear Factor of Activated T-cells (NFAT)-reporter construct (data not shown), suggesting that it cannot stimulate the Ca2+-dependent WNT pathway in this system. FZD5 Can Function as a Component of the WNT7A Receptor Complex—WNT signaling through the canonical pathway involves the dismantling of the AXIN-APC·GSK3β macromolecular complex, stabilization of β-catenin, and stimulation of TCF-mediated transcription through the activation of a receptor complex comprised of members of the FZD and LRP protein families (2Miller, J. R. (2002) Genome Biol., 3, Reviews 3001Google Scholar). There are indications that distinct FZD proteins display different affinities for individual WNT ligands (23Holmen S.L. Salic A. Zylstra C.R. Kirschner M.W. Williams B.O. J. Biol. Chem. 2002; 277: 34727-34735Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 25Karasawa T. Yokokura H. Kitajewski J. Lombroso P.J. J. Biol. Chem. 2002; 277: 37479-37486Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Consequently, an appropriate combination of FZD·LRP proteins must be expressed in order for PC12 cells to respond to WNT7A. The expression of FZD genes in this system was therefore analyzed by RT-PCR. The results (Fig. 3A) indicate that FZD2, FZD5, and FZD7 are expressed in PC12 cells, in general agreement with a previous report where FZD expression in PC12 cells was analyzed by Northern blotting (47Chou A.H. Zheng S. Itsukaichi T. Howard B.D. Brain Res. Mol. Brain Res. 2000; 77: 232-245Crossref PubMed Scopus (19) Google Scholar). We reasoned that one or more of these FZD proteins would be responsible for mediating WNT7A signaling in PC12 cells. Therefore, the complete open reading frames for FZD2, FZD5, and FZD7 were obtained and cloned into expression constructs either individually or in tandem with the WNT7A cDNA as dicistronic constructs. These plasmids were first tested by Western blotting for appropriate expression of the encoded FZD proteins in PC12 cells (Fig. 3B) and then co-transfected in the TCF-luciferase reporter assay to evaluate a possible synergistic effect with WNT7A on TCF-mediated transcription, indicative of functional interaction. The results (Fig. 3C and Fig. 4) indicate that the most substantial synergy with WNT7A in the stimulation of TCF-mediated transcription is achieved by FZD5. Although a synergistic stimulation of canonical Wnt signaling by the FZD7·WNT7A combination was never observed, some activity was displayed by the FZD2·WNT7A combination in most experiments, but only when FZD2 and WNT7A were expressed from separate plasmids. On the other hand, the synergy between FZD5 and WNT7A was observed independently of whether the FZD5 and WNT7A cDNAs were encoded by the same (dicistronic) construct (Fig. 3C and also Fig. 5) or by separate expression plasmids (Fig. 4). Importantly, transfected FZD5 cDNA was incapable of stimulating the TCF reporter in the absence of co-transfected WNT7A expression plasmid. Overall, this suggests that FZD5 is the major component of the highest affinity WNT7A receptor for canonical Wnt signaling in PC12 cells.Fig. 4FZD5, but not FZD2 or FZD7, can synergize with WNT7A to potentiate activation of the TCF reporter. Expression plasmids comprising individual FZD cDNAs or their antisense sequences were co-tra
WNT factors represent key mediators of many processes in animal development and homeostasis and act through a receptor complex comprised of members of the Frizzled and low density lipoprotein-related receptors (LRP). In mammals, 19 genes encoding Wingless and Int-related factor (WNTs), 10 encoding Frizzled, and 2 encoding LRP proteins have been identified, but little is known of the identities of individual Frizzled-LRP combinations mediating the effects of specific WNT factors. Additionally, several secreted modulators of WNT signaling have been identified, including at least three members of the Dickkopf family. WNT7A is a WNT family member expressed in the vertebrate central nervous system capable of modulating aspects of neuronal plasticity. Gene knock-out models in the mouse have revealed that WNT7A plays a role in cerebellar maturation, although its function in the development of distal limb structures and of the reproductive tract have been more intensely studied. To identify a receptor complex for this WNT family member, we have analyzed the response of the rat pheochromocytoma cell line PC12 to WNT7A. We find that PC12 cells are capable of responding to WNT7A as measured by increased beta-catenin stability and activation of a T-cell factor-based luciferase reporter construct and that these cells express three members of the Frizzled family (Frizzled-2, -5, and -7) and LRP6. Our functional analysis indicates that WNT7A can specifically act via a Frizzled-5.LRP6 receptor complex in PC12 cells and that this activity can be antagonized by Dickkopf-1 and Dickkopf-3.
