The Galanin Receptor Type 2 Initiates Multiple Signaling Pathways in Small Cell Lung Cancer Cells by Coupling to Gq, Gi And

Oncogene (2000) 19, 4199 ± 4209 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc The galanin receptor type 2 initiates multiple signaling pathways in small cell lung cancer cells by coupling to Gq,Gi and G12 proteins

Norbert Wittau1, Robert Grosse1, Frank Kalkbrenner1,3, Antje Gohla1,GuÈ nter Schultz1 and Thomas Gudermann*,2

1Institut fuÈr Pharmakologie, UniversitaÈtsklinikum Benjamin Franklin, Freie UniversitaÈt Berlin, 14195 Berlin, Germany; 2Institut fuÈr Pharmakologie und Toxikologie, Fachbereich Humanmedizin, Philipps-UniversitaÈt Marburg, Karl-von-Frisch-Str. 1, 35033 Marburg, Germany

Neuropeptides like galanin produced and released by mutations or overexpression of receptor tyrosine small cell lung cancer (SCLC) cells are considered kinases are usually not encountered in SCLC, and the principal mitogens in these tumors. We identi®ed the expression of GTPase-de®cient Ras mutants or of a galanin receptor type 2 (GALR2) as the only galanin constitutively active Raf kinase induces growth arrest receptor expressed in H69 and H510 cells. Photoanity and apoptosis (Dhanasekaran et al., 1995; Ravi et al., labeling of G proteins in H69 cell membranes revealed 1998). These ®ndings lend credibility to the belief that that GALR2 activates G proteins of three subfamilies: the main driving force for growth and proliferation of 2+ Gq,Gi, and G12. In H69 cells, galanin-induced Ca this subtype of cancer is represented by various mobilization was pertussis toxin-insensitive. While neuropeptides, e.g. bombesin/gastrin-releasing peptide phorbol ester-induced extracellular signal-regulated ki- (GRP), bradykinin, cholecystokinin, gastrin, neuroten- nase (ERK) activation required protein kinase C (PKC) sin, vasopressin and galanin which stimulate SCLC cells activity, preincubation of H69 cells with the PKC- via multiple auto- and paracrine loops (Rozengurt, inhibitor GF109203X had no e€ect on galanin-dependent 1999). Consequently, neuropeptide-induced signal ERK activity. A rise of the intracellular calcium transduction pathways have attracted considerable concentration was necessary and sucient to mediate attention as they may provide potential targets for galanin-induced ERK activation. In support of Gi novel therapeutic strategies aimed at interfering with coupling, stimulation of GALR2 expressed in HEK293 growth stimulatory autocrine loops. Many neuropep- cells inhibited isoproterenol-induced cAMP accumulation tides and their corresponding cell surface receptors are and raised cAMP levels in COS-7 cells when coex- expressed in SCLC cells (Bepler et al., 1988; Moody et pressed with a chimeric GaS-Gai protein. In H69 cells, al., 1985). Various SCLC cell lines, like H69 and H510, galanin activated the monomeric GTPase RhoA and are generally employed as suitable in vitro models to induced stress ®ber formation in Swiss 3T3 cells study the molecular mechanisms of the mitogenic expressing GALR2. Thus, we provide the ®rst direct activity of neuropeptides in SCLC. evidence that in SCLC the mitogenic neuropeptide Neuropeptides exert their cellular e€ects by binding galanin, interacting with GALR2, simultaneously acti- to G-protein-coupled receptors (GPCRs), a large and vates multiple classes of G proteins and signals through diverse superfamily of proteins characterized by a the Gq phospholipase C/calcium sequence and a G12/Rho seven-transmembrane structure (Gudermann et al., pathway. Oncogene (2000) 19, 4199 ± 4209. 1997; SchoÈ neberg et al., 1999; Strader et al., 1994). Agonist-bound neuropeptide receptors transmit infor- Keywords: G-protein-coupled receptor; mitogenic sig- mation into the cell by activating regulatory guanine naling; multiple signaling; neuropeptides nucleotide-binding proteins (G proteins) composed of a, b, and g subunits. G proteins are classi®ed based upon primary sequence similarity of their a subunits Introduction and are subdivided into four families: Gs,Gi,Gq,G12 (Simon et al., 1991). Receptor activation initiates the Approximately 25% of all primary carcinomas of the exchange of GDP for GTP in the G-protein a subunit lung are classi®ed histologically as small cell lung cancer followed by dissociation of the G-protein heterotrimer (SCLC), a subtype which is characterized by rapid into a GTP-bound a subunit and a bg dimer. Both, Ga- growth and a high metastatic potential, giving rise to a GTP and Gbg are signaling molecules in their own 5-year survival rate of only 5% despite initial radio- and right and activate a diverse array of e€ector systems chemosensitivity (Lassen et al., 1995). Therefore, such as enzymes and ion channels (Birnbaumer et al., considerable e€ort is currently being devoted to the 1990; Clapham and Neer, 1997; Gautam et al., 1998; design of novel therapeutic approaches, which are based Hepler and Gilman, 1992). In recent years it has on an advanced understanding of the tumor biology of become evident that receptor/G protein systems play SCLC. In contrast to non-SCLC, transforming Ras an important role in cell di€erentiation, proliferation and even in transformation by engaging signal transduction pathways like MAPK cascades which are classically employed by growth factors such as *Correspondence: T Gudermann EGF or PDGF (Dhanasekaran et al., 1995, 1998; 3 Current address: Atemwegsforschung, Boehringer Ingelheim Pharma Gutkind, 1998; van Biesen et al., 1996). In addition, KG, D-55216 Ingelheim, Germany Received 9 December 1999; revised 23 June 2000; accepted 3 July GTPase-de®cient G-protein a subunits can induce a 2000 transformed phenotype in certain ®broblast cell lines, Galanin signal transduction in SCLC cells N Wittau et al 4200 and mutationally activated G-protein a subunits have 1991a). In order to determine which galanin receptor been identi®ed in human tumors (Dhanasekaran et al., subtypes, GALR1, GALR2 or GALR3, are expressed 1995, 1998). in these cells, total RNA from H69 and H510 cells was The neuropeptide galanin was among the ®rst isolated, and a reverse transcription polymerase chain peptide growth factors for which a mitogenic e€ect reaction was performed. Ampli®cation products of the on SCLC cells was demonstrated (Sethi et al., 1992; expected size were obtained only with primers speci®c Sethi and Rozengurt, 1991a,b). Galanin is a 29-amino for the GALR2 (Figure 1, lanes 4 and 5). The primer acid (30 in men) polypeptide which is abundantly combinations used were tested by amplifying the expressed in brain as well as in peripheral tissues. The corresponding receptor fragments using a human brain regulation of a variety of physiological processes like library (Superscript cDNA library, Gibco ± BRL) as a stimulation of food intake, nociception, memory, template (data not shown), and the identity of the PCR depression, and control of neuroendocrine hypothala- products was con®rmed by direct DNA sequencing. In mic-pituitary circuits has been correlated with galanin the two tumor cell lines examined, neither GALR1 nor and its receptors (Bartfai et al., 1993; Bedecs et al., GALR3 were detected (Figure 1, lanes 1, 2 and 7, 8 1995; Kask et al., 1995; Wang and Gustafson, 1998). respectively) suggesting that galanin-mediated e€ects in In pituitary GH3 cells and in the pancreatic b-cell these SCLC cells are exclusively mediated by GALR2. model RINm5F, galanin interferes with hormone release by inhibition of voltage-gated Ca2+ channels Photoaffinity labeling of galanin-activated G-proteins in (Kalkbrenner et al., 1995). In H69 and H510 SCLC H69 cells cells, galanin challenge does not a€ect the membrane potential but causes a rapid and transient rise of the To identify the G proteins which couple to the agonist- 2+ 2+ intracellular Ca concentration [Ca ]i in a pertussis bound GALR2 in H69 cells, we resorted to the toxin-insensitive manner and stimulates the clonal approach of photoanity labeling of receptor-activated growth of SCLC cells in semisolid medium (Sethi and G-proteins in conjunction with selective immunopreci- Rozengurt, 1991a). The growth-stimulatory e€ect of pitation of radioactively labeled G-protein a subunits galanin on SCLC cells is mediated by the ERK (Laugwitz et al., 1994). Membranes of H69 cells were subfamily of mitogen-activated protein kinases incubated with or without 1 mM galanin in the presence (MAPKs), which are activated by galanin in a PKC- of the non-hydrolysable GTP-analog [a-32P]GTP azi- dependent fashion (Seu€erlein and Rozengurt, 1996). doanilide. After UV cross-linking of the photolabel, G- The fact that galanin initiates an entirely di€erent set protein a subunits were immunoprecipitated by speci®c of early events in SCLC cells as compared to pituitary antisera, resolved on SDS polyacrylamide gels and or pancreatic endocrine cells, supports the hypothesis visualized by autoradiography. As shown in Figure 2a, that galanin receptors expressed in SCLC cells are treatment of H69 cell membranes with galanin resulted distinct from those mediating the inhibitory e€ects on in increased incorporation of radioactivity into Gaq/11 neurotransmission and secretion in neuronal and proteins. In addition, galanin challenge of H69 endocrine cells, respectively. As yet, three galanin membranes caused signi®cant labeling of Ga12, minor 32 receptor subtypes, denoted GALR1, GALR2, and [a- P]GTP azidoanilide binding to Ga13 and small but GALR3, have been cloned which di€er in their ligand reproducible agonist-induced photo anity labeling of binding and signal transduction characteristics (Wang Gai. The reduced incorporation of radioactivity into and Gustafson, 1998). So far, the molecular identity Ga13 as compared to Ga12 proteins cannot be simply and the G-protein coupling pro®le of the galanin explained by reduced Ga13 protein expression in H69 receptor expressed in SCLC cells remain elusive. membranes, since both, Ga12 and Ga13, were clearly In the present study, we show that GALR2 is the detectable by immunoblotting (Figure 2b). Likewise, in only galanin receptor subtype expressed in SCLC cells. the same membrane preparation Gas,Gaq/11, and Gai For the ®rst time, direct evidence is presented that in SCLC cells as well as in several heterologous cell systems GALR2 couples to members of three G- protein sub-families, i.e. Gq,Gi and G12. In SCLC cells, activation of Gq/11 proteins results in elevated 2+ [Ca ]i sucient to stimulate ERK activity even in the absence of PKC activity. In the same tumor cell model, galanin stimulation results in activation of the monomeric GTPase Rho most likely via G12/13.As Rho proteins are able to profoundly modify the growth properties of SCLC cells (Tokman et al., 1997), our results add to a deeper understanding of SCLC tumor biology and may highlight novel therapeutic targets.

