Neuron,

Vol. 9, 1067-1079, December,

1992, Copyright

0 1992 by Cell Press

Nerve G rowth Factor Mediates Signal Transduction through trk Homodimer Receptors Shuqian jing, Peter Tapley, and Mariano Barbacid Department of Molecular Biology Bristol-Myers Squibb Pharmaceutical Research Institute Princeton, New Jersey 08543-4000

Summary

We have investigated the molecular nature of the high affinity nerve growth factor (NCF) receptors by using cell lines expressing gp751NGFRand gp140th. Our results suggest that gp751NGFRand gp140’& interact with NGF independently and that only gp140’* mediates NGF signaling. NCF binds to gp140’& with picomolar affinity and induces its phosphorylation on tyrosine residues regardless of the presence of gp751NGFR.NGF-gplrlO’& complexes display the slow dissociation rate and rapid internalization characteristics of high affinity NGF receptors. Cross-linking studies reveal the existence of gp751NCF’and gp140td homodimers. However, we were unable to detect gp75LNCFR-gp140”C heterodimers. Coexpression in COS cells of wild-type and kinase deficient mutants reveals that gp140’* receptors can undergo intermolecular phosphorylation, indicating the formation of functional homodimers. Moreover, these kinase deficient mutants inhibit NGF-induced signaling through wild-type gp140”’ receptors. These results indicate that the functional high affinity NGF receptors consist of gpl 40fd homodimeric (or oligomeric) complexes. Introduction The nerve growth factor (NGF) family of neurotrophinsthat includes NGF, brainderived neurotrophicfactor (BDNF), neurotrophin3 (NT-3), and NT-4/5 is thought to play a central role in the development, growth, and maintenance of the nervous system. These neurotrophins recognizetwoclassesof cell surface receptors that can be identified by their distinct low(&=lnM)andhigh(&=lOpM)bindingaffinities. It is generally accepted that only the high affinity receptors mediate the biological properties of the NGF family of neurotrophins. The low affinity NGF receptors (LNGFR) have been identified as 75 kd glycoproteins, designated as gp751NCFR(Johnson et al., 1986; Radeke et al., 1987), with limited structural homology to the tumor necrosis factor receptors I and II (Loetscher et al., 1990; Schall et al., 1990; Smith et al., 1990); the lymphocyte surface antigens CD30 (Diirkop et al., 1992), CD40 (Stamenkovic et al., 1989), and OX40 (Mallett et al., 1990); and the apoptosis-mediating Fas cell surface antigen (Itoh et al., 1991). In addition, gp751NCFR recognizes each of the known members of this neurotrophin family with similar nanomolar affinities (Rodriguez-TCbar et al., 1990, 1992; Ernfors et al., 1990;

Hallb86k et al., 1991). gp751NCFRreceptors have been identified in cells of neuronal origin, including a limited subset of neurons. However, most of these receptors are expressed in a wide variety of nonneuronal cells, not known to respond to NCF (Bothwell, 1990). This lack of correlation between receptor expression and responsiveness to their cognate ligands has also been observed with the related tumor necrosis factor receptors (Tsujimoto et al., 1985). The molecular nature of the functional high affinity receptors remains to be elucidated. Recent studies haveshown thattheNGFfamilyof neurotrophinsalso binds to the trk family of tyrosine kinase receptors (reviewed in Barbacid et al., 1991; Bothwell, 1991; Ragsdale and Woodgett, 1991; Barbacid, 1993). Unlike the trk receptors interact with these neurogp75 LNGFR, trophins in a highly specific manner. For instance, whereas NGF only binds to the trk proto-oncogene product gp+MYk (Hempstead et al., 1991; Kaplan et al., 1991a, 1991b; Klein et al., 1991a), BDNF exclusively recognizes those receptors encoded by the related trkB locus (Klein et al., 1991b; Soppet et al., 1991; Squint0 et al., 1991). Accumulating evidence indicates that the trk receptors participate in the signaling processes initiated by the NGF family of neurotrophins. NGF induces a strong mitogenic response in mouse fibroblasts and mediates the meiotic maturation of Xenopus oocytes when these cells express gp140trk receptors (Cordon-Card0 et al., 1991; Nebreda et al., 1991). Moreover, transfection of cDNA clones encoding gp140frk receptors can restore NGF responsiveness in mutant PC12 cells resistant to NGF (Loeb et al., 1991). Similar results have been obtained when BDNF and NT-3 interact with their respective gp145*‘k* and gp145’rkC receptors (Glass et al., 1991; Klein et al., 1991 b, 1992; Squint0 et al., 1991; Lamballe et al., 1991). The relative contribution of the trk tyrosine kinases to the high affinity receptors for these neurotrophins remains to be established. A widely accepted model proposes that such high affinity receptors are heteromerit complexes of gp7SLNCFRand the various trk receptors (Bothwell, 1991). This model is primarily based on [1251]NGF binding studies in which COS cell membranes containing gp140trk receptors alone bind NGF with low affinity (Kaplan et al., 1991b; Hempstead et al., 1991). However, when these membranes were fused to those containing gp751NCFR,a small percentage of high affinity NGF binding sites indistinguishable from those present in NCF-responsive cells were detected (Hempstead et al., 1991). These results ate consistent with previous gene transfer studies in which transfection of NGF-nonresponsive cells with led to the generation of the gene encoding gp75 LNCFR high affinity NCF receptors and rendered these cells at least partially responsive to NGF (Hempstead et al., 1989,199O; Matsushima and Bogenmann, 1990; Pleasure et al., 1990).

Neuron 1068

Emerging evidence, however, argues against the concept that gp7SLNCFRis an integral part of the functional receptors for the NGF family of neurotrophins. We have previously reported that NIH 3T3 cells expressing gp140tik, but not gp75LNCFR,receptors bind [i251]NGF with the high affinity characteristic of its functional receptors (Klein et al., 1991a). Similarly, Meakin et al. (1992) have shown that NGF-gp140wk complexes exhibit the slow dissociation kinetics characteristic of NGF high affinity receptors. What is more important,gp140”kaswelI as thegp145tfkBand gpl4Srkc receptors can mediate neurotrophin-induced signal transduction in the absence of gp75LNCFR(CordonCardo et al., 1991; Glass et al., 1991; Klein et al., 1991b, 1992; Lamballe et al., 1991; Nebreda et al., 1991; Squint0 et al., 1991). Likewise, inhibition of NGF bindin either PC12 cells or peripheral sening to gp75 LNCFR sory neurons by specific monoclonal antibodies did not eliminate the high affinity NGF receptors or the responsiveness of these cells to NGF (Weskamp and Reichardt, 1991). More recently, Ib&iez et al. (1992) have shown that a mutant NCF molecule that binds to gp140Vk, but not to gp75LNCFR,retains most of its biological activity (IbBriez et al., 1992). In the present studies, we provideexperimental evidence that argues against the proposed heterodimerit gp75 LNGFR-gp140”k high affinity NGF receptor model. Instead, our results support the concept that NGF may mediate signal transduction through theformation of gp140 *rk homodimers (or oligomers) in a fashion highly reminiscent of other members of the tyrosine kinase family of cell surface receptors.

A a

B b

a

C b

a

D b

a

b

MW

w. - 200K gpl4O’h ---) b -97K gp7S LNQFR+ - 69K

- 46K

Figure 1. Expression NIH 3T3 Cells

of gp751NGFRand gp140”

NCF Receptors

in

(A) NIH 3T3cellsexpressing(B)gp75LNGFR(Z91-38cells), (C)gp140” (E2542 cells) or(D) gp75LNCFR,and gp140@k (R7-42) NGF receptors were metabolically labeled with [?3]methioninelcysteine as indicated in Experimental Procedures, lysed, normalized for incorporation of 35.Slabel, and immunoprecipitated with antisera specific for (a) gp140” (anti-trk COOH) or (b) gp75LNCFR.The resulting immunoprecipitates were fractionated by 8% SDS-PAGE, and the gel was subjected to fluorography, dried, and exposed to KodakX-OMATfilmat-70Vfor 18 hrwith the helpofan intensifying screen. The migration of the gp751NCfR and gp+W recep tors is indicated by arrows. The open arrowhead indicates the migration of a partially glycosylated precursor of gp14W (MartinZanca et al., 1989). Coelectrophoresed molecularweight markers included myosin (200,000), phosphorylase b (97,000), bovine serum albumin (69,000), and ovalbumin (46,000).

