Nerve growth factor induces neurite outgrowth of PC12 cells by promoting Gβγ-microtubule interaction

BMC Neuroscience

Sukla Roychowdhury²  Nazarius S Lamango³  Manuel Miranda²  Arshad M Khan²  Armando Varela-Ramirez²  Ellen M Walker²  Jessica Martinez-Jurado²  Omar Najera²  Jorge A Sierra-Fonseca¹ 

[1]Present Address: Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston 02115, MA, USA

[2]Department of Biological Sciences, University of Texas, El Paso 79968, TX, USA

[3]College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee 32307, FL, USA

关键词: Tubulin;    Heterotrimeric G proteins;    Gβγ;    Microtubules;    Neurite outgrowth;   

Others  :  1090208
DOI  :  10.1186/s12868-014-0132-4

 received in 2014-11-10, accepted in 2014-11-27,  发布年份 2014

【 摘 要 】

Background

Assembly and disassembly of microtubules (MTs) is critical for neurite outgrowth and differentiation. Evidence suggests that nerve growth factor (NGF) induces neurite outgrowth from PC12 cells by activating the receptor tyrosine kinase, TrkA. G protein-coupled receptors (GPCRs) as well as heterotrimeric G proteins are also involved in regulating neurite outgrowth. However, the possible connection between these pathways and how they might ultimately converge to regulate the assembly and organization of MTs during neurite outgrowth is not well understood.

Results

Here, we report that Gβγ, an important component of the GPCR pathway, is critical for NGF-induced neuronal differentiation of PC12 cells. We have found that NGF promoted the interaction of Gβγ with MTs and stimulated MT assembly. While Gβγ-sequestering peptide GRK2i inhibited neurite formation, disrupted MTs, and induced neurite damage, the Gβγ activator mSIRK stimulated neurite outgrowth, which indicates the involvement of Gβγ in this process. Because we have shown earlier that prenylation and subsequent methylation/demethylation of γ subunits are required for the Gβγ-MTs interaction in vitro, small-molecule inhibitors (L-28 and L-23) targeting prenylated methylated protein methyl esterase (PMPMEase) were tested in the current study. We found that these inhibitors disrupted Gβγ and ΜΤ organization and affected cellular morphology and neurite outgrowth. In further support of a role of Gβγ-MT interaction in neuronal differentiation, it was observed that overexpression of Gβγ in PC12 cells induced neurite outgrowth in the absence of added NGF. Moreover, overexpressed Gβγ exhibited a pattern of association with MTs similar to that observed in NGF-differentiated cells.

Conclusions

Altogether, our results demonstrate that βγ subunit of heterotrimeric G proteins play a critical role in neurite outgrowth and differentiation by interacting with MTs and modulating MT rearrangement.