Figure 1 GALR2 expression in SCLC cell lines H69 and H510. Results Reverse transcription polymerase chain reaction (RT ± PCR) experiments were performed with total RNA from H69 cells (lanes 1, 4, 7), H510 cells (lanes 2, 5, 8), or as negative control Expression of galanin receptor subtypes in H69 and H510 water (lanes 3, 6, 9) using receptor subtype-speci®c primers as cells indicated. Aliquots of the RT-PCR mixture were separated on a 1% agarose gel, and ampli®cation products were visualized by Galanin has been shown to stimulate the clonal growth ethidium bromide staining. DNA size standards (100-bp ladder) of H69 and H510 SCLC cells (Sethi and Rozengurt, are shown (M) as base pairs (bp) on the right

Oncogene Galanin signal transduction in SCLC cells N Wittau et al 4201 proteins were visible as 52/44, 42, and 40 kDa proteins, transduction pathways speci®c for individual G-protein respectively (see Figure 2b). families activated by GALR2 in H69 cells as well as in To be able to characterize the signal transduction several other cell systems. properties of GALR2 in more detail, we isolated the GALR2 cDNA from H69 cells employing the RACE The effect of galanin on intracellular calcium technique (rapid ampli®cation of cDNA ends) as concentration in H69 cells described in the Materials and methods section. The cloned GALR2 cDNA sequence was identical to the Activated G-proteins of the Gq family stimulate one recently isolated from human small intestine certain PLC-b isoforms which in turn catalyze (Bloomquist et al., 1998). phosphoinositide breakdown producing the second In order to see how the G-protein coupling pro®le of messengers inositol-1,4,5-trisphosphate (IP3) and dia- GALR2 in H69 cells would compare to that in a cylglycerol (DAG). While DAG activates conven- 2+ di€erent cellular background, we transiently expressed tional and novel PKC isoforms, IP3 releases Ca the recombinant human GALR2 in COS-7 cells and from internal stores. The galanin-induced increase in 2+ performed photoanity labeling experiments with cell [Ca ]i was determined in H69 cells after loading of membranes. In essence, we obtained the same results as cells with the ¯uorescent dye fura-2. Stimulation of for H69 cell membranes except for the lack of GALR2- H69 cells with 100 nM galanin rapidly increased 2+ dependent G13 activation in COS-7 cells (Figure 2c). [Ca ]i reaching peak values about 20 s after 2+ Collectively, these results directly demonstrate that the hormone addition (Figure 3a). Thereafter, [Ca ]i GALR2 endogenously expressed in H69 cells as well as slowly declined but exceeded unstimulated basal levels heterologously expressed in COS-7 cells has the ability throughout the observation period of 3 min after the to activate G-proteins of the Gq,Gi, and G12 families. galanin stimulus. Pretreatment of cells with pertussis To further assess the biological relevance of our G- toxin (100 ng/ml for 20 h) prior to the stimulation protein coupling data, we set out to analyse signal with galanin had no inhibitory e€ect at all, demonstrating an exclusive involvement of pertussis toxin-insensitive G-proteins in galanin-induced Ca2+ transients (Figure 3b). For the most part, the 2+ 2+ observed increase in [Ca ]i resulted from Ca release from internal stores, since chelating of extracellular Ca2+ by EGTA (®nal concentration in the cuvette: 3.9 mM) prior to stimulation with galanin did not diminish galanin-dependent Ca2+ rises in H69 cells (data not shown). In conclusion, these results

Figure 2 Photoanity labeling of G-protein a subunits in H69 and COS-7 cell membranes. Aliquots of H69 (a) or COS-7 (c) cell membranes (100 mg for Gaq/11,Gai; 150 mg for Ga12,Ga13) were incubated with the photoreactive [a32P]GTP azidoanilide in the absence or presence of galanin (7/+) for 3 ± 30 min depending on the G protein studied (see Materials and methods) and UV- irradiated for 15 s. Photolabeled G-protein a subunits were immunoprecipitated with appropriate antibodies, subjected to SDS ± PAGE and visualized by autoradiography or phosphoima- Figure 3 Pertussis toxin-insensitive increase in the intracellular ging. Representative autoradiographs out of at least three Ca2+ concentration in H69 cells by galanin. H69 cells were left independent experiments with two di€erent membrane prepara- untreated (a) or were pretreated (b) with pertussis toxin (100 ng tions are shown. (b) Membrane proteins of H69 cells (50 mg per for 20 h), loaded with the ¯uorescent calcium indicator fura-2, lane) were resolved on 10% SDS polyacrylamide gels and AM and transferred to a ¯uorescence spectrophotometer. Cells subsequently blotted onto nitrocellulose. Membranes were cut were stimulated by the addition of 100 nM galanin as indicated by into strips and incubated with antisera against Ga subunits as the arrows. Fluorescence was monitored by dual wavelength indicated. Positions of the 42 kD molecular weight marker excitation at 378C, and ¯uorescence ratios were converted into 2+ 2+ protein are shown on the left intracellular Ca concentrations ([Ca ]i)

