Neuroscience Letters, 127 (1991) 219-222 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 A DON1S 030439409100308 F

219

NSL 07823

Small GTP-binding proteins in human neuroblastoma cell lines A n n a M. Giudici, Chiara Bisiani, Antonia Zanini and Francesco Clementi CNR Center of Cytopharmacology, Department of Medical Pharmacology, University of Milan, Milan (Italy) (Received 8 January 1991; Revised version received 11 February 1991; Accepted 11 February 1991)

Key words: Small G protein; Human neuroblastoma; Neuronal differentiation; Dibutyryl-cAMP; 5-Bromodeoxyuridine; Retinoic acid The presence of small G proteins was investigated by [y-3sS]GTP-binding in 3 human neuroblastoma cell lines, IMR-32, SK-N-BE and SH-SY5Y, before and after treatment with differentiating agents (dibutyryl-cAMP, 5-bromodeoxyuridine or retinoic acid) which induce the appearance of secretory organdies. One major component of about 24 kDa and 3 minor components of smaller Mr were found to bind specifically D,-3sS]GTP in all 3 cell lines already before differentiation. Differentiation did not affect the expression of small G proteins in IMR-32 cells and only modestly affected it in the other two cell lines. The possibility that the expression of small G proteins in neuroblastoma cells is not coupled with the assembly of secretory organdies is discussed.

We have recently demonstrated that, after in vitro differentiation in response to pharmacological agents, IMR-32 human neuroblastoma cells undergo a profound reorganization of their cytoplasmic structure and, in particular, acquire a regulated secretory pathway shown by the expression of secretory organelles and secretory organelle markers [20]. Small G proteins, the class of regulatory GTP-binding proteins characterized over the last few years by molecular cloning in eukaryotes [7, 12, 13, 21], have been described in secretory cells and neurons; they may play an important role in a variety of processes related to membrane traffic [2, 9, 10, 19] and, in particular, in rapid axonal transport of neuronal organelles [1] and in exocytosis [6]. In the present study we analyzed different human neuroblastoma cell lines (IMR-32, SK-N-BE, SH-SY5Y [16]) for the presence of small G proteins and we investigated whether their expression could be modulated during cellular differentiation, in parallel with the maturation of the secretory pathway. The IMR-32 cell line (obtained from the American Type Culture Collection, Rockville, MD); the SK-N-BE cell line, kindly provided by Dr. G. Melino (Rome, Italy), and the SH-SY5Y cell line, kindly provided by Dr. M. Toselli (Pavia, Italy), were grown as previously described [20]. To induce differentiation IMR-32 cells

Correspondence: A. Zanini, CNR Center of Cytopharmacology, Department of Medical Pharmacology, University of Milan, via Vanvitelli 32, 20129 Milan, Italy. Fax: (39) (2) 7490937.

were treated with 1 mM dibutyryl-cAMP (BhcAMP) or 2.5 /aM 5-bromodeoxyuridine (BrdU) [20]. SK-N-BE and SH-SY5Y cells were differentiated by means of 20 /aM retinoic acid (RA) [15]. Drugs were added every 3 days at each medium change and the cells were used 10-12 days later when numerous neurites were evident. Secretogranin II (SglI), a secretory organelle marker [11], was immunolocalized in the neuroblastoma cells by immunofluorescence as reported [20]. Antibodies against bovine SglI were purified by affinity chromatography and characterized as previously described [17]. Neuroblastoma cell homogenates were prepared as reported [20]. Protein concentration was determined using the Bradford protein assay (Bio-Rad Laboratories, Richmond, CA). Homogenates were analyzed by SDS-PAGE as previously described [4]. 13% polyacrylamide and a Mini Protean II cell (Bio-Rad Laboratories, Richmond, CA) were used. Gels were stained with Coomassie brilliant blue R-250. To detect GTP-binding proteins, proteins were electrophoretically transferred to nitrocellulose membranes at 300 mA for 5 h in 25 mM Tris/192 mM glycine buffer containing 20% methanol and no SDS. Incubation of nitrocellulose blots with [~-asS]GTP (New England Nuclear DuPont Ltd, UK: spec. act. 1320 Ci/mmol) was performed as reported [3]. The nitrocellulose blots, preequilibrated for 10 min in incubation buffer (50 mM Tris-HCl, pH 7.5,,0.3% (v/v) Tween 20, 12 pM MgCI2, 1 mM dithiothreitol) were transferred to fresh incubation buffer containing 10/zM ATP and 2/aCi/ml of [~,35S]GTP. After 20-min incubation at room temperature