【 授权许可】

   
2014 Sierra-Fonseca et al.; licensee BioMed Central.

【 图 表 】

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【 参考文献 】

• [1]Conde C, Cáceres A: Microtubule assembly, organization and dynamics in axons and dendrites. Nat Rev Neurosci 2009, 10:319-332.
• [2]Mitchison T, Kirschner M: Cytoskeletal dynamics and nerve growth. Neuron 1988, 1:761-772.
• [3]Bulinski JC, Gundersen GG: Stabilization of post-translational modification of microtubules during cellular morphogenesis. Bioessays 1991, 13:285-293.
• [4]Greene LA, Tischler AS: Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci 1976, 73:2424-2428.
• [5]Patapoutian A, Reichardt LF: Trk receptors: mediators of neurotrophin action. Curr Opin Neurobiol 2001, 11:272-280.
• [6]Cantley LC: The phosphoinositide 3-kinase pathway. Science 2002, 296:1655-1657.
• [7]Zhou FQ, Zhou J, Dedhar S, Wu YH, Snider WD: NGF-induced axon growth is mediated by localized inactivation of GSK-3beta and functions of the microtubule plus end binding protein APC. Neuron 2004, 42:897-912.
• [8]Hur E,M, Zhou FQ: GSK3 signaling in neuronal development. Nat Rev Neurosci 2010, 11:539-551.
• [9]Govek EE, Newey SE, Van Aelst L: The role of the Rho GTPases in neuronal development. Genes Dev 2005, 19:1-49.
• [10]Hall A, Lalli G: Rho and Ras GTPases in axon growth, guidance, and branching. Cold Spring Harb Perspect Biol 2010, 2:a001818.
• [11]Igarashi M, Strittmatter S, Vartanian T, Fishman MC: Mediation by G proteins of signals that cause collapse of growth cones. Science 1993, 259:77-84.
• [12]Reinoso BS, Undie AS, Levitt P: Dopamine receptors mediate differential morphological effects on cerebral cortical neurons in vitro. J Neurosci Res 1996, 43:439-453.
• [13]Kwon JH, Vogt Weisenhorn DM, Downen M, Roback L, Joshi H, Wainer BH: Beta-adrenergic and fibroblast growth factor receptors induce neuronal process outgrowth through different mechanisms. Eur J Neurosci 1998, 10:2776-2789.
• [14]Lotto B, Upton L, Price DJ, Gaspar P: Serotonin receptor activation enhances neurite outgrowth of thalamic neurons in rodents. Neurosci Lett 1999, 269:87-90.
• [15]Sarma T, Voyno-Yasenetskaya T, Hope TJ, Rasenick MM: Heterotrimeric G-proteins associate with microtubules during differentiation in PC12 pheochromocytoma cells. FASEB J 2003, 17:848-859.
• [16]He JC, Neves SR, Jordon JD, Iyengar R: Role of the Go/i signaling network in the regulation of neurite outgrowth. Can J Physiol Pharmacol 2006, 84:687-694.
• [17]Sachdev P, Menon S, Kastner DB, Chuang JZ, Yeh TY, Conde C, Caceres A, Sung CH, Sakmar TP: G protein beta gamma subunit interaction with the dynein light-chain component Tctex-1 regulates neurite outgrowth. EMBO J 2007, 26:2621-2632.
• [18]Yu JZ, Dave RH, Allen JA, Sarma T, Rasenick MM: Cytosolic G{alpha}s acts as an intracellular messenger to increase microtubule dynamics and promote neurite outgrowth. J Biol Chem 2009, 284:10462-10472.
• [19]Wang K, Wong YH: G protein signaling controls the differentiation of multiple cell lineages. Biofactors 2009, 35:232-238.
• [20]Stoyanov B, Volinia S, Hanck T, Rubio I, Loubtchenkov M, Malek D, Stoyanova S, Vanhaesebroeck B, Dhand R, Nurnberg B, Gierschik P, Seedorf K, Justin Hsuan J, Waterfield MD, Wetzker : Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase. Science 1995, 269:690-693.
• [21]Stephens LR, Eguinoa A, Erdjument-Bromage H, Lui M, Cooke F, Coadwell J, Smrcka AS, Thelen M, Cadwallader K, Tempst P, Hawkins PT: The G beta gamma sensitivity of a PI3K is dependent upon a tightly associated adaptor, p101. Cell 1997, 89:105-114.
• [22]Wu EH, Wong YH: Involvement of G i/o proteins in nerve growth factor-stimulated phosphorylation and degradation of tuberin in PC-12 cells and cortical neurons. Mol Pharmacol 2005, 67:1195-1205.
• [23]Wu EH, Wong YH: Pertussis toxin-sensitive Gi/o proteins are involved in nerve growth factor-induced pro-survival Akt signaling cascade in PC12 cells. Cell Signal 2005, 17:881-890.
• [24]Roychowdhury S, Rasenick MM: G protein beta1gamma2 subunits promote microtubule assembly. J Biol Chem 1997, 272:31476-31581.
