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Lmmunocytochemical Localization of the Guanine Nucleotide-Binding Protein G, in Primary Cultures of Neuronal and Glial Cells

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Lmmunocytochemical Localization of the Guanine Nucleotide-Binding Protein G, in Primary Cultures of Neuronal and Glial Cells The Journal of Neuroscience, February 1988, 8(2): 701-708 lmmunocytochemical Localization of the Guanine Nucleotide-Binding Protein G, in Primary Cultures of Neuronal and Glial Cells P. Brabet, A. Dumuis, M. Sebben, C. Pantaloni, J. Bockaett, and V. Homburger Centre CNRS-INSERM de Pharmacologic-Endocrinologie, 34094 Montpelier Cedex, France We have localized the guanine nucleotide-binding protein, 1981). Another protein, termed G,, was initially isolated from G,, in primary cultures of pure neuronal and glial cells pre- bovine brain (Neer et al., 1984; Stemweisand Robishaw, 1984). pared from different mouse brain areas. lmmunoblotting ex- This protein exhibits structural and biochemical properties sim- periments with selective affinity-purified polyclonal rabbit ilar to the previously purified G onesthat justify its designation antibodies to the 39 kDa alpha subunit of G, (G, alpha) in- as a member of the family (Huff et al., 1985; Pines et al., 1985). dicated that G, is distributed in both neurons and glial cells. Its precise function has not yet been defined, although recent G, alpha accounts for 0.3% of total membrane proteins in evidence suggeststhe involvement of G proteins and perhaps striatal neurons. High specific G, immunoreactivity was also the G, protein in the neurotransmitter coupling to neuronal detected in cortical neurons and cerebellar granule cells. calcium channels (Holz et al., 1986; Lewis et al., 1986; Scott Similarly, striatal glial cells contain large amounts of G, (0.2% and Dolphin, 1986; Hescheleret al., 1987). of total membrane proteins), as do glial cells from cerebral Like G, and Gi, G, is a membrane-associatedheterotrimer cortex and colliculi. Surprisingly, G, was barely detectable consisting of alpha, beta, and gamma subunits. The beta and in cerebellar glial cells. 32P-ADP-ribosylation of the same gamma subunits are common components, which constitute a neuronal and glial cell membranes with pertussis toxin in- tightly associatedand hydrophobic dimer that could have a dicated the presence of at least 3 substrates related to G, regulatory function (Manning and Gilman, 1983; Stemweis, alpha, G, alpha (41 kDa), and a 40 kDa protein. This 40 kDa 1986). In SDS-PAGE analysis, the beta subunit can be resolved protein is the major pertussis toxin substrate in glial cells, into a doublet of 36 and 35 kDa, while the gamma subunit while G, alpha is predominant in neuronal membranes. Con- migrates at about 8 kDa. The alpha subunits differ for each G firming immunoblotting, no labeled band was detected at 39 protein; they possessthe guanine nucleotide-binding site, a kDa in cerebellar glial cells with pertussis toxin. Indirect GTPase activity, and can be ADP-ribosylated with bacterial immunofluorescence staining of cerebellar granule cells and toxins (Roof et al., 1985; Itoh et al., 1986). G,, alpha subunits striatal neurons with purified G, alpha antibodies was pro- (2 forms at 45-52 kDa) are substratesfor cholera toxin, while nounced at the plasma membrane level, particularly at cell- both G, alpha (39 kDa) and Gi alpha (41 kDa) subunits are cell contact areas, and in neurite arborization. More discrete susceptibleto covalent modification by ADP-ribosylating Bor- staining was also apparent in the cytoplasm, whereas nuclei detella pertussistoxin (Bokoch et al., 1983; Kahn and Gilman, remained unstained. In striatal glial cells, specific immuno- 1984; Katada et al., 1986). labeling was more diffused over the whole cell, and dense Severalstudies evidenced that G, is a highly conservedprotein around the nucleus. The localization of G, suggests that this that is predominantly localized in neuronal tissues(Neer et al., protein must perform important functions in both the neu- 1984; Stemweis and Robishaw, 1984; Gierschik et al., 1986a; ronal and glial cells that are discussed. Hornburger et al., 1987). Immunohistochemical observations in rat brain indicated a selective enrichment of G, in neuropil The contribution ofguanine nucleotide-binding proteins (G pro- areas (Worley et al., 1986). However, nothing is known about teins) in signal-transducingmechanisms is now firmly estab- the cellular localization of G, in the CNS cells. lished (Stryer and Bourne, 1986; Dolphin, 1987). G,, Gi, and We have produced polyclonal rabbit antibodies against the G, (transducin) are the functionally identified members be- purified bovine brain G, alpha subunit and obtained very se- longing to the G protein family. G, and G,, respectively, mediate lective probes towards the G, alpha subunit. Furthermore, cul- the stimulation and inhibition of adenylate cyclaseactivity (Hil- ture methods were developed in our laboratory to obtain highly debrandt et al., 1983; Gilman, 1984; Neer et al., 1984). G, is differentiated neurons and glial cells in separatedprimary