Results Contribution of gp75LNCFuand gpl40’& to the High Affinity NGF Receptors To assess the relative contribution of gp75LNCFRto the high affinity NGF receptors, we generated NIH 3T3 cell lines expressing either gp75LNGFR(291-38 cells) and gp+KPk (E25-42 cells) receptors alone or in combination (R7-42 cells). As illustrated in Figure 1, each of these cell lines expresses similar levels of the corresponding NGF receptors. Table 1 summarizes the dissociation constants and number of receptor sites in these cell lines as determined by Scatchard plot analysis of our [1251]NGF binding assays to monolayer cultures Uing et al., 1990). 291-38 cells express about 50,000 gp75LNCFRreceptors with the expected low affinity in the nanomolar range (b = 1.20 f 0.50 nM) (Table 1; Figure 2A). In contrast, the gp140”rk-expressing E25-42 cells possess two classes of dGF-binding sites (Figure 2B). About 2,000 of these NGF-binding sites exhibit high affinity in the low picomolar range (16 = 8.4 f 1.2 PM). The other NGF-binding sites (1.1 f 0.1 x IO5 sites per cell) have a significantly lower dissociation constant (Kd = 0.71 f 0.39 nM), about 2-fold higher than that observed for the gp75LNCFRreceptors (& = 1.20 f 0.50 nM) (Table 1). These results are in agreement with our

previous observations using two independent NIH 3T3 cell lines that expressed higher levels of gp14Urk receptors (Klein et al., 1991a). Coexpression of LNCFRand gp140trk receptors in R7-42 cells resulted gp75 in high (b = 7.6 f 2.0 pM) and low (b = 0.59 + 0.14 nM) affinity NGF-binding sites similar to those found in gpl4Otrk-expressing E25-42 cells (Table 1; Figures 28 and 2C). In control experiments, NGF binding to PC12 cells yielded the expected high (& = 8.4 f 4.6 pM; 1.1 f 0.5 x IO3 sites per cell) and low (Kd = 0.79 f 0.46 nM; 0.4 f 0.1 x 105 sites per cell) affinity NGF receptors (Table 1; Figure 2D). Similar results were obtained in preliminaryexperiments using commercial (Amersham) immobilized receptor assays that do not require separation of bound from unbound ligand (see Experimental Procedures). Whereas the 291-38 cells (gp75LNCFRexpressors) depicted a single class of low affinity binding sites (& = 0.55 nM), E25-42 cells (gp140*” expressors) revealed two classes of NGF-binding sites with high (b = 5.0 pM) and low (Kd = 0.38 nM) affinities. What is more important, the Kd of these high affinity binding sites was not significantly affected (b = 4.2 pM) by the presence of gp75LNCFRreceptors in the dual-expressor R7-42 cells. These results indicate that gp140rrk binds NGF with the picomolar dissociation constant ex-

trk Homodimers 1069

Mediate

Table 1. NCF Binding

NCF Signal Transduction

to Cells Expressing

gp75LNCFRand gpl4W

Receptors

High Affinity

Low Affinity

Receptors

Cell Line

NGF Receptor

Number of Experiments

Kd (PM)

Sites per Cell (x10-‘)

Kd (nM)

291-38 E25-42 R7-42 PC12

gp75LNG’R gp140’” gp75LNCFR + gp+W gp75LNCFR + gpl4W

4 3 4 4

a.4 + 1.2 7.6 f 2.0 8.4 f 4.6

1.7 f 0.8 1.7 * 1.0 1.1 * 0.5

1.20 0.71 0.59 0.79

Summary of the Scatchard the LICAND program.

plot analysis

of [l”l]NGF

binding

to cell lines expressing

petted for the functional high affinity receptors identified in NCF-responsive cells. Moreover, the high affinity binding of NGF to gp140rlk is not affected by coexpression of gp751NGFRreceptors. Dissociation Kinetics Functional NGF receptors have also been characterized by their slow dissociation rate of bound [1251]NCF (Sutter et al., 1979). As shown in Figure 3, gp751NCFRexpressing 291-38 cells release bound [1251]NGFwith a half-dissociation time (td of about 5 min (5.7 + 1.7 min), whereas the gpl4O@k-containing E25-42 cells exhibited a tla of over 30 min (31.7 f 7.7 min). The R7-42 cells, which express both receptors, exhibit two phase kinetics with an initial fast rate of dissociation (presumably due to the gp751NCFRreceptors) followed by a slower rate (presumably due to the NCF-gp140rrk complexes) that closely resembles that of PC12 cells (Figure 3). More importantly, their slow dissociation phase parallels that of E25-42 cells (Figure 3). These results suggest that coexpression of gp751NCFRrecep-

A

0.05 P

0

B

Trk

D

PC12 0.06

L!!

.

.

0

NGF BOUND (fmoles/l

2

05CELLS)

* * f f

0.50 0.39 0.14 0.46

gp75LNGFRand gp1W”

0.5 1.1 1.2 0.4 receptors

It f f f

0.1 0.1 0.1 0.1

with the help of

of the

Receptor Internalization It is generally accepted that initiation of NGF signaling is accompanied by ligand internalization (Green et al., 1986; Hosang and Shooter, 1987). Both gp140rrk (Cordon-Card0 et al., 1991; Kaplan et al., 1991a, 1991b; Klein et al., 1991a; Loeb et al., 1991) and gp75LNCFR (Hempstead et al., 1989, 1990; Pleasure et al., 1990; Berg et al., 1991; Yan et al., 1991) have been implicated in mediating NGF signal transduction. Whereas endocytosis of gp751NCFRhas not been detected (Green et al., 1986; Hosang and Shooter, 1987), there is no information regarding internalization of gp140trk receptors. In the present studies, we have examined the internalization response of both of these receptors upon NGF binding to NIH 3T3 and PC12 cells. As shown in Figure 4, NIH 3T3 cells ectopically expressing gp140”rk receptors (E25-42 cells) internalize [1251]NGFwithin seconds upon shifting from 4°C to 37OC. Moreover, over 50%

0.3

0.03

Sites per CelT (x10-5)

tors does not affect the slow rate of dissociation NGF-gp140trk complexes.

.

LNGFR

Receptors

4

. 6

Figure 2. Scatchard Plot Analysis of the Equilibrium Binding of rXl]NCF to gp75LNCrR and gp14W Receptors Expressed in NIH 3T3 and PC12 Cells Binding of [‘ZsI]NCF to (A) NIH 3T3 cells expressing gp7SLNGFR (291-38 cells), (B) NIH 3T3 cells expressing gp140”k (E25-42 cells), (C) NIH 3T3 cells coexpressing gp751NGFR and gp1401* (R7-42 cells), and (D) PC12 cells was carried out as described in Experimental Procedures. Duplicate determinations were made for each point. Nonspecific binding (lo%-25% of the total [1251]NCF bound) was measured by addition of 500 nM unlabeled NGF and was subtracted in all cases. The data were analyzed and transformed into Scatchard plots by using the LIGAND program. Binding data obtained with low concentrations of [‘2SI]NCF are expanded in the inserts.

NeUrOn 1070

60

0

20

40

AA “V 10”

60

TIME (min)

0

4

A

3

u

10

4 ” ii

PClPnnr5 n n

0 0

30

40

20

NIH3T3

Figure 3. Dissociation Kinetics of the [‘V]NGF Bound to PC12 Cells and NIH 3T3 Cells Expressing gp75LNGFRand gp14W Receptors

Figure 4. Internalization

Monolayers of NIH 3T3 cells expressing gp751NGFR(291-36 cells) (open circles), NIH 3T3 cells expressing gp+W (E25-42 cells) (closed circles), NIH 3T3cellscoexpressinggp7YNCFRand gp+Wk (R7-42 cells) (open triangles), and PC12 cells (closed triangles) were incubated with 2 nM [‘V]NGF to equilibrium at 4°C for 2 hr. Dissociation of cell bound [‘ZSI]NGF was determined as indicated in Experimental Procedures. Duplicate determinations were made at each time point. The data are expressed as the percentage of the initial specific binding.

