Nerve Growth Factor Stimulation of p42/p44 Mitogen-Activated Protein Kinase in PC12 Cells: Role of Gi/o, G Protein-Coupled Receptor Kinase 2, b-Arrestin I, and Endocytic Processing

In this study, we have shown that nerve growth factor (NGF)dependent activation of the p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) pathway in PC12 cells can be partially blocked by pertussis toxin (which inactivates the G proteins Gi/o). This suggests that the Trk A receptor may use a G protein-coupled receptor pathway to signal to p42/p44 MAPK. This was supported by data showing that the NGF-dependent activation of p42/p44 MAPK is potentiated in cells transfected with G protein-coupled receptor kinase 2 (GRK2) or b-arrestin I. Moreover, GRK2 is constitutively bound with the Trk A receptor, whereas NGF stimulates the pertussis toxin-sensitive binding of b-arrestin I to the TrkA receptor-GRK2 complex. Both GRK2 and b-arrestin I are involved in clathrin-mediated endocytic signaling to p42/p44 MAPK. Indeed, inhibitors of clathrinmediated endocytosis (e.g., monodansylcadaverine, concanavalin A, and hyperosmolar sucrose) reduced the NGFdependent activation of p42/p44 MAPK. Finally, we have found that the G protein-coupled receptor-dependent component regulating p42/p44 MAPK is required for NGF-induced differentiation of PC12 cells. Thus, NGF-dependent inhibition of DNA synthesis was partially blocked by PD098059 (inhibitor of MAPK kinase-1 activation) and pertussis toxin. Our findings are the first to show that the Trk A receptor uses a classic G protein-coupled receptor-signaling pathway to promote differentiation of PC12 cells. Nerve growth factor (NGF) promotes the survival and differentiation of sensory and sympathetic neurons. NGF also induces growth arrest of PC12 cells, which then accumulate in the G1 phase of the cell cycle and subsequently undergo differentiation. NGF binds to a specific high-affinity tyrosine kinase receptor, Trk A. Binding of NGF to Trk A induces autophosphorylation of the receptor on specific tyrosine residues. The subsequently phosphorylated sites on the receptor act as acceptors for the recruitment and assembly of signaling complexes, such as Grb-2, phospholipase C g, and PI3K to elicit intracellular responses. For instance, the binding of the SH2 containing protein Shc, Grb-2, and mSos site to phospho-Tyr-490 on the Trk A receptor elicits Ras-dependent activation of the p42/p44 MAPK pathway. The novel neuronal substrate FRS2 also uses the same docking site on the Trk A receptor as the SH2-containing protein Shc and has been implicated in the stimulation of the Ras-dependent p42/p44 MAPK pathway by forming a complex with the tyrosine phosphatase SHP-2, and associated adaptor proteins Grb-2, mSos, and Crk. This is achieved via its association with C3G and leads to the sustained activation of the small G protein Rap1. Rap1 then complexes to and activates B-raf, resulting in subsequent downstream stimulation of the p42/p44 MAPK pathway (York et al., 1998). Because NGF activates Ras and c-Raf transiently and B-Raf in a sustained manner, the prevalent view is that transient activation of p42/p44 MAPK leads to cell proliferation, whereas a more prolonged activation of this kinase pathway by NGF promotes cell differentiation (Marshall, 1995; Tombes et al., 1998). Recent studies have shown that the insulin-like growth factor-1 (IGF-1) can use the G proteins, Gi/o to stimulate activation of p42/p44 MAPK in fibroblasts (Luttrell et al. 1995). This was established using pertussis toxin (which inactivates Gi/o) and the C-terminal domain of b-adrenergic This study was supported by grants from the Wellcome Trust and the Strathclyde University Research and Development fund. S.P. is a Wellcome Trust Senior Fellow. ABBREVIATIONS: NGF, nerve growth factor; MAPK, mitogen-activated protein kinase; IGF, insulin-like growth factor; GRK, G protein-coupled receptor kinase; Grb-2, growth factor receptor binding protein; PI3K, phosphoinositide 3-kinase; LPA, lysophosphatidic acid; PDGF, plateletderived growth factor; Gi, inhibitory G protein; Gab1, growth factor receptor binding protein associated binder; HRP, horseradish peroxidase; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; NP-40, Nonidet P-40; EGF, epidermal growth factor; GPCR, G proteincoupled receptor; mSos, son of sevenless; c-Src, cellular Src tyrosine kinase; MDC, monodansylcadaverine. 