Oncogene Galanin signal transduction in SCLC cells N Wittau et al 4202 can be explained by coupling of GALR2 to Gq/11 cells. Therefore, galanin-dependent ERK activation can proteins in H69 SCLC cells (see Figure 2a). proceed through alternative pathways independent of PKC. SCLC exhibits neuroendocrine features and is Activation of extracellular signal-regulated kinases characterized by the presence of cytoplasmic neurosec- The neuropeptide-induced activation of ERK-MAP retory granules. In neuroendocrine PC12 cells, Ca2+ kinases is an essential component of mitogenic signals transients alone are sucient to trigger ERK activation arising from the agonist-bound galanin receptor in (Rosen et al., 1994). Therefore, we tested whether an 2+ SCLC cells (Seu€erlein and Rozengurt, 1996). There- increase in [Ca ]i would suce to stimulate ERK fore, we set out to further dissect the signaling activity in H69 cells. Treatment of SCLC cells with the pathways underlying the ability of galanin to stimulate calcium ionophore ionomycin (1 mM) caused an ERK activity in H69 cells. Treatment of H69 cells with increase in ERK activity (1.47+0.2-fold) comparable galanin led to a moderate (1.55+0.19-fold) stimulation with that observed after stimulation with galanin (see of ERK activity (Figure 4). The stimulatory e€ect of Figure 4). When Ca2+ transients were precluded by galanin was nearly abolished by pretreatment of cells preincubation of cells with the cell permeable Ca2+ with 1 mM staurosporine, indicating participation of chelator BAPTA, galanin-mediated ERK activity was serine/threonine kinases, e.g. PKC isozymes, in abrogated (1.08+0.13-fold). Collectively, these data 2+ galanin-induced ERK activation. However, a 30-min show that a galanin-mediated elevation of [Ca ]i alone pretreatment of H69 cells with the speci®c PKC is sucient to mediate ERK activation in H69 cells. inhibitor bisindolylmaleimide I (GF109203X) at 1 mM had no e€ect on galanin-induced ERK activation (see Effect of GALR2-activation on cAMP formation and Figure 4). At the same time, preincubation of H69 cells inositol phosphate accumulation with the same batch and concentration of GF109203X for 30 min caused an almost complete inhibition of The majority of G-protein-coupled receptors has the phorbol-12-myristate-13-acetate (PMA)-induced activa- ability to couple to more than one G protein tion of ERK (Figure 4, inset) demonstrating that the (Gudermann et al., 1996). Signaling pathways linking protein kinase C inhibitor used was biologically active G-protein-coupled receptors to ERKs can only be and could inhibit PKC-dependent ERK activity in dissected meaningfully if the G protein coupled by the these cells. These results would indicate that phorbol receptors and the functional relevance of each receptor/ ester-stimulated PKC activity is sucient to cause G-protein interaction is known. To study whether ERK activation in SCLC cells, but that PKC activity is coupling of GALR2 to Gq/11 and to Gi proteins in H69 not necessary for galanin to signal to ERK in H69 cell membranes as determined by photoanity labeling of receptor-activated G-proteins (see Figure 2) is mirrored by the engagement of typical Gq/11-andGi- mediated signal transduction pathways, we transiently expressed the GALR2 in COS-7 cells, a frequently employed cellular model, and analysed its signaling properties. In transfected COS-7 cells, galanin stimula- tion resulted in a concentration-dependent increase in the production of inositol phosphates with an EC50 value of 22+9nM (Figure 5a). Inositol phosphate accumulation was pertussis toxin-insensitive (data not shown) and most likely re¯ected Gq/11-mediated activation of phospholipase C-b isoforms. Next we analysed Gi-mediated coupling of GALR2 to adenylyl cyclase by coexpressing GALR2 with Gas-i5, a chimeric G-protein a subunit in which the C-terminal 5 amino acids of Gas are replaced by the corresponding amino acids of Gai2. This G-protein chimera has been shown to eciently redirect signaling of Gi-coupled receptors from inhibition to stimulation of adenylyl cyclase (Komatsuzaki et al., 1997). In cells Figure 4 Galanin-stimulated ERK activation in H69 cells transiently transfected with GALR2 cDNA, galanin- requires elevation of intracellular calcium. Aliquots of H69 cells stimulated cAMP formation did not di€er from basal 6 (3610 cells) were stimulated with 1 mM ionomycin or pretreated levels, exemplifying the inability of GALR2 to interact with staurosporine (1 mM, 30 min), GF109203X (1 mM, 30 min), or BAPTA-AM (1 mM, 30 min) and subsequently stimulated with with Gs proteins (Figure 5b). Similarly, despite 100 nM galanin for 5 min as indicated. ERK1/2 were immuno- moderately elevated basal cAMP levels in cells precipitated, and immune complex kinase assays were performed transiently transfected with Gasi-5 cDNA alone, galanin as described in Materials and methods. The data are presented as did not promote agonist-induced cAMP formation (see means+s.d. of at least three independent experiments performed in duplicate and are expressed as -fold stimulation relative to Figure 5b). However, galanin-dependent cAMP forma- mean basal ERK activity. Inset. Aliquots of H69 cells (36106 tion was clearly discernable in COS-7 cells co- cells) were pretreated with GF109203X (1 mM, 30 min) and transfected with GALR2 and Gas-i5 cDNA additionally stimulated with 1 mM phorbol-12-myristate-13-acetate (PMA) for proving the capability of GALR2 coupling to G- 5 min. Control cells were treated with solvent (DMSO). ERK proteins of the G class (see Figure 5b). activity was determined as described in Materials and methods, i and data are presented as counts per minute (c.p.m.)+R of one In a second approach we assessed the ability of the representative experiment out of two performed in duplicate GALR2 to inhibit cAMP formation in HEK293 cells.

Oncogene Galanin signal transduction in SCLC cells N Wittau et al 4203 In the latter cells, Gi-mediated inhibition of adenylyl cyclase is not confounded by the simultaneous activation of a Gbg-stimulable adenylyl cyclase like in COS cells (Wong, 1994). Thus, GALR2 was transiently coexpressed with the human b2-adrenoceptor (b2AR), and cAMP formation was stimulated via the b2AR receptor by incubation of the cells with 10 mM isoproterenol. Under these conditions addition of 1 mM galanin inhibited cAMP formation by 25.5+9.7% (Figure 5c). As a positive control, we coexpressed the b2AR with the Gi-coupled M2 muscarinic acetylcholine receptor instead of GALR2. In this case, isoproterenol-stimulated cAMP formation was suppressed by 67+6.1% following the addition of 100 mM carbachol (see Figure 5c). In summary, these ®ndings in COS-7 and HEK293 cells are in line with the results of our photolabel experiments in H69 SCLC cells (see Figure 2) and show that coupling of GALR2 to Gq/11 and Gi proteins engages corresponding classical signal transduction pathways in di€erent cell systems.

Rho activation and actin stress fiber formation mediated by GALR2 Photoanity labeling experiments with H69 and COS- 7 cell membranes revealed an interaction between GALR2 and G12 proteins. G12/13 proteins have been suggested to link G-protein-coupled receptors to the monomeric GTPase Rho and to reorganization of the actin cytoskeleton (Buhl et al., 1995). To see whether galanin challenge of SCLC cells would lead to activation of Rho GTPases, we used a GST-ROKa fusion protein (Kranenburg et al., 1999) as a bait for GTP-loaded RhoA. H69 and H510 cells were in- cubated either with galanin or as a positive control with lysophosphatidic acid (LPA), which is known to regulate the assembly of stress ®bers and focal adhesions via RhoA (Ridley and Hall, 1992). GTP- loaded RhoA bound to GST-ROKa was detected in cell lysates with a monoclonal anti-RhoA antibody and quanti®ed by ELISA. In H69 and H510 cells galanin stimulation resulted in a moderate increase in the GTP- loaded fraction of the monomeric GTPase (Figure 6a,b). The magnitude in the increase of Rho-GTP binding to GST-ROKa was comparable for galanin and LPA stimulation of SCLC cells. Immunoblot analysis of a portion of SCLC cell lysates used in the ELISAs con®rmed the expression of RhoA in both cell lines (Figure 6c). Figure 5 Functional coupling of GALR2 to Gaq/11 and Ga1 proteins. (a) COS-7 cells, transiently transfected with GALR2 To unequivocally show that activation of RhoA was were stimulated with increasing concentrations of galanin for due to stimulation of GALR2, we examined galanin- 60 min as indicated. Formation of [3H]inositol phosphates was dependent stress ®ber formation in Swiss 3T3 ®bro- determined by anion exchange chromatography and subsequent blasts which are a well characterized cellular model liquid scintillation counting. (b) COS-7 cells were transiently transfected either with GALR2 (1 mg/well), the chimeric G- system to study agonist-induced and Rho-dependent stress ®ber formation. These cells do not express protein Gas-i5 (1 mg/well), or both (0.5 mg/well for each construct). Cells were stimulated for 60 min with 1 mM galanin endogenous galanin receptors, and thus the human (&) or solvent (&), and [3H]cAMP accumulation was GALR2 was expressed in quiescent, serum-starved determined by anion exchange chromatography. Data in ®gures Swiss 3T3 cells by means of intranuclear injection of a and b are presented as means+R of one representative experiment out of three each performed in triplicate. (c) HEK293 cDNA. Stimulation of microinjected Swiss 3T3 cells cells were transfected with the b2-adrenoceptor (b2AR) together with GALR2 (&) or together with the M2 muscarinic acetylcho- line receptor (M2)(&) (0.5 mg/well for each construct) and were treated as indicated (10 mM isoproterenol, 1 mM galanin, 100 mM carbachol). Formation of cAMP was determined as described above and is expressed as percentage of the mean of represented as mean+s.d. of four independent experiments, each isoproterenol-stimulated cAMP formation (100%). Data are performed in triplicate