• [25]Roychowdhury S, Martinez L, Salgado L, Das S, Rasenick MM: G protein activation is a prerequisite for functional coupling between Galpha/Gbetagamma and tubulin/microtubules. Biochem Biophys Res Commun 2006, 340:441-448.
• [26]Montoya V, Gutierrez C, Najera O, Leony D, Varela-Ramirez A, Popova J, Rasenick MM, Das S, Roychowdhury S: G protein betagamma subunits interact with alphabeta- and gamma-tubulin and play a role in microtubule assembly in PC12 cells. Cell Motil Cytoskeleton 2007, 64:936-950.
• [27]Aguilar B, Amissah F, Duverna R, Lamango NS: Polyisoprenylation potentiates the inhibition of polyisoprenylated methylated protein methyl esterase and the cell degenerative effects of sulfonyl fluorides. Curr Cancer Drug Targets 2011, 11:752-762.
• [28]Marklund U, Larsson N, Gradin HM, Brattsand G, Gullberg M: Oncoprotein 18 is a phosphorylation-responsive regulator of microtubule dynamics. EMBO J 1996, 15:5290-5298.
• [29]Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 15:680-685.
• [30]Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci 1970, 76:4350-4354.
• [31]Azpiazu I, Gautam N: A fluorescence resonance energy transfer-based sensor indicates that receptor access to a G protein is unrestricted in a living mammalian cell. J Biol Chem 2004, 279:27709-27718.
• [32]Azpiazu I, Gautam N: Receptor-mediated reversible translocation of the G protein βγ complex from the plasma membrane to the Golgi complex. J Biol Chem 2004, 279:51541-51544.
• [33]Manders EMM, Verbeek FJ, Aten JA: Measurement of co-localization of objects in dual-colour confocal images. J Microsc 1993, 169:375-382.
• [34]Banker G, Goslin K: Culturing Nerve Cells. MIT Press, Cambridge; 1998.
• [35]Gomez LL, Alam S, Smith KE, Horne E, Dell’Acqua ML: Regulation of A-kinase anchoring protein 79/150-cAMP-dependent protein kinase postsynaptic targeting by NMDA receptor activation of calcineurin and remodeling of dendritic actin. J Neurosci 2002, 22:7027-7044.
• [36]Lema C, Varela-Ramirez A, Aguilera RJ: Differential nuclear staining assay for high-throughput screening to identify cytotoxic compounds. Curr Cell Biochem 2011, 1:1-14.
• [37]Koch WJ, Hawes BE, Inglese J, Luttrell LM, Lefkowitz RJ: Cellular expression of the carboxyl terminus of a G protein-coupled receptor kinase attenuates G beta gamma-mediated signaling. J Biol Chem 1994, 269:6193-6197.
• [38]Macrez N, Morel JL, Kalkbrenner F, Viard P, Schultz G, Mironneau J: A βγ dimer derived from G13 transduces the angiotensin AT1 receptor signal to stimulation of Ca2+ channels in rat portal vein myocytes. J Biol Chem 1997, 272:23180.
• [39]Dang VC, Napier IA, Christie MJ: Two distinct mechanisms mediate acute mu-opioid receptor desensitization in native neurons. J Neurosci 2009, 29:3322-3327.
• [40]Stern CM, Luoma JI, Meitzen J, Mermelstein PG: Corticotropin releasing factor-induced CREB activation in Striatal neurons occurs via a novel Gβγ signaling pathway. PLoS ONE 2011, 6:e18114.
• [41]Tummala H, Khalil HS, Mohammad R, Islam MR, Sarah J, Jones SJ, Ian R, Ellis IR, D’Ascanio I, Zhelev N, Lester DH: The alternate GNB3 splice variant, Gβ3s, exhibits an altered signalling response to EGF stimulation, which leads to enhanced cell migration. Biodiscovery 2013, 9:3.
• [42]Goubaeva F, Ghosh M, Malik S, Yang J, Hinkle PM, Griendling KK, Neubig RR, Smrcka AV: Stimulation of cellular signaling and G protein subunit dissociation by G protein betagamma subunit-binding peptides. J Biol Chem 2003, 278:19634-19641.
• [43]Garcia-Olivares J, Torres-Salazar D, Owens WA, Baust T, Siderovski DP, Amara SG, Zhu J, Daws LC, Torres GE: Inhibition of dopamine transporter activity by G protein βγ subunits. PLoS One 2013, 8:e59788.
• [44]Harrill JA, Mundy WR: Quantitative assessment of neurite outgrowth in PC12 cells. Methods Mol Biol 2011, 758:331-348.
• [45]Lamango NS: Liver prenylated methylated protein methyl esterase is an organophosphate-sensitive enzyme. J Biochem Mol Toxicol 2005, 19:347-357.
• [46]Winter-Vann AM, Casey PJ: Post-prenylation-processing enzymes as new targets in oncogenesis. Nat Rev Cancer 2005, 5:405-412.