NIH 3T3 cells (open squares), NIH 3T3 cells expressing gp751NCFR (291-38) (open triangles), NIH 3T3 cells expressing gp140’” (E2542) (closed triangles), PC12 cells (closed circles), and mutant PC12nnr5 cells (open circles) were incubated with 2 nM [lZ51]NCF to equilibrium at 4°C for 2 hr. Internalization of surface bound [lZSI]NCF was carried out at 37OC for the indicated periods of time as described in Experimental Procedures. Duplicate determinations were made at each time point. Data are expressed as the amount of radioactivity (cpm) internalized by 2 x IO5 cells.

of the total radioactivity bound to the cell surface could be localized intracellularly within IO min (data not shown). Finally, the internalization kinetics observed in these cells were indistinguishable from those of PC12 cells, a cell line known to possess functional high affinity NGF receptors (Figure 4). In contrast, thegp75LNCFR receptors expressed in the NIH 3T3-derived 291-38 cells failed to internalize significant amountsof [Y]NGF even after a 40 min incubation at 37°C. To eliminate the possibility that the was a property of NIH lack ofg’p751NCFRinternaliztition 3T3 cells, we performed ti similar experiment using PC12nnr5cells (Green et al., 1986),an NGF nonresponsive‘PC12 mutant cell line known to express gp75LNGFR but not gp140*rk receptors (Loeb et al., 1991). In agreement with previous studies (Green et al., 1986), the endogenous gp751NCFR receptors present in these PC12nnr5 cells also failed to internalize [1251]NGF(Figure 4).

cells to determine whether NGF could also be crosslinked to gp75LNGFR-gp%@ heterodimers. Incubation of R7-42 cells with the cross-linking agent in the presence of [12sl]NGF induced the efficient formation of gp75LNGFRand gp140Lrk homodimers. However, and within the limits of detection of this assay, we could not identify the presence of gp75LNGFR-gp140’rkheterodimers (Figure 5).

Cross-linking Studies The biologically active form of NGF is thought to be a stable dimer of the mature 118 amino acid long NGF polypeptide. This dimeric structure raises the possibility that NGF may bind to either receptor homodiTo test mers or to gp75 LNCFR-gp140*rkheterodimers. this possibility, we incubated 291-38 and E2542 cells with [1251]NGFin the presence of cross-linking agents. As shown in Figure 5, [1251]NGFcould be readily crosslinked to gp751NCFRand gp+MYrk homodimers in these cell lines. Similar results have been recently obtained by Meakin and Shooter (1991). Next, we used R7-42

Time (min) Kineticsof

Cell SurfaceBound

[‘“IINGF

Tyrosine Phosphorylation Binding of NGF to gp140rfk receptors induces their rapid phosphorylation on tyrosine residues (Kaplan et al., 1991a, 1991b; Klein et al., 1991a). The experiment shown in Figure 6 compares the levels of tyrosine phosphorylation of gp140’rk receptors present in E2542, R7-42, and PC12 cells when exposed to various concentrations of NGF. Increasing NGF concentrations (&IO rig/ml) effectively induced the phosphorylation of gp14Q*rk on tyrosine residues in E25-42 cells, as determined by Western blot analysis using antiphosphotyrosine antibodies (Figure 6). Interestingly, the response of gpl4Vto NGF in thedual expressor R7-42 cells was similar to that observed in E25-42 cells, thus indicating that the presence of gp75LNCFRreceptors does not enhance the activation of gp140trk receptors by NGF in these NIH 3T3-derived cell lines. What is more important, the levels of tyrosine phosphorylation of the ectopically expressed gp140”k receptors present in the E2542 and R742 cell lines were comparable to those observed in the NGF-responsive PC12 cells. These results indicate that biochemical activa-

trk Homodimers 1071

Mediate

NGF Signal Transduction

tion of gp140rrk receptors by NGF is not enhanced the presence of gp75LNCFRreceptors.

A

by

Receptor Signaling We have recently shown that NGF is a powerful mitogen for NIH 3T3 cells ectopically expressing gp140’rk receptors (Cordon-Card0 et al., 1991). Moreover, continuous activation of gp14CVrk, by either an autocrine loop or addition of exogenous NGF, results in the efficient morphologic transformation of these fibroblasts (Cordon-Card0 et al., 1991). To assess the biological significance of the interaction of NCF with gp140tik receptors in NIH 3T3 cells, we transfected these cells with a trk proto-oncogene expression plasmid (pDM69) and cultivated them in the presence of various amounts of NGF for 2 weeks. We used freshly transfected NIH 3T3 cells instead of E25-42 cells, since NIH 3T3-derived cell lines often acquire an altered phenotype that makes it difficult to score morphologic transformation. As shown in Table 2, NGF was able to elicit high levels of morphologic transformation when supplied at nanomolar concentrations. More importantly, picomolar concentrations of NGF, well within the range expected from its high affinity dissociation constant for gp14Vrk receptors, also elicited a moderate transformation response (Table 2). NCF Induces Intermolecular Phosphorylation of gpl 40hl Receptors Tyrosine protein kinase receptors are thought to become activated by a ligand-dependent oligomerization step followed bythe intermolecular phosphorylation on tyrosine residues of their respective cytoplasmic domains (Ullrich and Schlessinger, 1990). The existence of gp140tik homodimer complexes in E25-42 and R7-42 cells (Figure 5) raised the pdssibility that these structures may correspond to the functional high affinity NGF receptors. To test this hypothesis, we constructed pSJ4, a plasmid that allows efficient expression of gp140tik receptors in COS7 cells (see Experimental Procedures). Next, we generated two gp140’rk tyrosine kinase defective mutants to determine whether they could serve as substrates for the wild-type receptors. One of these plasmids (pSJ8) lacks sequences encoding 77 amino acid residues of the gp140rrk kinase domain. The second mutant plasmid (pSJ15)containsa miscoding mutation that results in the replacement of the lysine residue (KS%)of the ATP-binding site by alanine (Figure 7). These mutant plasmids also contain sequences encoding al3 amino acid long “tagging” sequence derived from the trkB TK- gp95 noncatalytic receptor (Klein et al., 1990), which can be recognized by specific polyclonal antibodies (anti-trkB TK-) (Figure 7). In pSJ8, these tagging sequences replaced its normal carboxyl terminus. In pSJ15, they were added to the end of the molecule (Figure 7). As illustrated in Figure 8, these wild-type (pSJ4) and tyrosine kinase deficient (pSJ8 and pSJ15) receptors could be efficiently expressed in COS7

la---

gp1406”dimers

IS

gp75LNGFR dimers m

B a

b

C c

a

b

c

MW

-3lOK - 200K

gpl4oM__) gp75 LNoFR-

-97K

-69K

Figure 5. Chemical Cross-linking Expressing gp75LNGFRand gp140”

of [‘251]NCF to NIH 3T3 Cells Receptors

(A) NIH 3T3 cells expressing gp75LNCFR(Z91-36), (B) NIH 3T3 cells expressing gp140” (E25-42 cells), and (C) NIH 3T3cells coexpressing g~75~“~” and gplwfk (R7-42 cells) were incubated with 1 nM [lZSI]NGF at 4OC for 2 hr and cross-linked as described in Experimental Procedures. [lzzl]labeled cells were lysed in RIPAE buffer, and the cell lysates were immunoprecipitated with (a) normal rabbit serum (b), anti-gp751NCFR, and (c) anti-trk COOH antisera, respectively. The resulting immunoprecipitates were analyzed by SDS-PAGE using a 7% resolving gel with a 2oO:l acrylamide:bisacrylamide ratio. The gel was fixed, dried, and exposed to Kodak X-OMAT film at -70°C for either (A) 5 days or (B and 02dayswith the helpof intensifying screens. The migration of the cross-linked monomeric receptors is indicated by arrows. The open arrowheads and the small arrowheads indicate the migration of the cross-linked gp751NGFR and gp140” dimers. Coelectrophoresed molecular weight markers are those described in the legend to Figure 1.

cells. Moreover, they were specifically immunoprecipitated by either the anti-trk C O O H (wild-type pSJ4 receptors) or anti-trkB TK- (kinase defective pSJ8 and pSJl5 mutants) antibodies. Interestingly, the pSJ15 kinase defective mutant cannot be recognized by the anti-trk C O O H antibodies despite the fact that it contains the normal gp140’rk carboxy-terminal sequences recognized by these antibodies (Figure 8). This observation suggests that the tagging sequences added to pSJ15 either mask the epitope recognized by the antitrk C O O H antibodies or alter the normal conformation of its carboxy-terminal tail. COS7 cells were next transfected with pSJ4, pSJ8, and pSJl5 DNAs either alone or in combination; made quiescent as described in Experimental Procedures; and incubated in the presence or absence of NGF (100 rig/ml) for 10 min. Cell extracts were immunoprecipitated with the corresponding specific antibodies and submitted to Western blot analysis using an antiphosphotyrosine monoclonal antibody (Figure 9). As shown in FiguregA, NGF induced tyrosine phosphorylation of the wild-type gp140trk receptors regardless of whether they were expressed alone or in the presence of the kinase defective mutants. In contrast, the trk

