Proc. Nati. Acad. Sci. USAVol. 90, pp. 8717-8721, September 1993Neurobiology

Chimeric tumor necrosis factor-TrkA receptors reveal thatligand-dependent activation of the TrkA tyrosine kinase issufficient for differentiation and survival of PC12 cellsGIoRGIo ROVELLI*, RENU A. HELLERt, MARCO CANOSSA*, AND ERic M. SHOOTER**Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305-5401; and tInstitute of Biochemistry and Cell Biology, SyntexDiscovery Research, Palo Alto, CA 94304

Communicated by Harden M. McConnell, June 4, 1993

ABSTRACT To elucidate the function of the two nervegrowth factor (NGF) receptors, p75NGFR and pl40*, chimericmoecules were constructed oftumor necrosis factor (TNF) andNGF receptors. Rat PC12 pheochromocytoma cels tnentlytransfcd with TNF-p140r chimeras, which conain theextracellular domain of TNF receptor and the ransmembraneand cytoplasmic domains of pl40t, showed TNF-dependentneuronal differentiation and cell ival. The activity ofTNF-pl4HO chimeras was completely blocked by the tyrosinekinase inhibitor K252a, and TNF was unable to induce neuriteelonation In PC12 cells tansfected with a tyrosine kinase-defective chimeric receptor. The TNF-p75NGR chimeras,which contain the cytoplasic domain of p75NGFR, were non-functinal. Our results suggest that pl40" may function aslgand-activated homodimers and that lgand-mediated activa-tion of the cytoplasmic domain of pl40 alone b sfcient forinducing a neuronal phenotpe.

Nerve growth factor (NGF) plays a role in determining thesurvival of specific neuronal populations in the central andperipheral nervous systems during development and in es-tablishing and maintaining their differentiated phenotype (1).Two classes of NGF receptors, of low and high affinity,respectively, were identified pharmacologically (2). In chickembryonic sensory neurons, dose-response experimentssuggest that only the high-affinity receptors are required tomediate NGF's biological actions (2, 3). Two NGF receptorshave now been cloned, p75NGm, a 75-kDa protein with asingle transmembrane domain and unknown signal transduc-tion mechanism (4, 5), and p140t&, a 140-kDa tyrosine kinasereceptor whose activity and autophosphorylation on tyrosineresidues are stimulated byNGF binding (6, 7). p75NGFR is thelow-affinity receptor defined pharmacologically (5), whilep140t exhibits properties characteristic ofhigh-affinity NGFbinding (7-9).

It has been suggested, however, on the basis of bindingexperiments (10, 11), that the high-affinity NGF receptorcombines p140r and p75NGFR. This view is supported bytransfection studies, in which introduction of p75NGPR re-stores high-affinity binding in mutant cells lacking this proteinand also allows NGF-induced protein phosphorylation (12-14). On the other hand, attempts at crosslinking the tworeceptors have so far failed (15), and they do not coimmu-noprecipitate (9, 16). Furthermore, in heterologous systems,p140* can promote cell growth in the absence of p75NGPRand it does so at picomolar concentrations of NGF (17-19).We have now extended this analysis to show that the

p140t tyrosine kinase domain can, by itself, modulate neu-rite outgrowth in a neuronal cell line. To do this we haveconstructed chimeric receptors having the ligand-binding

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

domain of a tumor necrosis factor (TNF) receptor-i.e.,p55ThFR or p70TNh (20-22)-attached to the transmembraneand cytoplasmic domains of p140k. Because of the homol-ogy in their binding domains (20, 21), similar constructs havebeen made between the TNF receptors and p75NGFR.

MATERIALS AND METHODSAntibodies. The anti-p70TNm' antiserum was raised against

a fragment of the extracellular domain expressed as a bac-terial fusion protein. The antisera against the carboxyl ter-minus of p140t and p75NGFR were generous gifts of D.Kaplan (National Cancer Institute-Frederick Cancer Re-search Center) and P. Barker (Stanford University).DNA Cloning and Construction of the Chineric Receptors.