Oncogene Galanin signal transduction in SCLC cells N Wittau et al 4204

Figure 7 Stimulation of GALR2 causes the reorganization of the actin cytoskeleton in Swiss 3T3 ®broblasts. Swiss 3T3 cells were cultured on glass coverslips, serum-starved and microinjected with expression plasmids coding either for GALR2 (a) or the functionally inactive receptor mutant GALR2D218-230 (b). Cells were further incubated for 24 h, subsequently treated with galanin (100 nM) for 15 min, and ®xed in paraformaldehyde. The actin cytoskeleton was stained with FITC-phalloidin and visualized on an inverted microscope at a 400-fold magni®cation

texas red was coinjected with the receptor cDNAs (Figure 7c,d).

Discussion

Neuropeptides produced and released by SCLC cells are regarded as the principal mitogens in these tumors (Dhanasekaran et al., 1995; Rozengurt, 1999). Galanin was among the ®rst Ca2+ mobilizing peptides for which a mitogenic e€ect on SCLC cells was clearly demon- strated (Sethi and Rozengurt, 1991a). Early signaling events initiated by galanin in SCLC cells are notably disparate from those induced by the same agonist in Figure 6 RhoA activation by galanin and LPA in SCLC cells. pituitary, pancreatic, and other neuronal cells (Kalk- Aliquots of H69 (a) or H510 (b) cells (26106 cells) were cultured brenner et al., 1995; Sethi and Rozengurt, 1991b; in HITESA medium for 3 days. Stimulations with vehicle (basal), Seu€erlein and Rozengurt, 1996; Wang & Gustafson, 100 nM galanin or 10 mM LPA were carried out at 378C for 5 min 1998). These observations either re¯ect a cell-type- and reactions were stopped on ice. Cells were collected by speci®c expression of crucial signaling proteins or centrifugation and subsequently lysed at 08C. Lysates were cleared by centrifugation and added to anti-GST/GST-ROKa indicate that the galanin receptors mediating clonal precoated 96-well plates. GTP-RhoA binding to GST-ROKa was growth in SCLC cells are molecular entities separate quantitatively determined by applying an ELISA technique (see from those responsible for galanin-induced Ca2+ Materials and methods). Data are represented as means+s.e.m. channel inhibition. However, the issue concerning the of three independent experiments performed in quadruplicate. (c) Expression of RhoA in lysates of H510 (lanes 1 ± 3) or H69 cells molecular identity of galanin receptors expressed in (lanes 4 ± 6) was con®rmed by SDS ± PAGE and immunoblotting SCLC cells remained unresolved. using a monoclonal anti-RhoA antibody Molecular cloning e€orts in the past few years revealed that there are at least three genes coding for heptahelical receptors, which bind galanin with high with 100 nM galanin led to formation of actin stress anity (Wang and Gustafson, 1998). GALR1 is most ®bers within 15 min (Figure 7a). Galanin-induced abundantly expressed in brain and in the spinal cord stress ®ber formation critically depended on the and is a classical Gi-coupled receptor (Habert-Ortoli et expression of functionally intact galanin receptors, al., 1994). GALR2 is more widely distributed in because neuropeptide-induced cytoskeletal rearrange- peripheral tissues and to a lesser extent in the central ments were abrogated when a GALR2 deletion mutant nervous system (Wang and Gustafson, 1998). Most designated GALR2-D218 ± 230 was expressed instead authors describe the receptor's ability to activate of wild-type GALR2 (Figure 7b). In this deletion phospholipase C-b in a pertussis toxin-insensitive mutant, 13 amino acids within the third intracellular manner, representing indirect evidence for coupling to loop were deleted, rendering the receptor incapable of Gq proteins (Pang et al., 1998; Smith et al., 1997; G-protein interaction (data not shown). To aid Wang et al., 1998). GALR3 is prominently expressed in identi®cation of microinjected cells, dextran-conjugated spleen, heart and testis as well as in several regions of