0026-895X/01/6001-63–70$3.00 MOLECULAR PHARMACOLOGY Vol. 60, No. 1 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 522/904844 Mol Pharmacol 60:63–70, 2001 Printed in U.S.A. 63 at A PE T Jornals on M ay 9, 2017 m oharm .aspeurnals.org D ow nladed from kinase (which sequesters Gbg subunits). Both reduced the IGF-1-dependent activation of p42/p44 MAPK. These agents also reduced fibroblast growth factor-dependent activation of p42/p44 MAPK in fibroblasts and promoted differentiation (Fedorov et al. 1998). Recent studies have also implicated the involvement of G protein-coupled receptor kinase 2 (GRK2) and b-arrestin I and II in regulating IGF-1and b-adrenergic receptor-stimulated p42/p44 MAPK activation via a process that involves clathrin-mediated endocytosis of receptor-signal complexes (Daaka et al. 1998; Ahn et al. 1999; Lin et al. 1999). GRK2 is activated in an agonistand G protein-dependent manner. b-arrestin I/II are clathrin adaptor proteins that promote dynamin II-mediated internalization of receptor signal complexes containing c-Raf-MAPK kinase-1 for subsequent activation of p42/p44 MAPK. Certain G protein-coupled receptor agonists have also been shown to stimulate the tyrosine phosphorylation/trans-activation of growth factor receptors. The subsequently phosphorylated sites on the receptor act as acceptors for the recruitment and assembly of signaling complexes such as Grb-2, phospholipase C g, and PI3K to elicit mitogenic responses. For instance, LPA has been shown to trans-activate the EGF receptor and p185 to stimulate p42/p44 MAPK activation in Cos-7 cells (Daub et al., 1996), whereas angiotensin II can induce platelet-derived growth factor (PDGF) receptor trans-activation in vascular smooth muscle (Linsemen et al. 1995). We have shown that the PDGF can also use Gi-dependent and -independent routes to promote stimulation of c-Src and p42/p44 MAPK in cultured airway smooth muscle cells (Conway et al. 1999; Rakhit et al. 2000). Furthermore, c-Src inhibitors abolished the PDGF-dependent activation of p42/ p44 MAPK in these cells. We have suggested that Gi might recruit c-Src near the PDGF receptor tyrosine kinase for activation. Furthermore, PDGF stimulates a Gi-mediated tyrosine phosphorylation of the Grb-2 associated binding protein, Gab1. This promotes the binding of tyrosine-phosphorylated PI3K1a to Gab1 and is required for dynamin II mediated endocytic stimulation of the p42/p44 MAPK pathway (Rakhit et al. 2000). Moreover, Gab1 binds to and activates PI3K (Kaplan and Millar, 1997; Korhonen et al., 1999) in response to NGF in neuronal cells. This raises the possibility that the Trk A receptor may also use classic GPCRdependent signaling to regulate activation of the p42/p44 MAPK pathway. However, to date there is no direct evidence to support this proposed model. Therefore, in this article, we have investigated whether the NGF-dependent activation of p42/p44 MAPK involves a classic GPCR signaling pathway. We have also evaluated whether this pathway has an important role in regulating the differentiation of PC12 cells. Experimental Procedures Materials. All biochemicals, including NGF, were from Sigma Chemical Co. (Dorset, UK). [H]Thymidine, [g-P]ATP (3000 Ci/ mol), MAPK Biotrak assay kits, and enhanced chemiluminescence reagents were from Amersham Pharmacia Biotech (Bucks, UK). Cell culture supplies were from Life Technologies (Paisley, UK). Antiphospho-p42/p44 MAPK antibodies were from New England Biolabs (Beverly, MA). Anti-p42/p44 MAPK and HRP-linked anti-phosphotyrosine antibodies were from Transduction Laboratories (Lexington, KY). Anti-Trk A phospho-Tyr-490 and Trk A