Specific complementary oligonucleotides containing eitherthe first 17 coding nucleotides for theTNFreceptor or the last17 coding nucleotides for NGF receptor and a Xho I restric-tion site were synthesized. The hinge regions ofthe chimerasbearing the extracellular domain of TNF receptor and thetransmembrane and cytoplasmic domain of NGF receptorwere made by using sense and the corresponding antisenseoligonucleotides containing both TNF and NGF receptorcomplementary sequences. The TNF and NGF receptorchimeras were generated by a two-step polymerase chainreaction (PCR) essentially as previously described (23). Theresulting chimeras were cloned in the appropriate orientationof the mammalian expression vector pCDM8. Chimera p70-trkLK * RI, where Lys-547 is replaced by an arginine residue,was made by PCR-based overlap extension mutagenesis. Thecytoplasmic domain of pl40r (i.e., trkcD) was made by asingle PCR; the sense oligonucleotide contained a newlyintroduced translation initiation codon and the first 18 nu-cleotides of the putative cytoplasmic domain of pl40trk. Thesequences of the entire coding regions duplicated by PCRwere verified by automated dideoxynucleotide DNA se-quencing.Receptor Binding Assay. Binding of human recombinant

1251-labeled TNF (125I-TNF) (DuPont) to intact cells wasassayed as described by Heller et aL (21).

Transient Expression In COS 7 Cells. Transfection, immu-noprecipitation, and detection ofthe chimeric receptors wereperformed as described (19, 24).

Bioogical Assays. Electroporation. Rat PC12 pheochromo-cytoma cells were electroporated (17), replated on a 150-mmdish, and incubated for 24 hr in medium supplemented withserum.Neurite outgrowth. Viable transfected cells were seeded at

a density of 1 x 104 cells per well in 24-well plates or 1.5 x105 cells in 35-mm plastic dishes. One day later, cells werewashed twice with serum-free medium and exposed to either

Abbreviations: NGF, nerve growth factor; TNF, tumor necrosisfactor.

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BTNF NGF

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Fio. 1. Human TNF does not affect gene expression in PC12 cells. Northern blot analysis of total RNA isolated from cells, exposed to eitherTNF orNGF (100 ng/ml) for the indicated times (min). Blots were hybridized with cDNA for c-fos (A) or c-jun (B). (C) RNA was isolated fromPC12 cells cultured for 48 hr in medium containing either TNF or NGF (100 ng/ml), and a blot was prepared and hybridized with a probe fortransin. (D) Specific binding of 125I-TNF to naive PC12 cells or HeLa cells.

TNF (Genzyme) or NGF (Bioproducts for Science, India-napolis).

Cell survival. Viable transfected cells were replated in96-well plates (1.5 x 104 cells per well). One day later, wellswere washed twice with serum-free medium and incubated for72 hr in the presence of 1 pM cytosine (3-D-arabinonucleoside(to eliminate dividing cells) in serum-free medium or mediumsupplemented with either TNF or NGF. Percentages of viablecells were determined by using a Promega CellTiter kit.

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FIG. 2. Representation of TNF and NGF chimeras. The trans-membrane region is represented by a vertical bar. The extracellulardomains of p55ThfR and p70NR are represented by open andstippled bars, respectively; vertical and diagonal stripes correspondto the cytoplasmic domains of p75NGFR and p1404, respectively.The position of Lys-547, mutated to an arginine residue, is markedby a vertical bar.