Neuron 1072

A NGF [nglml]

B

C I

IO-

0

1

10 50 100

MW

Figure 6. Tyrosine Phosphorylation of gpWYrk Receptors in Cells Coexpressing gp75LNGFRReceptors

I 0

1 10 50 100

-2OOKgp140"-

'/

crc~

e-gp140"

-97K>69K-

7 E25-42

R?-42

urk)

(trk+ LNGFR)

46K-

PC12

mutant receptors only became phosphorylated on tyrosine residues when coexpressed with wild-type gp14.W (Figure 98). The relatively high levels of phosphorylation observed in the absence of NGF are due to the presence of sodium vanadate used to stabilize the phosphorylated tyrosine residues and to the high levels of receptors expressed in these cells. These results establish that gp140*rk receptors can undergo intermolecular phosphorylation, presumably through the formation of dimeric (or oligomeric) complexes. trk Tyrosine Kinase Mutants Act as Dominant Negative Suppressors To determine the biological significance of the above results, we transfected NIH 3T3 cells with either the kinase defective mutants pSJ8 and pSJ15 alone or in combination with the wild-type pSJ4 plasmid. As a control, cells were also transfected with the parental

Table 2. gp140” Receptors Transduction at Picomolar

Mediate NGF-Induced Signal Concentrations of NGF

Transfected DNA”

NGF Concentration

None pDM69 pDM69 pDM69 pDM69 pDM69 pDM69 pDM69

100 q/ml 10 q/ml 2.5 @ml 1 rig/ml 0.25 q/ml 0.1 @ml -

4nM ~0 100 40 10 4 -

PM pM PM pM PM

Number FocP

of

0 >500 >500 >SCXl 292 142 56 1

0 >500 >500 >500 274 196 68 2

a NIH 3T3 cells (1.5 x IIY cells per 10 cm plate) were transfected as described in Experimental Procedures with 100 ng of pDM69, a pMEXneoderived trk expression plasmid (Martin-Zanca et al., 1989), and incubated in the presence of the indicated concentrations of NGF. b Foci were scored after 14 days.

Quiescent (A) NIH 3T3 cells expressing gp140rrk (E25-42 cells) and (B) NIH 3T3 cells coexpressing gp140’” and gp7YGFR recep tors (R7-42 cells) and (C) exponentially growing PC12 cells were incubated in the presence of the indicated amounts of NGF for either 10 min (A and B) or 5 min (C), lysed in P-Tyr buffer, and immunoprecipitated with anti-trk COOH antiserum. The resulting immunoprecipitates were fractionated by 8% SDS-PAGE, transferred to a nitrocellulose filter, and blotted with the anti-phosphotyrosine monoclonal antibody 4ClO. Filters were exposed to Kodak X-OMAT film at -70°C for either 18 hr (A), 8 hr (B), or 24 hr (C) with the help of intensifying screens. The migration of thetyrosine phosphorylated gpl40” receptors is indicated by arrows. Coelectrophoresed molecular weight markers are those described in the legend to Figure 1.

expression vector pREX2N (see Experimental Procedures). Transfected cells were incubated in the presence of NGF for 2 weeks, and the levels of signal transduction mediated by the various trk receptors were measured by the appearance of foci of morphologically transformed cells. As expected, cells transfected with pSJ4 had multiple foci of transformed cells, whereas those transfected with either pSJ8, pSJ15, or pREX2N did not exhibit detectable levels of morphologic transformation (Table 3). However, when these plasmids were cotransfected with pSJ4 DNA, both pSJ8 and pSJ15, but not pREXZN, significantly inhibited NGF-dependent cell transformation (Table 3). In control experiments, neither pSJ8 nor pSJl5 DNAs had inhibitory activity on transformation assays using the unrelated ras oncogene (data not shown). These observations indicate that the formation of gp140frk homodimers (or homo-oligomers) is a functional requirement to mediate the biological properties of NGF in these cells. Discussion The recent discovery that the trk family of tyrosine kinases can bind and mediate signal transduction for the NGF neurotrophin family has represented an important step toward the elucidation of the molecular nature of their functional high affinity receptors (reviewed in Barbacid, 1993). However, these findings have not resolved whether the trk tyrosine kinases are sufficient to mediate the biological properties of these neurotrophins or they are the signaling subunits of more complex cell surface receptors. Last year, Hempstead et al. (1991) proposed that the high affinity NGF receptors require the presence of gp751NCFR and gp14o’lk, presumably forming heterodimeric complexes. This model was primarily based on binding studies in which the formation of high affinity NGF

trk Homodimers 1073

Mediate

NCF Signal Transduction

phologic transformation of NIH 3T3 cells transfected with a trk expression vector. Moreover, coexpression of equal numbers of gp751NCFRand gp140trk receptors had no significant effect on either the dissociation constant or the relative percentage of low and high affinity NGF-binding sites displayed by the gp140tik receptors. The reason for the discrepancy between our binding studies and those of Hempstead et al. (1991) remains to be resolved. The functional NGF receptors present in neurons have also been characterized by their slow dissociation and rapid internalization kinetics (Sutter et al., 1979). Our resultsareconsistentwith the recent observations of Meakin et al. (1992), indicating that gplWrkNGF complexes display the slow rate of dissociation expected for the functional NCF receptors. Moreover, the NGF bound to gp+Wrk, but not to gp751NGFR,receptors was rapidly internalized. These properties were not significantly affected by coexpression of equal numbers of gp751NGFRreceptors. Cross-linking studies in cells expressing both receptors revealed the existence of both gp75LNCFRand gp14Wk homodimers. However, we were unable to detect gp7SLNCFR-gp14Wk heterodimers. Similar results have been recently obtained by Meakin et al. (1992). These findings, however, do not exclude the possibility that upon NGF receptors may inbinding, the gp751NCFRand gpl4V teract (in a fashion that may escape detection by crosslinking studies) and cooperate in mediating the biological activity of this neurotrophin. Previous studies have shown that upon NGF binding, the gpVWk receptors become rapidly phosphorylated on tyrosine residues (Kaplan et al., 199la, 1991b; Klein et al., 1991a), a step thought to be required for activation of downstream signaling pathways (Ullrich and Schlessinger, 1990). Our comparative studies, using NIH 3T3 cells expressing gp140th alone or in combination with gp75LNCFR,have indicated that the presence of these low affinity receptors does not sig-

(A). pSJ4

pSJ8

pSJ15

Figure 7. Schematic Representation of Mutant ing Tyrosine Kinase Deficient trk Receptors

trk cDNAs

Encod-

The initiation (ATG) and terminator (TAG) codons define the cDNAs’ coding sequences. These sequences include those encoding the signal peptide (light stippling), cysteine clusters (dots), leucine-rich motifs (cross-hatching), immunoglobulin C2like domains (loops), transmembrane region (open diamonds), and the kinase domain (dark stippling). The”tagging” sequences encoding the 13 amino acid carboxy-terminal sequences of gp9Sfrm (Klein et al., 1990) are represented by the open box. Sequences deleted in pSJ8 are indicated. The K5”+ A” miscoding mutation of pSJl5 is indicated by an asterisk. Noncoding trk cDNA sequences are represented by the wavy line. The locations of the restriction endonuclease sites utilized for construction of the plasmids (see Experimental Procedures) are also shown.

binding sites in heterologous COS cells required coexpression of gp751NCFRand gp140’rk. These results were at variancewith our own observations indicating that gpl4O%?xpressing NIH 3T3 cells displayed high affinity NGF-binding sites (Klein et al., 1991a). At that time, however, we did not examine the possible contribution of the gp751NCFRreceptors to the interaction between NGF and gp%Wk. In the present studies, we have confirmed our previous observations that a percentage of gp14W receptors can bind NGF with high affinity in the physiological picomolar range (Klein et al., 1991a). This interaction is likelyto have biological relevance, since picomolar concentrations of NGF induced the mor-

C

E

Figure 8. Expression of Wild-Type and Kinase Defective trk Receptors in COS Cells

---mm

abcabcabcabcabc

MW

-

200K

gpl4OPk< E

- 97K

-69K

4

-46K ---II PsJ4

PSJB

psJ15

pSJ4+pSJB

pSJ4+pSJ15

COS7 cells were transfected with (A) pSJ4, (W pSJ8, (0 pSJ15,(W pSJ4 plus pSJ8, or (E) pSJ4 plus pSJ15, as described in Experimental Procedures. Transfected cells were metabolically labeled with [US]methionine/ cysteine, lysed in RIPAE buffer, and immunoprecipitated with (a) normal rabbit serum, (b) anti-trkI3 TK-, and (c) antitrk COOH antisera. The resulting immunoprecipitates were fractionated by 8% SDS-PAGE, and the gel was subjected to fluorography, dried, and exposed to Kodak X-OMAT film at -70°C for 18 hr with the help of an intensifying screen. The migration of the wildtype and mutant gp140vf receptors is indicated by arrows. The open arrowheads indicate the migration of the partially glycosylated precursor proteins. Coelectrophe resed molecular weight markers are those described in the legend to Figure 1.