antibodies were from New England Biolabs (Beverly, MA). Anti-FLAG antibody was from Stratagene (La Jolla, CA). Anti-b-arrestin I antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Reporter HRP-antimouse/rabbit antibodies were from the Scottish Antibody Production Unit (Carluke, Scotland). pRK5-GRK2 and pcDNA3-barr1 FLAG cDNA plasmid constructs and anti-GRK2 antibodies were kind gifts from Professor R. Lefkowitz (Duke University, Durham, NC). Cell Culture. PC12 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS). Cells were placed in DMEM supplemented with 0.1% (v/v) fetal calf serum (FCS) for 24 h before experimentation. In some cases, pertussis toxin (0.1 mg/ml) was added to the DMEM supplemented with 0.1% (v/v) FCS. Transfection. PC12 cells were transiently transfected with b-arrestin I or GRK2 plasmid constructs. Cells at 90% confluence were placed in DMEM containing 2% (v/v) FCS and transfected with 2 to 4 mg of plasmid construct after complex formation with LipofectAMINE 2000, according to the Manufacturer’s instructions. The cDNA containing media was then removed after 24 h at 37°C, and the cells incubated for a further 24 h in DMEM supplemented with 0.1% (v/v) FCS before addition of agonists. p42/p44 MAPK Assays. The phosphorylation and activation of p42/p44 MAPK was detected by Western blotting using an antiphospho-p42/p44 MAPK antibody. p42/p44 MAPK activity was also measured in PC12 cell lysates using a specific p42/p44 MAPK peptide substrate (EGFR peptide synthesized to contain one phosphorylation site) as we described previously (Conway et al. 1999). Blotting. Immunoblotting was performed as we described previously (Conway et al., 1999; Rakhit et al. 1999). Briefly, nitrocellulose membranes were blocked for 2 h at 4°C in 10 mM phosphate-buffered saline and 0.1% (v/v) Tween-20 containing 5% (w/v) nonfat dried milk and 0.001% (w/v) thimerosal. The nitrocellulose sheets were then incubated overnight at 4°C in blocking solution containing antibodies. The sheets were then washed with phosphate-buffered saline and 0.1% (v/v) Tween-20 before incubation with HRP-linked antirabbit mouse antibodies in blocking solution for 2 h at room temperature. In the case of HRP-linked anti-phosphotyrosine antibodies, no secondary antibody was used. After washing the blots as above, immunoreactive proteins were visualized using the enhanced chemiluminescence detection kit and were quantified using densitometry. Immunoprecipitation of Trk A. The medium was removed and cells lysed in ice-cold immunoprecipitation buffer (1 ml) containing 20 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 1% (v/v) Nonidet P-40 (NP-40), 10% (v/v) glycerol, 1 mg/ml bovine serum albumin, 0.5 mM sodium orthovanadate, 0.2 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, antipain, pepstatin, and aprotinin; pH 8) for 10 min at 4°C. The material was harvested, centrifuged at 22,000g for 5 min at 4°C and 200 ml of cell lysate supernatant (equalized for protein, 0.5 to 1 mg/ml) taken for immunoprecipitation with antibodies (5 mg of anti-Trk A-490 phosphotyrosine or Trk A antibodies and 30 ml of 1 part immunoprecipitation buffer and 1 part protein A Sepharose CL4B, pre-equilibrated with lysis buffer. After agitation for 2 h at 4°C, the immune complex was collected by centrifugation at 22,000g for 15 s at 4°C. Immunoprecipitates were washed twice with buffer A [containing 10 mM HEPES, pH 7, 100 mM NaCl, 0.2 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 20 mg/ml aprotinin, and 0.5% (v/v) NP-40] and once in buffer A without NP-40. Samples were taken for Western blotting with HRP-linked anti-phosphotyrosine or anti-GRK2 or b-arrestin I antibodies DNA Synthesis. [H]Thymidine incorporation studies were performed as described by Rakhit et al. (1999). cAMP Assays. Intracellular cAMP was measured using a cAMP enzyme-linked immunosorbent assay kit as described by the manufacturer (Amersham Pharmacia Biotech). 64 Rakhit et al. at A PE T Jornals on M ay 9, 2017 m oharm .aspeurnals.org D ow nladed from