RNA Analysis. RNA isolation, electrophoresis, and transferand cDNA-mRNA hybridizations were performed using stan-dard techniques. 32P-labeled c-fos and c-jun cDNAs weremade by using a random primer labeling kit (Boehringer

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FIG. 3. Receptor protein expression. (A) Transfected COS 7 cellswere solubijized, receptors were immunoprecipitated with antibod-ies against pl4rk, and chimeras were resolved by SDS/PAGE in11% gels. Western blots were developed by using antibodies againstp7OTfNl (lanes a-c) or p140t (lanes d-g). Lanes a and d, vectorpCDM8 alone; lane b, p70-trk; lane c, p70-trk[K - RI; lane e, p55-trk;lane f, p55D'trk; and lane g, trkcD. The band at 50 kDa is theimmunoglobulin heavy chain. (B) Western blot of crude membranepreparations of transfected COS 7. Chimeras were detected onimmunoblots after SDS/PAGE in 11% gels using an antibody di-rected against the carboxyl terninus of p75NGFR. Lane a, vectorpCDM8 alone; lane b, p70-NGER; lane c, p70'-NGFR; and lane d,p55-NGFR.

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Mannheim). Digoxigenin-UTP- (Boehringer Mannheim) la-beled transin was generated by using a Promega Riboprobe kit.

RESULTSLack of Effect ofTNF on PC12 Cells. Human TNF does not

induce cell death in rat PC12 cells nor does it modify theoutgrowth ofneurites promoted by NGF (results not shown).Consistently, exposure of naive PC12 cells to TNF does notincrease mRNA levels of the protooncogenes c-fos and c-jun(Fig. 1 A and B). Also, TNF is unable to induce the transinprotease gene (Fig. 1C), which accompanies NGF-inducedneuronal differentiation in PC12 cells (25). The binding ofm'5I-TNF to PC12 cells is about 1/12 of that to HeLa cells(Fig. 1D), and no crosslinking of 125I-TNF to any membranecomponents of PC12 cells was observed (S. 0. Meakin,personal communication).

Expression of the TNF andNGF Chimeric Receptor ProteinsInCOS 7 Cells. The chimeric receptors (Fig. 2) were built withthe extracellular domain of either p55TNPR or p70TNFR, fusedto the transmembrane and cytoplasmic domains of p75NGFR(chimeras p55-NGFR and p70-NGFR) or p140" (chimerasp55-trk and p70-trk). Chimera p55rm-trk was made from

A ! B _

p55-trk by replacing the transmembrane domain of pl40'with the corresponding region of the p55TNFR. Chimerap70-trkK RI was derived from p70-trk by replacing Lys-547 byan arginine residue. Chimera p70A-NGFR was derived fromp70-NGFR by deleting from this region the 26 residues mostimmediate to the transmembrane domain of p70TNFR andreplacing them with the corresponding region of p75NGFR.Chimeras p70-trk or p70-trk[K- RI and p55-trk or p55Th-trkhave apparent molecular masses of 70 and 65 kDa, respec-tively, and the cytoplasmic domain of pl40k, trkCD, has amass of 40 kDa (Fig. 3A). Chimeras p70-NGFR (or p70O-NGFR) and p55-NGFR have molecular masses of approxi-mately 55 and 46 kDa, respectively (Fig. 3B). Ligand-bindingactivity of these receptors was demonstrated by flow cyto-metric analysis using a fluoresceinated derivative of TNF(results not shown).

p70-trk Induces Neuronal Differentiation and Survival inPC12 Cells. Since PC12 cells have been extensively used toanalyze the biological effects of NGF, we have used them asa model system to study neurite outgrowth and cell survivalaction of the chimeric receptors. TNF did not induce anyneurite outgrowth in cells transfected with the pCDM8 vectoralone (Fig. 4 A-C). In contrast, TNF promoted a pronounced

FIG. 4. TNF induces neurite outgrowth in p70-trk transfectants. Transfected PC12 cells were seeded on plastic dishes coated with collagenand cultured in serum-free medium supplemented with TNF at 50 ng/ml (A-F) or NGF at 50 ng/ml (G-1). (A-C) PC12 cells transfected withvector pCDM8 alone. (D-F) Cells transfected with chimera p70-trk. (G-1) Wild-type PC12 cells. Representative fields were photographed at12 hr (A, D, and G), 24 hr (B, E, and H), and 48 hr (C, F, and I) of incubation with the growth factor. (x40.)

