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. Photoanity 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 eect 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 sucient 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 eects 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 eector systems chemosensitivity (Lassen et al., 1995). Therefore, such as enzymes and ion channels (Birnbaumer et al., considerable eort 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 dierentiation, 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 eect 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 eects 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 aect 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 photoanity 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 eect 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 (Seuerlein and Rozengurt, 1996). doanilide. After UV cross-linking of the photolabel, G- The fact that galanin initiates an entirely dierent 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 eects 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 dier in their ligand reproducible agonist-induced photo anity 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.