NWUXl

1074

B NGF:

-

+

-

+

-

+

-

+

-

-+-+-+-+-+

+

_‘ _a *“;

MW

I

7200Kgpl4O’h

+ ‘

b

7

g7K--

7

69 K-

7

46 K-

”

uuuuu pSJ4

uuuuu pSJ6

pSJ4 ps;6

Figure 9. Transphosphorylation

pSJ15

p&J4

PsJ4

pSJ6

ps;15

of Kinase Deficient

pk.94 ps;6

trk Proteins

by Wild-Type

pSJ15

pSJ4 ,Sk 5

gp140frk Receptors

COS7 cells were transiently transfected with plasmids encoding the wild-type gp140rrk (pSJ4) or the kinase defective trk proteins (pSJ8 and pSJl5) either alone or in combination as indicated. Duplicate cultures were quiesced and incubated either (A) without or(B) with 50 u M sodium vanadate for 3 hr prior to treatment with either 108 rig/ml NGF (+) or buffer (-) for IO min. Cells were lysed in P-Tyr buffer and immunoprecipitated with either (A) anti-trk COOH or (8) anti-trkB TK- antisera. The resulting immunoprecipitates were fractionated by 8 % SDS-PAGE, transferred to nitrocellulose filters, and blotted with the anti-phosphotyrosine monoclonal antibody 4GlO. Filters were exposed to Kodak X-OMAT film at -70°C for (A) 18 hr or (B) 30 hr with the help of intensifying screens. The migration of the tyrosine-phosphorylated trk receptors is indicated by solid arrows. The open arrow indicates the migration of the phosphorylated precursors (Martin-Zanca et al., 1989). Coelectrophoresed molecular weight markers are those described in the legend to Figure 1.

nificantly increase the levels of tyrosine phosphorylation of gp14Ook as a response to a wide range of NGF concentrations. Interestingly, the response of the g~140’~~ receptors expressed in NIH 3T3 cells to NGF was comparable to that observed with the endogenous gp140 trk receptors present in PC12 cells. However, the gp14Urk receptors expressed in NIH 3T3 cells exhibited low levels of constitutive phosphorylation not observed in PC12 cells. Whether this NGFindependent phosphorylation is due to the nonneuronal nature of the NIH 3T3 cells or to the relatively high number of gp140*rk receptors expressed in these cells remains to be elucidated. Our results do not rule out a role of the low affinity receptors in mediating and/or potentiating NCF activity (see below). However, they strongly argue against the proposed role of gp751NoFRas an integral component of the functional high affinity NGF receptors (Hempstead et al., 1991). Recent evidence obtained in other laboratories also failed to support such a model. For instance, Weskamp and Reichardt (1991) have demonstrated that polyclonal antibodies capable of inhibiting NGF binding to gp751NCFRreceptors in PC12 cells and peripheral sensory neurons did not eliminate their high affinity receptors or their responsiveness to NGF. Similarly, lbdriez et al. (1992) have recently reported that a mutated form of NGF, which binds to gp140rk but not to gp75LNGFRreceptors, retains much of its biological activity.

Available evidence supporting a role for gp75LNCFR in signal transduction has been derived, for the most part, from studies using the NGF nonresponsive NR18 cells, a PC12-derived cell line (Bothwell et al., 1980). Infection of NR18 cells with LNGFR retroviral vectors led to the appearance of high affinity receptors and limited NGF responsiveness, as determined by induction of c-fos expression (Hempstead et al., 1989) and increased tyrosine phosphorylation (Berg et al., 1991). In contrast, when these cells were infected with similar retroviral constructscarrying LNGFR mutants lacking cytoplasmic sequences, neither high affinity nor NGF responsiveness could be identified (Hempstead et al., 1990; Berg et al., 1991). In our laboratory, transfection of NR18 cells with plasmids encoding gp75LNGFRand gp140tik receptors, either alone or in combination, did not confer NGF responsiveness to thesecelIs,asdetermined bylackof neuriteformation and failure to survive in the absence of serum (unpublished data).Therefore, the biological relevance of the limited responses (e.g., c-fos induction) observed by Hempstead et al. (1989, 1990) in these gp75LNCFRexpressing NR18 cells remains to be determined. Additional evidence suggesting a role of gp75LNCFR in signal transduction has been provided by recent studies in which epidermal growth factor (EGF) could inducedifferentiationof PC12cellswhentheyexpress a chimeric EGFR-LNGFR molecule (Yan et al., 1991). These observations suggest that this chimeric recep-

trk Homodimers 1075

Mediate

NGF Signal Transduction

Table 3. Trk Tyrosine Kinase Mutants Inhibit NCF-Induced Signal Transduction through Wild-Type gp140”’ Receptors Experiment

#I

Experiment

#2

Transfected DNA

Number of Foci

Percentage Inhibition

Number of Foci

Percentage Inhibition

pSJ4 PSJS PSJT5 pSJ4 + pSJ8 pSJ4 + pSJl5 pSJ4 + pREX2N

88 f 14 0 0 26 f 6 27 f 2 82 f 8

NA NA NA 70% 69% 7%

87 it 2 ND ND 25*2 14+2 70 + 4

NA

71% 134% 19%

Transfection assays were carried out as described in Experimental Procedures using 50 ng (Experiment #I) or 10 ng (Experiment #2) of pSJ4 DNA either alone or in the presence of 500 ng of pSJ8, pSJ15, or pREX2N DNA. Transfected cells were incubated for 14 days in the presence of 50 rig/ml NGF. ND, not determined. NA, not applicable.

tor can mediate PC12 cell differentiation by either activating the cy-toplasmic sequences derived from gp751NcFRor inducing their interaction with other molecules, such as the resident gp14Vrk receptors. Both of these hypotheses are highly unlikely, since PC12 cells do not respond to other gp751NCFRligands, such as BDNF and NT-3 (Glass et al., 1991; Klein et al., 1992). Therefore, the ability of the ECFR-LNGFR chimera to induce neurite outgrowth must reside in its capacity to bind EGF, perhaps by allowing its interaction with wild-type EGF receptors. If this is the case, however, such interaction must uncouple the EGF receptors from their own signaling pathways, since EGF induces proliferation, but not differentiation, of these PC12 cells. In addition to serving as a receptor for NCF, gp75LNCFRalso binds the other members of this neurotrophin family, BDNF, NT-3, and NT4/5 with comparable low affinity (Rodriguez-TCbar et al., 1990, 1992; Hallbook et al., 1991). These neurotrophins mediate signal transduction through the gpl40”&-related gp145rrks (BDNF and NT-4) and gp145”kc (NT-3) tyrosine kinase receptors (Barbacid, 1993). Therefore, if gp75LNGFRand gp140trk were required to generate the functional high affinity NGF receptors as proposed by Hempstead et al. (1991), gp75LNCFRmight be expected to be an integral component of the functional receptors for the other neurotrophins along with either gp145”kflor gp145trkc. However, recent studies indicate that gp75LNCFRis coexpressed with gp14(rrk in the cholinergic neurons of the basal forebrain Cr. L. Steininger, B. H. Wainer, F. R. Klein, M. Barbacid, and H. C. Palfrey, submitted; C. Dreyfuss and I. Black, personal communication), but not in those of the striatum (Steininger et al., submitted). Similarly, BDNF-responsive hippocampal pyramidal neurons express significant levels of gp145trkn, but not gp75LNCFR,receptors. These observations raise serious questions regarding the putative role of gp75LNCFRin mediating the biological activity of the NGF family of neurotrophins, at least in these CNS neurons.