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neurite outgrowth in PC12 cells transfected with chimerap70-trk (Fig. 4 D-F). After 12 hr of exposure to TNF, somecells already had short processes; after 24 hr, long neuritesmeasuring a few cell diameters were observed; the fibersreached their maximal length after 2 days of treatment. Theneurites induced by TNF were considerably longer andthicker than the fibers observed in control cells treated withNGF alone (Fig. 4 G-).TNF induced neurite outgrowth in p70-trk transfectants

with an ED50 4.5 ng/ml. In contrast, TNF concentrationsas high as 100 ng/ml did not mediate outgrowth when PC12cells were transfected with chimeras p70-trk[K - RI or p70-NGFR (Fig. 5A) with full-length pl4otrk, p70TNER, orp55TNFR, or with chimeras p55-NGFR orp70A-NGFR (resultsnot shown). The tyrosine kinase inhibitor K252a completelyblocked the TNF-mediated neurite outgrowth in p70-trktransfectants (Fig. SC). The EDso for K252a of25-50nM wasvery similar to that previously reported with the p140Oreceptor (26).The ability ofTNF to mimic the survival effect of NGF in

PC12 cells expressing chimera p70-trk was tested by using acell survival assay (Fig. SD). Transfected cells were culturedfor 3 days in serum-free medium or in medium supplementedwith eitherTNF or NGF; the number of cells surviving in thepresence of NGF was set as 100%. Without growth factor,about 42%, 30%o, and 37% of the cells transfected withchimera p70-trk, p70-NGFR, and vectorpCDM8 were viable,

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FIG. 5. Chimeras p55-trk and p70-trk mediate neurite outgrowthand celi survival. (A and B) Neurite outgrowth assays. TransfectedPC12 cells were seeded on plastic wells coated with poly(L-ornithine)and exposed to increasing concentrations of TNF in serum-freemedium; 2 days later, cells were scored for neurite outgrowth. (A) e,

Chimera p70-trk; A, chimera p70-trk[K - RI; C, chimera p70-NGFR.(B) *, Chimera pSS-trk; a, chimera p55"t-trk; o, trkCD. (C) K252ablocks the TNF-dependent neurite outgrowth activity. p70-trk trans-fectants were exposed to TNF (50 ng/ml) and to increasing amountsof K252a (0-200 nM) in serum-free conditions. The number of cellsbearing neurites was scored 2 days later; 100%! reflects the numberofcells with neurites in the absence ofK252a. (D) Cell survival assay.PC12 cells transfected with chimeras p70-NGFR, p55-trk, p70-trk, orvector pCDM8 were cultured in serum-free medium (open bars) ormedium supplemented with TNF (50 ng/ml) (stippled bars); 3 dayslater, viable cells were scored by using a colorimetric assay. Survivalvalues obtained in presence of NGF (50 ng/ml) were set as 100%.

respectively. In the presence of TNF, about 80% of thep70-trk transfectants were viable, whereas no increase in cellsurvival was observed in p70-NGFR (29%) or vector pCDM8transfectants (35%).

pSS-trk Is Less Efficient Than p70-trk in Transducing theNGF Activity. Expression of the cytoplasmic domain ofp140" (i.e., trkcD) alone was unable to induce neuriteoutgrowth in PC12 cells (Fig. 5B). Since two TNF receptorshave been isolated, we wanted to compare the effect ofheterologous ligand-binding domains on the functionality ofp140" chimeras. As expected, both chimera pSS-trk andchimera p55mT-trk were able to mimic the biological activityofp70-trk; however, the number of cells bearing neurites wasreduced to about 1/3. The ED50 values for chimeras pS5-trkand p55Tm-trk, approximately 15.0 and 17.5 ng/mi, respec-tively, were about 4-fold higher than the ED50 for the corre-sponding p70-trk chimera. In agreement with these results,TNF caused only an 18% increase in viability of pSS-trktransfectants as compared with the 38% increase mediated incells transfected with p70-trk (Fig. SD).