Oncogene Galanin signal transduction in SCLC cells N Wittau et al 4205 the brain (Smith et al., 1998; Wang et al., 1997b). of the chimeric G-protein a subunit Gs-i5 characterized Functional data on GALR3 are scant; yet coexpression by the replacement of the C-terminal ®ve amino acids of the receptor with GIRK1 and GIRK4 potassium of Gs by the corresponding amino acids of Gi2, thus + channels resulted in inward K currents providing ®rst enabling a Gi-activating receptor to stimulate adenylyl evidence for GALR3 being a Gi-coupled receptor cyclase (Komatsuzaki et al., 1997). Transient expres- (Smith et al., 1998). sion in COS-7 cells of human GALR2 in conjunction In the present study we demonstrate that the with Gs-i5 conferred the ability upon the agonist-bound mitogenic e€ects of galanin on SCLC cells are receptor to initiate intracellular cAMP production. mediated by a single galanin receptor subtype, These data provide independent experimental evidence GALR2. In accord with earlier studies (Sethi et al., for the Gi-coupling potential of GALR2. So far, the 1992; Sethi and Rozengurt, 1991a,b) we observed that role of GALR2/Gi interaction in galanin-mediated galanin evokes a pertussis toxin-insensitive Ca2+ mitogenic signaling remains elusive. However, the mobilization in H69 cells, but in addition we directly failure of pertussis toxin to appreciably inhibit in vitro show galanin-dependent activation of Gq/11 proteins in growth of SCLC cells (Viallet et al., 1990) indicates SCLC cell membranes. Per de®nition, receptor-cata- that pertussis toxin-sensitive Gi proteins are unlikely to lyzed GDP/GTP exchange is the only direct measure to play a pivotal role. monitor G-protein activation, while second messenger The growth of SCLC cells critically depends on studies alone only yield circumstantial evidence. There- neuropeptide receptor/Gq/11-initiated phospholipase C fore, the experimental approach that was chosen to (Beekman et al., 1998) and ERK activity (Seu€erlein monitor GALR2/G-protein interaction consisted of a and Rozengurt, 1996). PKC isotypes are central combination of receptor-dependent photolabeling of signaling molecules coupling the pertussis toxin- G-protein a subunits with subsequent immunoprecipi- insensitive Gq/11 family of G-proteins to ERKs. While tation. This experimental design has previously been down-regulation of PKC-isoforms by phorbol ester adopted for the signaling analysis of various G-protein- pretreatment abrogated galanin- and phorbol ester- coupled receptors (Allgeier et al., 1994; Herrlich et al., induced ERK activation in H69 and H510 cells 1996; KuÈ hn et al., 1996; Laugwitz et al., 1994). (Seu€erlein and Rozengurt, 1996), in our hands the Photolabeling experiments employing the non-hydro- speci®c PKC inhibitor bisindolylmaleimide I only lyzable GTP analog [a-32P]GTP azidoanilide showed inhibited phorbol ester-induced ERK activity, but the that in membranes of H69 cells as well as in COS-7 galanin e€ect remained unaltered in H69 cells. In fact, 2+ cells, GALR2 couples to G12 and to a lesser extent to we found that a rise of Ca , the other second Gi proteins apart from interacting with Gq/11. Thus, messenger originating from phospholipase C activity, GALR2 has to be added to the ever growing list of was sucient to stimulate ERK activity in SCLC cells. heptahelical receptors with multiple G-protein coupling Thus, it appears that in H69 cells signals from Gq- potential (Gudermann et al., 1996, 1997). Two other coupled receptors to ERK can follow two independent G-protein-coupled receptors involved in the regulation routes, the one depending on PKC activity, the other of cell growth and di€erentiation, the thrombin relying on Ca2+. receptor and the neurokinin-1 receptor, are character- In CHO cells ERK activation via primarily Gq/11- ized by the same broad G-protein coupling pattern coupled receptors has been reported to involve Go (Barr et al., 1997; O€ermanns et al., 1994). However, proteins (van Biesen et al., 1996; Wang et al., 1998), to our knowledge the present study is the ®rst to while in most other cell systems Gq/11-coupled receptor directly demonstrate the activation of members of three signal to ERK in a strictly Gq/11/phospholipase C-b- distinct G-protein families, i.e. Gq,G12,andGi,bya dependent manner (Grosse et al., 2000a,b). As Go endogenously expressed neuropeptide receptor. proteins are not expressed in most other ®broblast cell We further analysed classical cellular signal transduc- lines used to express recombinant G-protein-coupled tion pathways initiated by the di€erent G-proteins receptors, the CHO cell model represents a rare activated by GALR2. While most authors agree on exception rather than the rule, but nevertheless the receptor's ability to couple to phospholipase C-b illustrates that signaling pathways can be a€ected by isoforms via Gq/11 proteins, the additional interaction of the cellular background. Thus, G-protein-coupling GALR2 with Gi or Go is discussed controversially studies on GALR2 expressed heterologously have to (Pang et al., 1998; Smith et al., 1997; Wang et al., 1997a, be supplemented by experiments on GALR2 endogen- 1998). The galanin-induced inhibition of isoproterenol- ously expressed in a native environment, e.g. SCLC dependent cAMP accumulation in HEK293 cells cells, before rigorous conclusions can be reached. coexpressing the human b2AR and GALR2 is con- In vascular smooth muscle cells and in neuroendo- 2+ siderably smaller than the e€ect of carbachol acting on crine PC12 cells, a rise in [Ca ]i appears to be the M2 muscarinic receptor. This e€ect is compatible sucient to engage the ERK cascade via growth with the fairly modest activation of Gi proteins via factor-independent tyrosine phosphorylation of recep- GALR2 as assessed by photoanity labeling experi- tor tyrosine kinases (Eguchi et al., 1998; Zwick et al., ments in H69 cell membranes and can therefore be 1997). In these cellular settings Pyk2, a focal adhesion regarded as a true re¯ection of the Gi coupling kinase related tyrosine kinase independently activated e€ectiveness of GALR2 in an intact cell setting. by Ca2+ and phorbol esters (Lev et al., 1995), has Mutagenesis studies have shown that the C-terminus recently been placed upstream of receptor tyrosine of G protein a subunits plays a pivotal role in de®ning kinases in the signaling pathway from Gq/11-coupled the speci®city of receptor/G-protein interaction (Liu et receptors to ERKs (Eguchi et al., 1999; Solto€, 1998). al., 1995). In particular, the C-terminal 5 amino acids To better understand mitogenic signaling by Gq/11- are sucient to switch the receptor speci®city of Gaq/ coupled receptors in SCLC cells, a detailed analysis of Gai2 chimeric proteins. Therefore, we took advantage the role of receptor tyrosine kinases like c-kit and of

Oncogene Galanin signal transduction in SCLC cells N Wittau et al 4206 non-receptor tyrosine kinases such as Pyk2 should be BRL. Transferrin, hydrocortisone, selenium, estradiol, insulin, enlightening in the future. and bovine serum albumin were from Sigma. Pertussis toxin, Di€erential e€ects of substance P-derived broad porcine galanin, GF109203X, and phorbol-12-myristate-13- spectrum antagonists on neuropeptide receptor signal- acetate were obtained from Calbiochem. Texas Red-coupled ing in Swiss 3T3 cells, a frequently used cellular dextran, BAPTA, AM and fura-2, AM were purchased from Molecular Probes. Antibodies against RhoA, Gai, and ERK1/2 substitute for SCLC cells, were interpreted to indicate (Sc-154) were from Santa Cruz Biotechnology. Enzyme-linked activation of G12/13 proteins by neuropeptide receptors secondary antibodies, anti GST-antibody and Protein A- (Dhanasekaran et al., 1998; Jarpe et al., 1998; Mitchell Sepharose were obtained from Sigma. [2,8-3H]adenine, et al., 1995). A second line of evidence is provided by [g-32P]ATP, and [a-32P]GTP were obtained from NEN Life 3 the demonstration that G12/13 proteins signal to the Sciences. Myo-[2- H]inositol was from Amersham. monomeric GTPase Rho, which is involved in the regulation of the cytoskeleton (Buhl et al., 1995), and Cell culture and transfection that neuropeptides like bombesin regulate Rho- mediated stress ®ber formation in Swiss 3T3 cells H69 and H510 cells were routinely cultured in RPMI 1640 (Ridley and Hall, 1994). However, by monitoring with 10% heat-inactivated fetal bovine serum (FBS) without antibiotics in a humidi®ed atmosphere containing 5% CO at 32 2 receptor-mediated binding of [a- P]GTP azidoanilide 378C. For experimental purposes H69 cells were cultured in to G proteins we present the ®rst direct evidence that HITESA medium (RPMI 1640 supplemented with 10 nM neuropeptide receptor like GALR2 in fact activate G12 hydrocortisone, 5 mg/ml insulin, 10 mg/ml transferrin, 10 nM proteins in SCLC cells. Furthermore, we show that the estradiol, 30 nM selenium and 0.25% bovine serum albumin) agonist-bound GALR2 stimulates the formation of as described (Sethi and Rozengurt, 1991a). COS-7 cells and Rho-GTP in H69 and H510 cells. The magnitude of Swiss 3T3 cells were cultured in Dulbecco's modi®ed Eagle's the increases in RhoA activation noted by us and by medium (DMEM) supplemented with 100 U/ml penicillin, others (Gohla et al., 1998) is usually less than twofold 100 mg/ml streptomycin, and 10% FBS in 7% CO2 at 378C. and is similar to the situation when Ras activation by HEK293 cells were cultured in essential minimal Eagle's G-protein-coupled receptors is measured. medium (MEM) with Earle's salts containing antibiotics and 10% FBS in a humidi®ed atmosphere containing 5% CO2 at The search for the causative oncogene of soft tissue 378C. For expression purposes, COS-7 cells were cultured in sarcoma led to the identi®cation of human Ga12 as a 12-well plates (200 000 cells/well), transfected by a standard putative oncogene in NIH3T3 cells (Chan et al., 1993), calcium-phosphate procedure and analysed 72 h later. and Rho family proteins contribute signi®cantly to the malignant growth properties of Ras-transformed cells Cloning of GALR2 (Zohn et al., 1998). As yet, the relevance of The cDNA coding for the human type 2 galanin receptor neuropeptide-induced G12 and Rho activation in SCLC cells is poorly understood. ADP-ribosylation of RhoA (GALR2) was isolated from H69 cells employing the rapid in SCLC cells by Clostridium botulinum C3 exoenzyme ampli®cation of cDNA ends technique (RACE), exactly leads to cadherin-mediated compaction and aggrega- following the procedure described by Frohman et al. (1988). Brie¯y, 2 mg of total RNA were reverse transcribed tion of SCLC cells (Tokman et al., 1997). Thus, Rho using the QT primer. For 3' RACE, a primer complementary proteins control the adhesive properties and probably to nucleotides 238 ± 259 of the rat GALR2 cDNA and primer also the metastatic potential of SCLC cells. The QO were used. A second PCR reaction was performed with neuropeptide-controlled growth of SCLC cells appears diluted ampli®cation products obtained in the ®rst step using to be controlled by a delicately orchestrated interplay a primer complementary to nucleotides 290 ± 315 of the rat between various signaling pathways initiated by G- GALR2 and the primer QI. The resulting PCR product was protein-coupled neuropeptide receptors. Overexpres- cloned into the T-easy vector (TA Cloning Kit, Invitrogen) sion of GTPase-de®cient G-protein a subunits of the and sequenced with Ampli-Taq DNA Polymerase FS (Perkin Elmer) using an automated DNA sequencer (ABI PRISM Gq/11 family, for instance, results in discordant signal transduction and growth inhibition (Heasley et al., 377, Perkin-Elmer). 5' RACE was performed with human brain Marathon- 1996). Likewise, expression of a constitutively active Ready cDNA and the Advantage-GC cDNA PCR Kit (both Raf kinase in SCLC cells leads to persistent ERK Clontech, Heidelberg). A ®rst ampli®cation was performed activation but inhibition of clonogenicity and growth using 5 ml of human brain cDNA, the supplier's adaptor arrest, most probably due to a marked induction of the primer 1, and an antisense primer complementary to the PCR cyclin-dependent kinase inhibitor p27kip1 (Ravi et al., product obtained above (corresponding to nucleotides 385 ± 1998). Simultaneous signaling of neuropeptide recep- 364 of GALR2 cDNA). A portion of the initial PCR reaction was diluted and ampli®ed with adaptor primer 2 and a tors to the Gq/PLC/Ras/ERK and a still ill-de®ned second antisense primer (nucleotides 340 ± 319). The PCR G12/Rho pathway may be required to regulate the expression of cyclin-dependent kinase inhibitors to product obtained was sequenced and speci®c primers for the allow cell cycle progression (Olson et al., 1998). Thus, 5'- and 3'-end of GALR2 were designed to amplify the full- length receptor from H69 cell cDNA. The exact sequence of the neuropeptide receptor/G12/Rho signaling sequence the GALR2 was determined by direct sequencing of three in SCLC cells may o€er novel therapeutic targets to independent ampli®cation products. Individual clones were interfere with the proliferation of these tumor cells. obtained using the TA Cloning Kit, analysed by sequencing, and a construct harboring the exact GALR2 cDNA was subcloned into the mammalian expression vectors pcDNA3 Materials and methods (Invitrogen) and pcD-PS. Materials RT ± PCR of galanin receptor subtypes COS-7, HEK293, and Swiss 3T3 cells were from ATCC (Manassas, Virginia, USA). Cell culture media were obtained Total RNA from H69 and H510 cells was isolated with from Biochrom, and fetal bovine serum was from Gibco ± Trizol reagent (Gibco ± BRL) and reverse transcribed (Super-