Recently, Lee et al. (1992) have generated strains of mice deficient in the LNGFR gene. These mice exhibit pathological deficiencies in a limited subset of sensory neurons. However, no abnormalities could be observed in the CNS or in other PNS structures, including sympathetic neurons, a major target for NCF (Lee et al., 1992). These observations also argue against an intrinsic role of gp75LNCFRin the generation of the functional high affinity receptors for the NGF neurotrophin family. Instead, gp75LNGFRmay contribute to the activation of the functional trk receptors by promoting ligand presentation or by participating in the recruitment of circulating neurotrophins (Figure 10). What is then the molecular nature of the functional high affinity receptors? Wewould like to propose that the NGF family of neurotrophins exerts its biological propertiesthrough homodimeric(oroligomeric)complexes of their cognate trk receptors (Figure 10). Crosslinking studies using [1251]NGFrevealed the existence of gp140rrk homodimers. Moreover, our studies using tyrosine kinase deficient mutants illustrated that the formation of such gp140trk homodimers is necessary for NGF-mediated signal transduction. Coexpression of these kinase mutants with wild-type gp140”‘k resulted in their efficient phosphorylation on tyrosine residues, thus demonstrating the ability of these receptors to undergo intermolecular autophosphorylation. Autophosphorylation of tyrosine kinase receptors is a step thought to be required for the catalytic activation of their cytoplasmic kinase and to facilitate its interaction with downstream signaling substrates (Ullrich and Schlessinger, 1990; Koch et al., 1991). Moreover, our gene transfer assays illustrate that coexpression of these kinase defective mutants with wild-type gp140rrk receptors results in a significant inhibition of NGF-mediated signal transduction. These

NGF

gpl4O”k

fl

SIGNAL TRANSDUCTION

I

gp75LNGFR

PRESENTATION? RECRUITMENT?

Figure 10. Diagrammatic Representation of the Proposed Molecular Structure of the High and Low Affinity NGF Receptors NCF is represented as a dimeric molecule. P-Y indicatestyrosinephosphorylated residues in thecatalytic kinase domain (shaded) of the gp%@ receptors.

NeLlKXl 1076

results indicate that the trk kinase deficient receptors behave as partial dominant negative suppressors, presumably by forming inactive wild-type-mutant gp140frk dimers. Similar results have been recently reported for kinase deficient mutants of the EGF and platelet-derived growth factor receptors (Kashles et al., 1991; Ueno et al., 1991; Redemann et al., 1992). These observations support the concept that thefunctional high affinity receptors known to mediate the biological activity of NGF are gp140Vk homodimers (or oligomers). Experimental

Procedures

Ceils, NCF, and Antisera Mouse NIH 3T3 Uainchill et al., 1969) and monkey COS7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% calf serum and 10% fetal calf serum, respectively. Rat pheochromocytoma PC12 cells (Greene and Tischler, 1976) and NCF-nonresponsive PC12nnr5 mutant cells (Green et al., 1986) were grown on collagen-coated (Vitrogen) dishes in DMEM containing 5% calf serum and 10% horse serum. NIH 3T3 cells expressing gp140frL (E25-42) have been described previously CTapley et al., 1992). NIH 3T3 cells expressing gp751NGFR alone (291-38) and in combination with gp140f’k (R7-42) were generated by transfection of NIH 3T3 and E25-42 cells, respectively, with pLL44, an expression plasmid containing the rat LNGFR cDNA (see below). NIH 3T3cells were transfected by thecalcium phosphate technique of Graham and van der Eb (1973) as modified by Wigler et al. (1978). COS7 cells were transfected by the DEAEdextranlchloroquine method as described by Seed and Aruffo (1987). Twenty-four hours after transfection, the COS7cells were split at a 1 to 3 dilution, and 24 hr later they were quiesced by a 48 hr incubation in the presence of DMEM containing 0.5% fetal calf serum before NGF treatment. Murine 2.5s NGF was purchased from Upstate Biotechnology, Inc. (New York). [‘251]NCF (1,500-2,000 Cilmmol) and Scintillation Proximity Assay beads were purchased from Amersham. Rabbit polyclonal antisera were raised against the 14 carboxy-terminal amino acid residues of human gp140” (anti-trk COOH) (Martin-Zanca et al., 1989) and the 13carboxy-terminal residues of the mouse tyrosine kinase negative gp9SfrkB protein (anti-trkB TK-) (Klein et al., 1990). A rabbit polyclonal antiserum elicited against the external domain of the rat gp75LNCFL protein was a gift of C. Weskamp and L. F. Reichardt. Plasmids The expression vectors pMEX and pMEXneo (Martin-Zanca et al., 1989) as well as the human trk proto-oncogene expression plasmid pDM69 (Martin-Zanca et al., 1989) have been described. The COS7 cell expression vector pREX2N is a derivative of pREX2 (Norman et al., 1992), which lacks the Ncol site in the SV40 origin of replication. pLL44 was generated by inserting the rat LNCFR cDNA clone (Radeke et al., 1987) of pGEM-NCFR (kindly provided by C. Weskamp and L. Reichardt) into pMEX. pSJ4 is an expression plasmid obtained by inserting the human trk protooncogene cDNA clone of pDM69 into pREX2N (Figure 6). pSJ8 and pSJl5 are two pSJ4derived expression plasmids encoding kinase defective gp140” receptors. To generate pSJ8, we removed the 1656 bp BamHI-EcoNI DNA fragment of pSJ4 (which encodes amino acid residues 228-779 of gp140”k) and replaced it by an equivalent 1425 bp BamHI-EcoNI DNAfragment isolated from pFRK78, a pDM69derived mutant (R. Klein et al., unpublished data) that carries a 231 bp deletion in the tyrosine kinase domain (amino acid residues 518-594). pSJ8 also carries a 47 bp EcoNI-Sal1 “tagging” sequence that encodes the 13 carboxyterminal residues and theTAG stop codon of the mouse noncatalytic gp9Sfrk8 receptor (Klein et al., 1990) (Figure 6). To generate pSJ15, we first constructed pSJ6 by polymerase chain reactionaided site-directed mutagenesis (Dubau et al., 1989; Ho et al., 1989). pSJ6 is identical to pSJ4 except for an AAC-GCT mutation