cul- the retinal rod outer-segmenttransducer that couplesrhodopsin tures from mousebrain (Ebersolt et al., 198 1; Weisset al., 1986). to cyclic GMP phosphodiesterase(Kuhn, 1980; Fung et al., These primary cultures provide ideal modelsfor examining the cellular physiology of nerve cells (Bockaert et al., 1986). In the present study, we first used these antibodies to inves- Received May 19, 1987; revised July 24, 1987; accepted July 29, 1987. tigate the G, alpha immunoreactivity in nerve cells and then The authors are very grateful to Dr. B. Rouot, who participated in the purifi- cation of G proteins, and to Dr. M. Toutant for constructive discussion. We wish described intracellular localization of G, by light immunocy- to thank A. Turner-Madeuf and K. Alarcon for typing the manuscript. tochemistry. Our results indicated that G, is distributed in neu- Correspondence should be addressed to Phillipe Brabet, Centre CNRS-INSERM de Pharmacologic-Endocrinologie, Rue de la Cardonille, 34094 Montpellier Ce- rons, as well as in some glial cells, and mainly, but not exclu- dex, France. sively, associatedwith the cytoplasmic surface of the plasma Copyright 0 1988 Society for Neuroscience 0270-6474/88/020701-08$02,00/O membrane. 702 Brabet et al. - G. Protein in Primary Cultures of Neurons and Glial Cells in SDS-PAGE was performed on 1.5-mm-thick slab gels with a 10% Materials and Methods a&amide, 0.13% bis-separating gel containing 0.1% SDS in 0.375 M Materials. Lubrol PX, ATP, GTP, and L-myristyl phosphatidylcholine Tris-HCl. DH 8.8. Electroohoresis was carried out at 4°C for about 90 were obtained from Sigma. Lubrol PX was deionized prior to use. CU-‘~P- min at 166 V, followed by 5 hr at 35 mA constant. Gels were then NAD and Y-protein A were purchased from New England Nuclear. stained with Coomassie blue and dried. Autoradiography was performed Pertussis toxin was provided from List Laboratories. All other chemicals using Kodak-X Omat AR5 film without intensifying screens for 24-96 were of high purity from commercial sources. hr. Cell cultures Immunoblotting Neuronal cell cultures. Neuronal cultures generated from the striatum Membrane proteins (100 pg) were pelleted for 10 min in an Eppendorf and cerebral cortex of 14-15d-old Swiss albino mouse fetuses were centrifuee and solubilized in 10 ul of 50 mM Tris-HCl. DH 6.8. 10% grown for about 7 d under conditions previously described by Weiss et SDS, and 0.5 mM DTT by incubation for 2 hr at 30°C. -The samples al. (1986). These cultures were grown in the absence of serum and were then alkylated with NEM, as described above, prior to electro- contained less than 10% of glial cells (Bockaert et al., 1986). Cerebellar phoresis. Analysis of the molecular weight of the proteins was performed granule cells were cultured from cerebella of 7-d-old mice, as originally on 10% polyacrylamide gels prepared according to Laemmli (1970). described by Messer (1977), with minor modification. Briefly, cells were Proteins were then transferred onto nitrocellulose sheets, as previously isolated by mechanical dissociation with a fire-narrowed Pasteur pipette described (Audigier et al., 1985). The nitrocellulose sheets were dried after 5 mm incubation in 1 mM EDTA buffer, pH 7.4. Granule cells and stained with red Ponceau S in order to assess the quality of the (1.5 x lo6 cells/ml) were arown for 8-14 d in Dulbecco’s modified transfer. After blocking nonspecific protein binding with 3% gelatin, Eagle’s medium plus Ham F- 12 nutrient (1: 1) containing 5% fetal calf blots were incubated overnight at RT in 10 mM Tris-HCl, pH 7.5, 500 serum and 5% horse serum. After 48 hr in culture, 40 PM cytosine mM NaCl containing 0.3% gelatin, and affinity-purified anti-G,, alpha arabinofuranoside (ara-C) was added, and after 6 d this medium was antibodies (6 @g/ml). The blots were then washed and incubated at RT changed. Mitotic inhibitors were present throughout the culture. with lZ51-protein A (100,000 cpm/ml) for 60 min. They were then thor- Non-neuronalcell cultures.Glial cells generated from cerebral cortex oughly washed, dried, and exposed to Kodak X-AR5 with DuPont of newborn Swiss mice were grown for 18-21 d in the same medium Cronex image-intensifying screens at -80°C for 16-48 hr. used for neuronal cultures, but containing 10% fetal calf serum, ac- cording to the methods described by Ebersolt et al. (198 1). More than 95% of the cells were identified as glial cells since they were stained Afinity pur$cation of antibodies with anti-GFA (glial fibrillary acidic protein) antibodies. Meningeal Characteristics of antisera against anti-G,, alpha have been described layers were prepared under the same conditions described by Ebersolt previously by Homburger et al. (1987). For purification of antisera, 2 et al. (198 1). Cultures of glial cells from the striatum and of 14-15-d- mg of a purified mixture of G, and G, (heptylamine Sepharose elution old Swiss mouse fetuses were grown for 18-2 1 d in the serum medium fraction) from bovine brain was coupled to activated CH-Sepharose described above at a concentration of 8 x lo5 cells/ml.
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