Oncogene Galanin signal transduction in SCLC cells N Wittau et al 4207 script II, Gibco ± BRL), followed by a digestion with RNase Rozengurt, 1991a) with modi®cations. Brie¯y, 4 ± 66106 H69 H (Promega) according to the manufacturer's instructions. cells, cultured in HITESA medium for 3 ± 5 days, were PCR-reactions were performed with the Advantage-GC washed in RPMI 1640 without fetal bovine serum and cDNA PCR Kit (Clontech, Heidelberg) using sense (S) and incubated for 2 h at 378C in 10 ml of fresh RPMI 1640. Cells antisense (AS) oligonucleotides for the cDNAs of GALR1 were further incubated with 1 mM fura-2, AM for 5 min, (nucleotides 249 ± 268 (S) / 802 ± 821 (AS)), GALR2 (290 ± centrifuged at 800 g for 15 s and resuspended in 2 ml of a 315 (S) / 767 ± 788 (AS)), and GALR3 (311 ± 336 (S) / 826 ± solution containing 140 mM NaCl, 5 mM KCl, 0.9 mM 850 (AS)). The cycling parameters for PCR were 30 s at MgCl2, 1.8 mM CaCl2,25mM glucose, 16 mM HEPES, 958C, 45 s at 558C (608C for GALR3), and 2 min at 688C for 6mM Tris, at pH 7.2. The cell suspension was transferred 40 cycles, with a pre- and post-incubation of 2 min at 958C to a cuvette, stirred continuously, and ¯uorescence was and 7 min at 688C, respectively. The resulting PCR products recorded at 378C in a Perkin-Elmer LS 50 B ¯uorescence were analysed on a 1% agarose gel. spectrophotometer with dual wavelength excitation of 340 and 380 nm and an emission wavelength of 510 nm. To remove extracellular Ca2+ aliquots of 200 mM EGTA were Immunoprecipitation procedure added sequentially (®nal concentration in the cuvette: H69 cells were cultured in HITESA for 3 ± 5 days and washed 3.9 mM) until a stable baseline of the F340/380 ratio was twice in RPMI 1640. Aliquots of approximately 36106 cells reached. Lysis of cells at the end of an experiment resulted in 2+ were incubated in fresh RPMI 1640 for 30 min at 378C and a further decline of the F340/380 ratio demonstrating that Ca subsequently treated as described. Cells were transferred onto interacting with intracellular fura-2 was chelated by the ice and lysed in 500 ml of ice-cold lysis bu€er (50 mM Tris- EGTA present in the incubation bu€er. Intracellular calcium HCl, 100 mM NaCl, 5 mM EDTA, 1 mM DTT, 40 mM concentrations were calculated as described (Kao, 1994). sodium pyrophosphate, 500 M sodium orthovanadate, 1% For the determination of inositol phosphates, cells were Triton X-100, 1 mM phenylmethylsulfonyl ¯uoride, 1 M transfected with GALR2, cultured for 48 h, and labeled with leupeptin and 0.1 M aprotinin, pH 7.5) for 15 min. Lysates 2 mCi/ml of myo-[2-3H]inositol for 20 ± 24 h. Cells were were cleared by centrifugation at 10 000 g for 15 min at 48C washed twice with serum-free culture medium without and supernatants were diluted 1 : 1 into lysis bu€er without antibiotics containing 10 mM LiCl and stimulated with the Triton X-100. Cleared lysates were then subjected to same medium containing agonists at variable concentrations immunoprecipitation with 2 mg of the appropriate antibody for 60 min. Reactions were terminated by aspiration of the overnight at 48C at constant rotation. Thirty ml of protein A- medium, and cells were lysed in 0.3 ml NaOH (0.1 M) for Sepharose beads (50%, vol/vol) were added, and lysates were 5 min, neutralized by addition of 0.2 ml of formic acid further incubated for 2 h at 48C. (0.2 M), and diluted with 2 ml of a solution consisting of 5mM Na-borate and 0.5 mM EDTA. Agonist-induced formation of intracellular inositol phosphates was determined Membrane preparation by anion-exchange chromatography as described (Berridge, H69 and COS-7 cells were washed with phosphate bu€ered 1983). saline (PBS), resuspended in mannitol bu€er (220 mM To determine cyclic AMP formation, COS-7 and HEK293 mannitol, 70 mM sucrose, 5 mM HEPES, 0.5 mM EDTA, cells were transfected with expression plasmids. Forty-eight pH 7.4) and ruptured by nitrogen cavitation. Nuclei and hours after transfection, 2 mCi/ml of [2,8-3H]adenine was cellular debris were removed by centrifugation at 600 g for added to the growth medium. After a labeling period of 20 ± 10 min. Membranes were pelleted by centrifugation at 48C 24 h, cells were washed twice with serum-free culture medium for 30 min at 40 000 g, resuspended in 10 mM triethanola- containing 1 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma) mine HCl (pH 7.4), and stored at 7708C. and stimulated with appropriate concentrations of agonist for 60 min as indicated. Reactions were terminated by aspiration of the medium and addition of 1 ml of ice-cold 5% Photolabeling of G-protein a subunits trichloroacetic acid containing 1 mM ATP and 1 mM cAMP. [a32P]GTP azidoanilide was synthesized and puri®ed as Intracellular [3H]cAMP was determined by anion-exchange described (O€ermanns et al., 1991). Photolabeling and chromatography as described (Salomon et al., 1974). immunoprecipitation of membrane G-proteins was performed as described (Laugwitz et al., 1994). Brie¯y, cell membranes (100 ± 150 mg) were incubated in the absence or presence of receptor agonist for 3 min (Gai), 10 min (Gas,q), or 30 min MAP kinase assay 32 (Ga12,13) with 10 nM [a P]GTP azidoanilide, followed by UV-irradiation at 254 nm for 10 s. Photolabeled G-proteins Endogenously expressed ERK1 and ERK2 were immunopre- were solubilized, and insoluble material was removed by cipitated from H69 cells by incubating cleared lysates with an centrifugation. Lysates were precleared by addition of protein antibody against ERK1/2 (Sc-154). Immune complexes were A-Sepharose beads, and subsequently 10 ml of either pelleted by centrifugation at 5000 g for 2 min and washed antiserum AS 86 (Gai), AS 370 (Gaq), AS 233 (Ga12), or three times with lysis bu€er and two times with kinase bu€er AS 343 (Ga13) was added to the supernatants for (40 mM HEPES, 5 mM magnesium acetate, 2 mM DTT, immunoprecipitation. The antibodies used were raised against 1mM EGTA and 200 mM sodium orthovanadate, pH 7.5). peptides corresponding to speci®c regions of G-protein a Kinase reactions were performed in 50 ml kinase bu€er subunits and have been described before (Allgeier et al., 1994; containing 250 mg/ml myelin basic protein (MBP, Sigma) Laugwitz et al., 1994). Immunoprecipitated G-protein a and 2 mM protein kinase A inhibitor (fragment 6-22 amide, subunits were separated on 10% SDS-polyacrylamide gels Sigma). Kinase reactions were started by adding 5 mlof and visualized by autoradiography of dried gels with Kodak 500 mM ATP containing 2 mCi [g-32P]ATP, incubated for X-Omat AR-5 ®lms or with a phosphoimaging system (Fuji 20 min at 258C, and stopped with the addition of 6 ml of 88% BAS1000). formic acid. The reaction mixture was spotted onto pieces of Whatman P81 chromatography paper (262 cm), and washed four times with 150 mM phosphoric acid. Radioactivity Determination of intracellular calcium concentration, inositol incorporated into MBP was measured by liquid scintillation phosphate formation and cyclic AMP accumulation counting. Basal counts ranged from 3000 to 7000 c.p.m. The Measurement of the intracellular calcium concentration radioactivity of reactions performed without immune-com- 2+ ([Ca ]i) was performed as described previously (Sethi and plex was between 150 and 250 c.p.m.