• [47]Gelb MH, Brunsveld L, Hrycyna CA, Michaelis S, Tamanoi F, Van Voorhis WC, Waldmann H: Therapeutic intervention based on protein prenylation and associated modifications. Nat Chem Biol 2006, 2:518-528.
• [48]Dotti CG, Sullivan CA, Banker GA: The establishment of polarity by hippocampal neurons in culture. J Neurosci 1988, 8:1454-1468.
• [49]Powell SK, Rivas RJ, Rodriguez-Boulan E, Hatten ME: Development of polarity in cerebellar granule neurons. J Neurobiol 1997, 32:223-236.
• [50]Tahirovic S, Bradke F: Neuronal polarity. Cold Spring Harb Perspect Biol 2009, 1:a001644.
• [51]Purves D, Augustine GJ, Fitzpatrick D, Hall WC, LaMantia AS, McNamara JO, White LE: Neuroscience. 4th edition. Sinauer Associates Inc, Sunderland; 2008.
• [52]Koehl M, Abrous DN: A new chapter in the field of memory: adult hippocampal neurogenesis. Eur J Neurosci 2011, 33:1101-1114.
• [53]Roychowdhury S, Rasenick MM: Submembraneous microtubule cytoskeleton: regulation of microtubule assembly by heterotrimeric G proteins. FEBS J 2008, 275:4654-4663.
• [54]Sanada K, Tsai TH: G protein betagamma subunits and AGS3 control spindle orientation and asymmetric cell fate of cerebral cortical progenitors. Cell 2005, 122:119-131.
• [55]Okae H, Iwakura Y: Neural tube defects and impaired neural progenitor cell proliferation in Gbeta1-deficient mice. Dev Dyn 2010, 239:1089-1101.
• [56]Cushman I, Casey PJ: RHO methylation matters: a role for isoprenylcysteine carboxylmethyltransferase in cell migration and adhesion. Cell Adh Migr 2011, 5:11-15.
• [57]Ibrahim MX, Sayin VI, Akula MK, Liu M, Fong LG, Young SG, Bergo MO: Targeting isoprenylcysteine methylation ameliorates disease in a mouse model of progeria. Science 2013, 340:1330-1333.
• [58]Lamango NS, Charlton CG: Farnesyl-L-cysteine analogs block SAM-induced Parkinson’s disease-like symptoms in rats. Pharmacol Biochem Behav 2000, 66:841-849.
• [59]Lamango NS, Ayuk-Takem LT, Nesby R, Zhao WQ, Charlton CG: Inhibition mechanism of S-adenosylmethionine-induced movements deficits by prenylcysteine analogs. Pharmacol Biochem Behav 2003, 76:433-442.
• [60]Blackmer T, Larsen EC, Bartleson C, Kowalchyk JA, Yoon EJ, Preininger AM, Alford S, Hamm HE, Martin TF: G protein betagamma directly regulates SNARE protein fusion machinery for secretory granule exocytosis. Nat Neurosci 2005, 8:421-425.
• [61]Yoon EJ, Gerachshenko T, Spiegelberg BD, Alford S, Hamm HE: G betagamma interferes with Ca2-dependent binding of synaptotagmin to the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex. Mol Pharmacol 2007, 72:1210-1219.
• [62]Cash AD, Aliev G, Siedlak SL, Nunomura A, Fujioka H, Zhu X, Raina A, Vinters HV, Tabaton M, Johnson AB, Paula-Barbosa M, Avila J, Jones PK, Castellani RJ, Smith MA, Perry G: Microtubule reduction in Alzheimer’s disease and aging is independent of tau filament formation. Am J Pathol 2003, 162:1623-1627.
• [63]Buxton GA, Siedlak SL, Perry G, Smith MA: Mathematical modeling of microtubule dynamics: Insights into physiology and disease. Prog Neurobiol 2010, 2010(92):478-483.
• [64]Cartelli D, Ronchi C, Maggioni MG, Rodighiero S, Giavini E, Cappelletti G: Microtubule dysfunction precedes transport impairment and mitochondria damage in MPP+ − induced neurodegeneration. J Neurochem 2010, 115:247-258.
• [65]Cartelli D, Casagrande F, Busceti CL, Bucci D, Molinaro G, Traficante A, Passarella D, Giavini E, Pezzoli G, Battaglia G, Cappelletti G: Microtubule alterations occur early in experimental parkinsonism and the microtubule stabilizer epothilone D is neuroprotective. Sci Rep 2013, 3:1837.
• [66]De Vos KJ, Grierson AJ, Ackerley S, Miller CC: Role of axonal transport in neurodegenerative diseases. Annu Rev Neurosci 2008, 31:151-173.
• [67]Millecamps S, Julien JP: Axonal transport deficits and neurodegenerative diseases. Nat Rev Neurosci 2013, 14:161-176.
• [68]Franker MA, Hoogenraad CC: Microtubule-based transport - basic mechanisms, traffic rules and role in neurological pathogenesis. J Cell Sci 2013, 126:2319-2329.