220 the strips were washed 3 times (for 20 rain each) in incubation buffer and air dried. When the specific binding of [~-3sS]GTP was studied, the nitrocellulose blots were pre-incubated with competing substrates (0.05-10/tM G T P or 10/~M ATP) in incubation buffer for 10 min. Labeled proteins were detected by autoradiography using Kodak X-Omat AR films and quantified by densitometric analysis of the autoradiograms in a laser densitometer (Ultroscan XL; LKB Instruments, Gaithersburg, MD). In all 3 neuroblastoma cell lines, differentiation induced clear morphological changes, as shown by both the appearance of numerous neurites and the high expression of the secretory organelle marker SglI (Fig.

l b, d, f). Moreover, the differentiated neuroblastoma cell lines had a protein composition, particularly at the level of low Mr components, different from that of non-differentiated cells (Fig. 2A). This is in agreement with the differences that we have previously reported at the level of neurotransmitter receptors and tyrosine hydroxylase [8]. After the incubation of nitrocellulose blots with [y3sS]GTP, autoradiography revealed a group of proteins consisting of one major component of about 24 kDa and of 3 minor components of smaller Mr in all of the neuroblastoma cell line homogenates (Fig. 2B). The specificity of G T P binding for the group of proteins identified in the 3 human neuroblastoma cell lines is shown in Fig. 3. Pre-incubation with 0.05 pM GTP (Fig. 3C) slightly decreased the binding of [y-3sS]GTP to the 4 proteins,

Fig. 1. Immunolocalizationof SglI in neuroblastomacells. (a) control IMR-32 cells; (b) IMR-32 cellsdifferentiatedwith BtzcAMP;(c) control SK-NBE cells; (d) RA-differentiatedSK-N-BE cells; (e) control SH-SY5Ycells; (f) RA°differentiatedSH-SY5Y cells. No neurites and very few labeled secretory organellescan be seen in control cells (a, c, e). In differentiatedcells (b, d, f) a typical punctate fluorescenceis evident in the Golgi area and in the neurites which, particularlyin SH-SY5Ycells, frequentlyappear ramified. Bars= 25 pm.

221

A

B

"Tr rK SK SK fK K ,K

1

2

3

4

5

6

7

8

1

2

3

4

5

6

7

Fig. 2. Coomassie blue-stained electrophoretic pattern of proteins separated from neuroblastoma cell homogenates (A) and autoradiograms of proteins from the same homogenates blotted onto nitrocellulose and incubated with D,-3sS]GTP(B). Lane 1, non-differentiated IMR-32 cells; lane 2, IMR-32 cells differentiated with B½cAMP; lane 3, IMR-32 cells differentiated with BrdU; lane 4, control SK-N-BE cells; lane 5, SK-N-BE cells treated with RA; lane 6, control SH-SY5Y; lane 7, SH-SY5Y cells treated with RA; lane 8, Mr standard. Protein loaded onto each lane: 14/tg. Film exposure time: 4 days. structures and functions small G proteins are involved [1, 2]. Furthermore, in PC12 p h e o c h r o m o c y t o m a cells it has been shown that the level of the small G protein Smg25A m R N A was increased after differentiation [18]. It was therefore conceivable to expect modifications in the expression of small G proteins which participate in membrane traffic and secretory mechanisms also in neuroblastoma cells. However, densitometric analysis of the autoradiograms of our gels indicated that there were no quantitative differences in the 24-kDa GTP-binding protein between untreated IMR-32 cells and IMR-32 cells treated either with Bt2cAMP or with BrdU (Fig. 2B, lanes 1, 2, 3). An increase of about 25% in the amount of 24-kDa GTP-binding protein was detected in RA-differentiated SK-N-BE (Fig. 2B, lanes 4, 5) and SH-SY5Y (Fig. 2B,