Oncogene Galanin signal transduction in SCLC cells N Wittau et al 4208 RhoA ELISA 1 M H2SO4 containing 0.05 M Na2SO3, and the color development was assessed bichromatically at 450 and For the assessment of Rho activation, a GST-Rho kinase a 630 nm with a Titertek Multiscan MCC/340 ELISA reader (GST-ROK) fusion protein known to bind the GTP-loaded (Labsystems). form of RhoA and Cdc42 (Kranenburg et al., 1999) was used. To quantitatively analyse the binding of GST-ROK to GTP-loaded RhoA, an ELISA-based method was established. Cytoskeletal reorganization The GST fusion protein was prepared as previously described Quiescent, serum starved Swiss 3T3 cells were grown on glass (Kranenburg et al., 1999) and stored at 7808C. Protein coverslips with imprinted squares to facilitate the localization preparations were stable for up to 2 weeks as determined by of injected cells. Expression plasmids coding for GALR2 and, anti-GST immunoblotting (data not shown). Ninety-six well as negative control, GALR2 D 218 ± 230 were microinjected plates were coated with 150 ml/well rabbit polyclonal anti- into the nucleus together with Texas Red-coupled dextran GST antibody (dilution 1 : 100) overnight at 48C. After (5 mg/ml) to visualize injected cells. After micro-injection, washing with phosphate bu€ered saline containing 0.1% cells were incubated overnight at 378C, and subsequently Triton X-100 (PBST), GST-ROK fusion protein was added stimulated with 100 n galanin 1 ± 29 for 15 min, ®xed in 4% (100 ml/well) and incubated for 1 h at room temperature M paraformaldehyde for 20 min, and permeabilized in 0.2% (RT). In parallel, stimulation of cells (26106 cells/tube) was Triton X-100 for 5 min. Actin ®laments were stained with carried out at 378C. Cells were lysed in 500 ml ice-cold lysis 0.5 mg/ml ¯uorescein isothiocyanate (FITC)-phalloidin (Sig- bu€er (150 m NaCl, 50 mM Tris pH 7.4, 5 mM MgCl , M 2 ma) for 40 min. The coverslips were mounted on microscope 0.1% Triton X-100, 10% glycerol) for 15 min, and insoluble slides and visualized on an inverted microscope (Zeiss material was removed by centrifugation. After washing plates Axiovert 100). with PBST, cell lysates were split, added to the 96-well plates (100 ml/well) and incubated for 45 min at 48C. Plates were then washed with PBST, and bound GTP-RhoA was analysed using a monoclonal anti-RhoA antibody (dilution Acknowledgments 1 : 400) followed by incubation with a horseradish peroxidase- We thank Rita Haubold for excellent technical assistance conjugated anti-mouse IgG antibody. Hydrogen peroxide and and Dr Enrique Rozengurt for providing the H69 and o-phenylenediamine (Sigma) (2.5 mM each in 0.1 M phos- H510 cells. We also thank Drs Onno Kranenburg and phate-citrate bu€er, pH 5.0) were then added to serve as Wouter Moolenaar for supplying the GST-ROK expression substrate and chromogen, respectively. After a 10 min plasmid and Dr JuÈ rgen Wess for providing the cDNAs of incubation at RT, the enzymatic reaction was stopped with the M2 receptor and Gas-i5.