(nucleotides 1696-1698 of the trk cDNA insert; Martin-Zanca et al., 1989), which results in the replacement of the ATP-binding site Lys-538 by Ala-538. To obtain pSJ15, we replaced the 814 bp Ncol-Sall DNA fragment of pSJ6 (a fragment that encompasses sequences encoding amino acid residues 598-790 and all 3’ noncoding sequences of the trk cDNA insert of pSJ4) with a 576 bp polymerase chain reaction-amplified Ncol-Aflll DNA fragment (encoding amino acid residues 598-789) plus a 48 bp Aflll-Sal1 DNA fragment containing the same gp9SrrkB “tagging” sequences present in pSJ8 (Figure 6). This synthetic 48 bp Aflll-Sall DNA fragment lacks the triplet encoding the carboxy-terminal residue (Cly-790) of gp140frk as well as its TAG terminator codon. The presence of the designed mutations and the added “tagging” sequences in pSJ8 and pSJ15 was confirmed by nucleotide sequence analysis. NCF Binding Thedissociation constant and number of NGF-binding siteswere determined by Scatchard plot analysis of [1251]NCF binding using two independent techniques. In conventional binding experiments (Jing et al., 1990; Klein et al., 1991a), NIH 3T3-derived cells were plated the day before the assay in 24well Costar tissue culture plates at a density of 7.5 x IO4 cells per cm*. Cells were washed with ice-cold binding buffer (10 m M HEPES [pH 7.0],137 m M NaCI, 4.7 m M KCI, 2.5 m M CaCI,, 1.2 m M KH2P04 [pH 7.41, 1.2 m M MgSO,, 1 mg/ml glucose, 1 mg/ml bovine serum albumin, and 1 m M phenylmethylsulfonyl flouride) (Meakin and Shooter, 1991) and incubated with 150 PI of binding buffer containing various concentrations (l-2,000 PM) of 1251-labeled NCF at 4OC for 2 hr. Cells were washed four times with ice-cold binding buffer and lysed with 1 M NaOH, and the radioactivity was counted in a G5500 y counter. 1251-labeled NGF binding to PC12 cells was conducted in a similar fashion but using suspension cells (2 x IO5 per sample) in a final volume of 150 ~1. Binding experiments were also performed using the Scintillation Proximity Assay protocol (Amersham). Cells were harvested, washed, and resuspended in ice-cold phosphate-buffered saline (PBS) containing 1 mg/ml bovine serum albumin and 0.02% sodium azide (PBS-bovine serum albumin) at a density of 2 x lo6 cells per ml. Cells were incubated with various concentrations of ‘=Ilabeled NGF (0.5-1000 pM) in a final reaction volume of 125 ~1 of PBS-bovine serum albumin in Bio-Vials (Beckman) and incubated at 4OC for 2 hr. These binding reactions were mixed with 25 ~1 of Scintillation Proximity Assay beads (Amersham) and incubated at room temperature for 1 hr, and the radioactivity was determined in a 2200CA TRI-CARB Liquid Scintillation Analyzer (Packard). In all assays, nonspecific binding was determined by using duplicate samples, one of which contained 500 nM unlabeled NGF. The levels of nonspecific binding varied from 10% to 25% of the binding measured in theabsenceof excess unlabeled NGF and were subtracted in each case. Binding data were analyzed with the help of the LICAND program. NCF Dissociation Kinetics PC12 cells and NIH 3T3 cells expressing gp7SLNGFR (Z91-38), gp140f’k (E25-42), and gp751NCFRand gp140rrk (R7-42) were seeded in 24well collagen-coated cluster plates the day before at a density of IO5 ceils per well. Cells were rinsed with ice-cold binding buffer and incubated with 150 ~1 of binding buffer containing 2 nM ‘2SI-labeled NGF at 4°C for 2 hr. Cells were washed twice with ice-cold binding bufferand incubated with 150~1 of binding buffer containing 200 nM unlabeled NGF at 4°C for various periods of time. After incubation, cells were washed three times with ice-cold binding buffer and lysed in 1 M NaOH, and the radioactivity was counted in a C5500 y counter. internalization Studies NIH 3T3 cells, NIH 3T3 cells expressing gp7SLNCFR(291-38) and gp140rti (E25-42) receptors, wild-type PC12 cells, and mutant PC12nnr5 cells (Green et al., 1986) were harvested, pelleted, and resuspended in ice-cold DMEM supplemented with 10% calf serum at a density of 2 x 106 cells per ml. [1251]NGF was added to a final concentration of 2 nM, and the cells were incubated at

trk Homodimers 1077

Mediate

NCF Signal Transduction

4’C for 2 hr. Cell suspensions (100 ~1 aliquots) were transferred to 1.5 ml Eppendorf tubes prewarmed at 37“C and incubated at 37OC for various time periods. Cells were washed two times with 0.5 ml of ice-cold binding buffer and treated two times, each for 2 min, with 0.5 ml of ice-cold sodium acetate buffer (0.2 M acetic acid, 0.5 M NaCl [pH 2.41) to remove the surface bound [lZSI]NGF Uing et al., 1990). Cells were spun down and rinsed once with ice-cold PBS, and the amount of internalized [?]NCF was determined in a C5500 y counter. Chemical Cross-linking Cells were harvested, pelleted, and resuspended in ice-cold binding buffer to a concentration of 2 x 106 cells per ml. ‘%Ilabeled NGF was added to a final concentration of 1 nM, and the cells were incubated at 4OC for 2 hr. The chemical cross-linker disuccinimidyl suberate (Pierce Chemical Co.) dissolved in dimethyl sulfoxide was added to a final concentration of 150 NM (in a final concentration of 0.5% dimethyl sulfoxide). The reaction was incubated at room temperature for 30 min and quenched by washing the cells three times with 5 ml of Tris-buffered saline (Maniatis et al., 1982). Cross-linked cells were lysed in RIPAE buffer (PBS [pH 7.21 containing 1% Triton X-100,0.1% SDS, 5 m M EDTA, 1% aprotinin, and 1% sodium deoxycholate) and subjected to immunoprecipitation analysis using anti-gpl40” and anti-gp75LNGFR rabbit polyclonal antibodies as described below. The resulting immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) using a 7% resolving gel with a 2oO:l acrylamide:bisacrylamide ratio. Immunoprecipitation and Western Blot Analysis Cells were labeled by incubation at 37OC for 4 hr with 100 uCi/ ml [“SJmethionine/cysteine (pS]Translabel; Du Pont NEN) in methionine/cysteine-free DMEM supplemented with 10% dialyzed calf serum. Labeled cells were lysed in RIPAE buffer, clarified, and incubated with the appropriate antisera. The resulting immune complexes were collected by precipitation with protein A-Sepharose and washed three times with RIPAE buffer, once with 1 M MgClz in 10 m M Tris-HCI (pH 7.5), and once with 0.5% Nonidet P-40,50 m M NaCl in 20 m M Tris-HCI (pH 7.5). Immunoprecipitates were dissolved in SDS-PAGE sample buffer, fractionated by 8% SDS-PAGE, and visualized as previously described (Harlow and Lane, 1988). Tyrosine phosphorylation was performed by Western blot analysis using anti-phosphotyrosine monoclonal antibodies as described elsewhere CTapley et al., 1992). Briefly, cells were incubated with or without NCF for either 5 min (PC12 cells) or IO min (all other cells), rinsed in cold PBS containing 0.1 m M sodium orthovanadate, and lysed in P-Tyr lysis buffer (50 m M HEPES [pH 7.5],1% Triton X-100,50 m M NaCI, 50 m M NaF, 10 m M sodium pyrophosphate, 5 m M EDTA, 0.5 m M sodium orthovanadate, and 0.5 m M phenylmethylsulfonyl fluoride). In some experiments, 50 BM sodium orthovanadate was added to the COS7 cell cultures 3 hr prior to NCF stimulation. Lysates were clarified and incubated with appropriate antisera, and the resulting immune complexes were washed with P-Tyr lysis buffer. These immunoprecipitates were fractionated by8% SDS-PAGE and transferred to nitrocellulosefilters (Harlow and Lane, 1988), and their level of tyrosine phosphorylation was determined by blotting with the anti-phosphotyrosine monoclonal antibody4GlO(Upstate Biotechnology, Inc.) as described (Tapley et al., 1992).

We are grateful to R. Klein, F. Lamballe, and V. Nanduri for help ful advice and discussions and to S. Bryant and N. Thomson for excellent technical assistance. We also wish to thank C. Weskamp and L. F. Reichardt for their anti-gp75LNGFR serum and the rat LNGFR cDNA clone and L. Greene for the mutant PC12nnr5 cells. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisemenr in accordance with 18 USC Section 1734 solely to indicate this fact.

Received

June 23,1992;

revised

September

2, 1992.

References Barbacid, M. (1993). The Trk family of neurotrophin receptors: molecular characterization and oncogenic activation in human tumors. In Molecular Genetics of Nervous System Tumors, A. J. Levineand H. H. Schmidek, eds. (NewYork: Wiley-Liss, Inc.), pp. 123-135. Barbacid, M., Lamballe, F., Pulido, D., and Klein, R. (1991). The trk family of tyrosine protein kinase receptors. Biochem. Biophys. Acta Rev. Cancer 1072, 115-127. Berg, M. M., Sternberg, D. W., Hempstead, B. L., and Chao, M. V. (1991). The low-affinity p75 nerve growth factor (NGF) receptor mediates NCF-induced tyrosine phosphorylation. Proc. Natl. Acad. Sci. USA 88, 7106-7110. Bothwell, M. (1990). Tissue localization of nerve growth factor and nerve growth factor receptors. Curr. Topics Microbial. Immunol. 765, 55-70. Bothwell, M. (1991). Keeping Cell 65, 915-918.

track

of neurotrophin

receptors.