whereas pre-incubation with 10/~M G T P (Fig. 3D) totally abolished it. On the contrary, pre-incubation with 10/tM A T P had no effect on the binding (Fig. 3E). A comparison between the G protein pattern obtained from IMR-32 cells (Fig. 3, lanes 2) and that obtained from homogenates prepared from bovine anterior pituitary (Fig. 3, lanes 3) shows that small G proteins with a M r similar to those of the major and at least two of the minor small G proteins iderktified in the human neuroblastoma cells are also present in endocrine cells. Differentiation of IMR-32 h u m a n neuroblastoma cells has been shown to cause a profound remodelling of their structure and, in particular, to induce the 'de novo' capacity of assembling secretory organelles ([20] and Fig. 1), storing [3H]dopamine and releasing this neurotransmitter in response t o secretagogue stimuli [20]. In these

A

B

C

D

E Mr

-97K -66K -45K w

-31 K -21 K -14K 123

2

3

2

3

2

3

2

3

Fig. 3. Specificityof binding of [7-35S]GTPto proteins from IMR-32 cells homogenates and comparison between small GTP-binding proteins from IMR-32 cells homogenates(lanes 2) and small G proteins from anterior pituitary homogenates (lanes 3). (A) Coomassieblue staining; (B) autoradiography after incubation in the presence of D,-3sS]GTP;(C-E) autoradiography after incubation in the presence of [y-3~S]GTPpreceded by pre-incubation either with 0.05/IM GTP (C), or with I0/tM GTP (D), or with 10/zM ATP (E). Lane 1, Mr standard. Protein loaded onto each lane: 20 /~g. Film exposure time: 2 days.

222 lanes 6, 7) cells. The other 3 proteins were hardly detectable a n d n o q u a n t i t a t i v e data were obtained. The results reported in the present paper d e m o n s t r a t e that small G proteins are already present in non-differentiated n e u r o b l a s t o m a cells, before the assembly of the secretory organelles, a n d that their expression is n o t affected, or is only slightly affected, by differentiating agents. This can be explained in two ways: (1) small G proteins are associated with secretory granules but their expression is n o t parallel to the assembly of the secretory organelles themselves a n d is i n d e p e n d e n t l y regulated or (2) small G proteins are n o t exclusively associated with secretory organelles b u t are also present in the cytosol a n d associated with other organelles, as has been s h o w n for other cells [1, 5, 9, 14]. I n this case, it is likely that the changes occurring in specific subtypes of small G proteins associated with the secretory organelles are hardly detectable. F u r t h e r studies, using antibodies specific for the different small G proteins, should give a n appropriate response to this point. The a u t h o r s wish to t h a n k Dr. E. Sher for critically reviewing the m a n u s c r i p t , Mr. P. Tinelli for preparing the micrographs a n d Mrs. Ida Ruffoni for her aid with the manuscript. 1 Bielinski, D.F., Morin, P.J., Dickey, B.F. and Fine R.E, Low molecular weight GTP-binding proteins are associated with neuronal organelles involved in rapid axonal transport and exocytosis, J. Biol. Chem., 264 (1989) 18363 18367. 2 Bourne, H., Do GTPases direct membrane traffic in secretion?, Cell, 53 (1988) 669q571. 3 Comerford, J.G., Gibson, J.R., Dawson, A.P. and Gibson, I., RASp21 and other Gn proteins are detected in mammalian cell lines by [y-3sS]GTPTSbinding, Biochem. Biophys. Res. Commun., 159 (1989) 1269 1274. 4 Cozzi, M.G. and Zanini, A., Sulfated LH subunits and a tyrosinesulfated secretory protein (secretogranin II) in femalerat adenohypophyses: changes with age and stimulation of release by LHRH, Mol. Cell. Endocrinol., 44 (1986) 47-54. 5 Darchen, F., Zahraoui, A., Hammel, F., Monteils, M.P., Tavitian, A. and Scherman, D., Association of the GTP-binding protein Rab3A with bovine adrenal chromaffin granules, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 5692 5696. 6 Fischer yon Mollard, G., Mignery, G.A., Baumert, M., Perin, M.S., Hanson, T.J., Burger, P.M., Jahn, R. and Siidhof, T.C., Rab3 is a small GTP-binding protein exclusively localized to synaptic vesicles, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 19881992.