References

Allgeier A, O€ermanns S, Van Sande J, Spicher K, Schultz G Frohman MA, Dush MK and Martin GR. (1988). Proc. and Dumont JE. (1994). J. Biol. Chem., 269, 13733 ± Natl. Acad. Sci. USA., 85, 8998 ± 9002. 13735. Gautam N, Downes GB, Yan K and Kisselev O. (1998). Cell Barr AJ, Brass LF and Manning DR. (1997). J. Biol. Chem., Signal, 10, 447 ± 455. 272, 2223 ± 2229. Gohla A, Harhammer R and Schultz G. (1998). J. Biol. Bartfai T, Hokfelt T and Langel U. (1993). Crit. Rev. Chem., 273, 4653 ± 4659. Neurobiol., 7, 229 ± 274. Grosse R, Schmid A, SchoÈ neberg T, Herrlich A, Muhn P, Bedecs K, Berthold M and Bartfai T. (1995). Int. J. Biochem. Schultz G and Gudermann T. (2000a). J. Biol. Chem., 275, Cell. Biol., 27, 337 ± 349. 9193 ± 9200. Beekman A, Helfrich B, Bunn Jr PA and Heasley LE. (1998). Grosse R, Roelle S, Herrlich A, HoÈ hn J and Gudermann T. Cancer. Res., 58, 910 ± 913. (2000b). J. Biol. Chem., 275, 12251 ± 12260. Bepler G, Rotsch M, Jaques G, Haeder M, Heymanns J, Gudermann T, Kalkbrenner F and Schultz G. (1996). Annu. Hartogh G, Kiefer P and Havemann K. (1988). J. Cancer. Rev. Pharmacol. Toxicol., 36, 429 ± 459. Res. Clin. Oncol., 114, 235 ± 244. Gudermann T, Schoneberg T and Schultz G. (1997). Annu. Berridge MJ. (1983). Biochem. J., 212, 849 ± 858. Rev. Neurosci., 20, 399 ± 427. Birnbaumer L, Abramowitz J and Brown AM. (1990). Gutkind JS. (1998). Oncogene, 17, 1331 ± 1342. Biochim. Biophys. Acta., 1031, 163 ± 224. Habert-Ortoli E, Amirano€ B, Loquet J, Laburthe M and Bloomquist BT, Beauchamp MR, Zhelnin L, Brown SE, Uayaux JF. (1994). Proc. Natl. Acad. Sci. USA, 91, 9780 ± Gore-Willse AR, Gregor P and Corn®eld LJ. (1998). 9783. Biochem. Biophys. Res. Commun., 243, 474 ± 479. Heasley LE, Zamarripa J, Storey B, Helfrich B, Mitchell Buhl AM, Johnson NL, Dhanasekaran N and Johnson GL. FM, Bunn Jr PA and Johnson GL. (1996). J. Biol. Chem., (1995). J. Biol. Chem., 270, 24631 ± 24634. 271, 349 ± 354. ChanAM,FlemingTP,McGovernES,ChedidM,MikiT Hepler JR and Gilman AG. (1992). Trends. Biochem. Sci., and Aaronson SA. (1993). Mol. Cell. Biol., 13, 762 ± 768. 17, 383 ± 387. Clapham DE and Neer EJ. (1997). Annu. Rev. Pharmacol. Herrlich A, Kuhn B, Grosse R, Schmid A, Schultz G and Toxicol., 37, 167 ± 203. Gudermann T. (1996). J. Biol. Chem., 271, 16764 ± 16772. Dhanasekaran N, Heasley LE and Johnson GL. (1995). JarpeMB,KnallC,MitchellFM,BuhlAM,DuzicEand Endocr. Rev., 16, 259 ± 270. Johnson GL. (1998). J. Biol. Chem., 273, 3097 ± 3104. Dhanasekaran N, Tsim ST, Dermott JM and Onesime D. Kalkbrenner F, Degtiar VE, Schenker M, Brendel S, Zobel (1998). Oncogene, 17, 1383 ± 1394. A, Heschler J, Wittig B and Schultz G. (1995). EMBO. J., Eguchi S, Iwasaki H, Inagami T, Numaguchi K, Yamakawa 14, 4728 ± 4737. T, Motley ED, Owada KM, Marumo F and Hirata Y. Kao JPY. (1994). A Practical Guide to the Study of Calcium in (1999). Hypertension, 33, 201 ± 206. Living Cells, Vol. 40: Methods in Cell Biology. Nuccitelli Eguchi S, Numaguchi K, Iwasaki H, Matsumoto T, R. (ed.). Academic Press Inc.: San Diego, pp. 155 ± 181. Yamakawa T, Utsunomiya H, Motley ED, Kawakatsu Kask K, Langel U and Bartfai T. (1995). Cell Mol. H, Owada KM, Hirata Y, Marumo F and Inagami T. Neurobiol., 15, 653 ± 673. (1998). J. Biol. Chem., 273, 8890 ± 8896.

Oncogene Galanin signal transduction in SCLC cells N Wittau et al 4209 Komatsuzaki K, Murayama Y, Giambarella U, Ogata E, SchoÈ neberg T, Schultz G and Gudermann T. (1999). Mol. SeinoSandNishimotoI.(1997).FEBS. Lett., 406, 165 ± Cell. Endocrinol., 151, 181 ± 193. 170. Sethi T, Langdon S, Smyth J and Rozengurt E. (1992). Kranenburg O, Poland M, van Horck FP, Drechsel D, Hall Cancer Res., 52, 2737s ± 2742s. A and Moolenaar WH. (1999). Mol. Biol. Cell, 10, 1851 ± Sethi T and Rozengurt E. (1991a). Cancer Res., 51, 1674 ± 1857. 1679. KuÈ hn B, Schmid A, Harteneck C, Gudermann T and Schultz Sethi T and Rozengurt E. (1991b). Cancer Res., 51, 3621 ± G. (1996). Mol. Endocrinol., 10, 1697 ± 1707. 3623. Lassen U, Osterlind K, Hansen M, Dombernowsky P, Seu€erlein T and Rozengurt E. (1996). Cancer Res., 56, Bergman B and Hansen HH. (1995). J. Clin. Oncol., 13, 5758 ± 5764. 1215 ± 1220. Simon MI, Strathmann MP and Gautam N. (1991). Science, Laugwitz KL, Spicher K, Schultz G and O€ermanns S. 252, 802 ± 808. (1994). Methods Enzymol., 237, 283 ± 294. Smith KE, Forray C, Walker MW, Jones KA, Tamm JA, LevS,MorenoH,MartinezR,CanollP,PelesE,Musacchio Bard J, Branchek TA, Linemeyer DL and Gerald C. JM, Plowman GD, Rudy B and Schlessinger J. (1995). (1997). J. Biol. Chem., 272, 24612 ± 24616. Nature, 376, 737 ± 745. Smith KE, Walker MW, Artymyshyn R, Bard J, Borowsky Liu J, Conklin BR, Blin N, Yun J and Wess J. (1995). Proc. B, Tamm JA, Yao WJ, Vaysse PJ, Branchek TA, Gerald C Natl. Acad. Sci. USA., 92, 11642 ± 11646. and Jones KA. (1998). J. Biol. Chem., 273, 23321 ± 23326. Mitchell FM, Heasley LE, Qian NX, Zamarripa J and Solto€ SP. (1998). J. Biol. Chem., 273, 23110 ± 23117. Johnson GL. (1995). J. Biol. Chem., 270, 8623 ± 8628. Strader CD, Fong TM, Tota MR, Underwood D and Dixon Moody TW, Carney DN, Cuttitta F, Quattrocchi K and RA. (1994). Annu. Rev. Biochem., 63, 101 ± 132. Minna JD. (1985). Life Sci., 37, 105 ± 113. Tokman MG, Porter RA and Williams CL. (1997). Cancer O€ermanns S, Laugwitz KL, Spicher K and Schultz G. Res., 57, 1785 ± 1793. (1994). Proc. Natl. Acad. Sci. USA., 91, 504 ± 508. van Biesen T, Luttrell LM, Hawes BE and Lefkowitz RJ. O€ermanns S, Schultz G and Rosenthal W. (1991). Methods (1996). Endocr. Rev., 17, 698 ± 714. Enzymol., 195, 286 ± 301. Viallet J, Sharoni Y, Frucht H, Jensen RT, Minna JD and Olson MF, Paterson HF and Marshall CJ. (1998). Nature, Sausville EA. (1990). J. Clin. Invest., 86, 1904 ± 1912. 394, 295 ± 299. Wang S and Gustafson EL. (1998). Drug News Perspect., 11, PangL,HashemiT,LeeHJ,MaguireM,GrazianoMP, 458 ± 468. Bayne M, Hawes B, Wong G and Wang S. (1998). J. Wang S, Hashemi T, Fried S, Clemmons AL and Hawes BE. Neurochem., 71, 2252 ± 2259. (1998). Biochemistry, 37, 6711 ± 6717. Ravi RK, Weber E, McMahon M, Williams JR, Baylin S, Wang S, Hashemi T, He C, Strader C and Bayne M. (1997a). Mal A, Harter ML, Dillehay LE, Claudio PP, Giordano Mol. Pharmacol., 52, 337 ± 343. A, Nelkin BD and Mabry M. (1998). J. Clin. Invest., 101, Wang S, He C, Hashemi T and Bayne M. (1997b). J. Biol. 153 ± 159. Chem., 272, 31949 ± 31952. Ridley AJ and Hall A. (1992). Cell, 70, 389 ± 399. Wong YH. (1994). Methods Enzymol., 238, 81 ± 94. Ridley AJ and Hall A. (1994). EMBO. J., 13, 2600 ± 2610. Zohn IM, Campbell SL, Khosravi-Far R, Rossman KL and Rosen LB, Ginty DD, Weber MJ and Greenberg ME. (1994). Der CJ. (1998). Oncogene, 17, 1415 ± 1438. Neuron., 12, 1207 ± 1221. ZwickE,DaubH,AokiN,Yamaguchi-AokiY,TinhoferI, Rozengurt E. (1999). Curr. Opin. Oncol., 11, 116 ± 122. Maly K and Ullrich A. (1997). J. Biol. Chem., 272, 24767 ± Salomon Y, Londos C and Rodbell M. (1974). Anal. 24770. Biochem., 58, 541 ± 548.

Oncogene