DISCUSSIONTNF is a cytokine that can act as a morphogen or as a growthfactor, but no neurite outgrowth or survival effects have beenascribed to it. We built chimeric receptors between thehuman TNF and the rat NGF receptors by replacing theextracellular regions (ligand-binding domains) of pl40" andp75NGFR with the corresponding regions of the TNF recep-tors. All the functional assays were carried out with cellstransiently expressing the chimeras (27), which allowed con-venient testing of a large number of constructs in a hetero-geneous population.The cytoplasmic domain of wild-type p70TNFR does not

contain consensus sequences for a protein tyrosine kinasedomain. However, our findings indicate that the extracellulardomain of p70TNh is able to activate the cytoplasmic domainofp140t1rk and that the TNF-induced neurite outgrowth and cellsurvival are a function of tyrosine phosphorylation ofchimerap70-trk. The tyrosine kinase inhibitor K252a (26) can com-pletely block the morphological changes induced by TNF inp70-trk transfectants, and TNF failed to produce the corre-sponding morphological changes in cells transfected withchimera p70trk[K - RI, in which Lys-547, considered to beessential for ATP binding (18), is mutated to arginine. Con-sistent with these results, TNF was unable to induce neuriteoutgrowth in cells transfected with full-length p55TNFR,p70oTN, p140", or p75NGFR. Expression of the cytoplasmicdomain ofp140tk (trkcD) alone or in the presence ofTNF wasalso totally ineffective in mediating neurite extension.Chimera p55-trk was not as efficient as p70-trk in trans-

ducing the activity of NGF, suggesting that the oligomeriza-tion signal given by TNF is transduced differentially by theextracellular domains ofp55TNFR and p70TmR. Interestingly,the 4-fold difference in the ED50 observed in the neuriteoutgrowth assay is of the same order of magnitude as thedifference reported for TNF binding to p55S and p70TNFR(28).The chimeras having the extracellular domain of a TNF

receptor fused to the transmembrane and cytoplasmic do-mains of p75NGFR were nonfunctional in our biological as-says. Unfortunately, it is difficult at present to measurep75NGFR activity and, thus, to assess the functionality ofthese chimeras, since little is known about the signalingpathways for p75NGFR or genes that are induced specificallyby activation of p75NGFR to serve as reporters for NGFaction. Stable PC12 cell transfectants, constitutively express-ing either p55-NGFR or p70-NGFR chimeras, may be usefulin this regard.

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Altogether, these results show that activation ofthe p140rktyrosine kinase signaling pathway is sufficient to induce aneuronal phenotype. The conclusion that p75NGFR is notrequired for either survival or neurite outgrowth from PC12cells agrees with previous data in which a NGF mutant thatfailed to bind p75NGFR still bound to p140r and induceddifferentiation and survival of these cells (29). Also, whilehomozygous mice lacking p75NGFR show sensory deficitsthey have normal sympathetic ganglia consistent with normalNGF-mediated development (30).

G.R. is indebted to Dr. Andrew Welcher for his technical advice.The authors are grateful to Dr. Philip Barker and Dr. Jack Snipes forcritical reading of this manuscript, to Kyung Song for her assistance,to Dr. John Dunne, Laura Chiu, and Forest Gray for help with cellsorting, and to Agnes Rovelli for her artwork. This research wassupported by grants from the National Institutes of Health(NS04270), the American Cancer Society (BE-47K), the Alzheimer'sAssociation (IIRG-92-138), and the Syntex Discovery ResearchInstitute of Biochemistry and Cell Biology. G.R. was supported bythe Swiss National Science Foundation and the Swiss Academy forMedical Science. M.C. was supported by the Fondazione MarinoGolinelli.

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