7 Gallwitz, D., Donath, C. and Sander, C., A yeast gene encoding a protein homologous to the human C-has/bas proto-oncogene product, Nature, 306 (1983) 704-707. 8 Gotti, C., Sher, E., Cabrini, D., Bondiolotti, G., Wanke, E., Mancinelli, E. and Clementi, F., Cholinergic receptors ion channels, neurotransmitter synthesis, and neurite outgrowth are independently regulated during the in vitro differentiation of a human neuroblastoma cell line, Differentiation, 34 (1987) 144-155. 9 Goud, B., Salminen, A., Walworth, N.C. and Novick, P.J., A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast, Cell, 53 (1988) 753-768. 10 Hall A., The cellular functions of small GTP-binding proteins, Science, 249 (1990) 635~i40. 11 Huttner, W.B., Gerdes, H. and Rosa, P., The granin (chromogranin/secretogranin) family, Trends Biochem. Sci., 16 (1991) 27-30. 12 Madaule, P. and Axel, R., A novel ras-related gene family, Cell, 41 (1985) 31-40. 13 Matsui, Y., Kikuchi, A., Kondo, J., Hishida, T., Teranishi, T and Takai, Y., Nucleotide and deduced aminoacid sequences of a GTP-binding protein family with molecular weights of 25,000 from bovine brain, J. Biol. Chem., 263 (1988) 11071 11074. 14 Matsuoka, I. and Dolly, J.O., Identification and localization of low-molecular-massGTP-binding proteins associated with synaptic vesicles and other membranes, Biochim. Biophys. Acta, 1026 (1990) 99-104. 15 P~hlman, S., Ruusala, A.I., Abrahamson, L., Mattsson, M.E,K. and Esscher, T., Retinoic acid induced differentiation of cultured human neuroblastoma ceils: a comparison with phorbol ester induced differentiation, Cell Differ., 14 ( 1984) 135-144. 16 Rettig, W.J., Spengler, B.A., Chesa, P.G., Old, L.J. and Biedler, J.L., Coordinate changes in neuronal phenotype and surface antigen expression in human neuroblastoma cell variants, Cancer Res., 47 (1987) 1383-1389. 17 Rosa, P., Fumagalli, G., Zanini, A. and Huttner, W.B., The major tyrosine-sulfated protein of the bovine anterior pituitary is a secretory protein present in gonadotrophs, thyrotrophs, mammotrophs and corticotrophs, J. Cell Biol., 100 (1985) 928-937. 18 Sano, K., Kikuchi, A., Matsui, Y., Teranishi, Y. and Takai, Y., Tissue-specificexpression of a novel GTP-binding protein (smgp 25A) mRNA and its increase by nerve growth factor and cyclic AMP in rat pheochromocytoma PC-12 cells, Biochem. Biophys. Res. Commun., 158 (1989) 377 385. 19 Segev, N., Mulholland, J. and Botstein, D., The yeast GTP-binding YPTI protein and a mammalian counterpart are associated with the secretion machinery, Cell, 52 (1988) 915-924. 20 Sher, E., Denis-Donini, S., Zanini, A., Bisiani, C. and Clementi, F., Human neuroblastoma cells acquire regulated secretory properties and different sensitivityto Ca2+ and ~-latrotoxin after exposure to differentiatingagents, J. Cell Biol., 108 (1989) 2291 2300. 21 Touchot, N., Chardin, P. and Tavitian, A., Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: molecular cloning of YPT-related cDNAs from a rat brain library, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 8210-8214.

Small GTP-binding proteins in human neuroblastoma cell lines.

The presence of small G proteins was investigated by [gamma-35S]GTP-binding in 3 human neuroblastoma cell lines. IMR-32, SK-N-BE and SH-SY5Y, before a...
461KB Sizes 0 Downloads 0 Views