Bothwell, M.A., Schechter,A. L., andvaughn, K. M. (1980). Clonal variants of PC12 pheochromocytoma cells with altered response to nerve growth factor. Cell 27, 857-866. Cordon-Cardo, C., Tapley, P., Jing, S., Nanduri, V., O’Rourke, E., Lamballe, F., Kovary, K., Klein, R., Jones, K. R., Reichardt, L. F., and Barbacid, M. (1991). The trk tyrosine kinase mediates the mitogenic propertiesof nervegrowth factorand neurotrophin-3. Cell 66, 173-183. Dubau, L., Cheyron, A., and Argle, M. (1989). Directed sis using PCR. Nucl. Acids Res. 77, 2873.

mutagene-

Diirkop, H., Latza, U., Hummel, M., Eitelbach, F., Seed, B., and Stein, H. (1992). Molecular cloning and expression of a new member of the nerve growth factor receptor family that is characteristic for Hodgkin’s disease. Cell 68, 421-427. Ernfors, P., Ibdtiez, C. F., Ebendal, T., Olson, L., and Persson, H. (1990). Molecular cloning and neurotrophic activities of a protein with structural similarities to nerve growth factor. Proc. Natl. Acad. Sci. USA. 87, 5454-5458. Glass, D. J., Nye, S. H., Hantzopoulos, P., Macchi, M. J., Squinto, S. P., Goldfarb, M., and Yancopoulos, C. D. (1991). T&B mediates BDNFINT-Zdependent survival and proliferation in fibroblasts lacking the low affinity NCF receptor. Cell 66, 405-413. Graham, F. L., and van der Eb, A. J. (1973). A new technique the assay of infectivity of human adenovirus 5 DNA. Virology 456-467.

for 52,

Green, S. H., Rydel, R. E., Connolly, J. L., and Greene, L. A. (1986). PC12 cell mutants that possess low- but not high-affinity nerve growth factor receptors neither respond to nor internalize nerve growth factor. J. Cell Biol. 702, 830-843. Greene, L. A., and Tischler, A. 5. (1976). Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nervegrowth factor. Proc. Natl. Acad. Sci. USA 73, 2424-2428. Hallbijijk, F., Ib&iez, C. F., and Persson, H. (1991). Evolutionary studies of the nerve growth factor family reveal a novel member abundantly expressed in Xenopus ovary. Neuron 6,845-858. Harlow, E., and Lane, D. (1988). Antibodies: A Laboratory Manual (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory). Hempstead, B. L., Schleifer, L. S., and Chao, M. V. (1989). Expression of functional nerve growth factor receptors after gene transfer. Science 243, 373-375. Hempstead, B. L., Patil, N., Thiel, B., and Chao, M. V. (1990). Deletion of cytoplasmic sequences of the nerve growth factor receptor leads to loss of high affinity ligand binding. J. Biol. Chem. 265, 9595-9598. Hempstead, B., Martin-Zanca, D., Kaplan, D., Parada, L., and Chao, M. (1991). High-affinity NGF binding requires co-

NeUrOIl

1078

expression of the trk proto-oncogene receptor. Nature 350, 678-683.

and the low-affinity

NCF

Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989). Site-directed mutagenesis byoverlapextension using the polymerase chain reaction. Gene 77, 51-59. Hosang, M., and Shooter, E. M. (1987). The internalization of nerve growth factor by high-affinity receptors on pheochromocytoma PC12 cells. EMBO J. 6,1197-1202. Ibatiez, C. F., Ebendal, T., Barbany, G., Murray-Rust, J., Blundell, T. L., and Persson, H. (1992). Disruption of the low affinity receptor-binding site in NGF allows neuronal survival and differentiation by binding to the trk gene product. Cell 69, 329-341. Itoh, N., Yonehara, S., Ishii, A., Yonehara, M., Mizushima, S.-l., Sameshima, M., Hase, A., Seto, Y., and Nagata, S. (1991). The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66, 233-243. Jainchill, J. L., Aaronson, S. A., and Todaro, C. J. (1969). Murine sarcoma and leukemia viruses: assay using clonal lines of contact-inhibited mouse cells. J. Virol. 4, 549-553. Jing, S., Spencer, T., Miller, K., Hopkins, C., and Trowbridge, I. S. (1990). Role of the human transferrin receptor cytoplasmic domain in endocytosis: localization of a specific signal sequence for internalization. J. Cell Biol. 110, 283-294. Johnson, D., Lanahan, A., Buck, C. R., Sehgal, A., Morgan, C., Mercer, E., Bothwell, M., and Chao, M. (1986). Expression and structure of the human NGF receptor. Cell 47, 545-554. Kaplan, D., Martin-Zanca, D., and Parada, L. (1991a). Tyrosine phosphorylation and tyrosine kinase activity of the trk protooncogene product induced by NCF. Nature 350,158-160. Kaplan, D., Hempstead, B., Martin-Zanca, D., Chao, M., and Parada, L. (1991b). The trk proto-oncogene product: a signal transducing receptor for nerve growth factor. Science 252, 554-558. Kashles, O., Yarden, J. (1991). A dominant of normal epidermal tion. Mol. Cell. Biol.

Y., Fischer, R., Ullrich,A., and Schlessinger, negative mutation suppresses the function growth factor receptors by heterodimeriza11, 1454-1463.

Klein, R., Conway, D., Parada, L. F., and Barbacid, M. (1990). The trkB tyrosine protein kinase gene codes for a second neurogenic receptor that lacks the catalytic kinase domain. Cell 61,647-656. Klein, R., Jing, S., Nanduri, V., O’Rourke, (1991a). The trk proto-oncogene encodes growth factor. Cell 65, 189-197.

E., and Barbacid, M. a receptor for nerve

Klein, R., Nanduri, V., Jing, S., Lamballe, F., Tapley, P., Bryant, S., Cordon-Cardo, C., Jones, K. R., Reichardt, L. F., and Barbacid, M. (1991b). The trkB tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3. Cell 66, 395403. Klein, R., Lamballe, F., Bryant, S., and Barbacid, M. (1992). The frkB tyrosine protein kinase is a receptor for neurotrophin-4. Neuron 8, 947-956. Koch, C. A., Anderson, D., Moran, M. F., Ellis, C., and Pawson, T. (1991). SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science 252, 666-674. Lamballe, F., Klein, R., and Barbacid, M. (1991). trkC, a new member of the trk family of tyrosine protein kinases, is a receptor for neurotrophin-3. Cell 66, 967-979. Lee, K.-F., Li, E., Huber, L. J., Landis, S. C., Sharpe, A. H., Chao, M. V., and Jaenisch, R. (1992). Targeted mutation of the gene encoding the low affinity NCF receptor p75 leads to deficits in the peripheral sensory nervous system. Cell 69, 737-749. Loeb, L. F., NGF lines.

D. M., Maragos, J., Martin-Zanca, D., Chao, M. V., Parada, and Greene, L. A. (1991). The frk proto-oncogene rescues responsiveness in mutant NGF-nonresponsive PC12 cell Cell 66, 961-966.

Loetscher, H., Pan, Y.-C. E., Lahm, H.-W., Gentz, R., Brockhaus, M., Tabuchi, H., and Lesslauer, W. (1990). Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor. Cell 61, 351-359.

Mallett, S., Fossum, S., and Barclay, A. N. (1990). Characterization of the MRC OX40 antigen of activated CD4 positive T lymphocytes-a molecule related to nervegrowth factor receptor. EMBO J. 9, 1063-1066. Maniatis,A. M., Fritsch, E. F., and Sambrook, Cloning: A Laboratory Manual (Cold Spring Cold Spring Harbor Laboratory).

J. (1982). Molecular Harbor, New York:

Martin-Zanca, D., Oskam, R., Mitra, T., Copeland, T., and Barbacid, M. (1989). Molecular and biochemical characterization of the human trk proto-oncogene. Mol. Cell. Biol. 9, 24-33. Martin-Zanca, D., Barbacid, M., and Parada, L. F. (1990). Expression of the trk proto-oncogene product is restricted to the sensory cranial and spinal ganglia of neural crest origin in mouse development. Genes Dev. 4, 683-694. Matsushima, H., and Bogenmann, E. (1990). Nerve growth factor (NGF) induces neuronal differentiation in neuroblastoma cells transfected with the NGF receptor cDNA. Mol. Cell. Biol. 10, 5015-5020. Meakin, S. O., and Shooter, E. M. (1991). Molecular on the high-affinity nervegrowth factor receptor. 163.

investigations Neuron 6,153-

Meakin, S. O., Suter, U., Drinkwater, C. C., Welcher, A. A., and Shooter, E. M. (1992). The rat trk proto-oncogene product exhibits properties characteristic of the slow nerve growth factor receptor. Proc. Natl. Acad. Sci. USA 89, 2374-2378. Nebreda, A. R., Martin-Zanca, Santos, E. (1991). Induction Xenopus oocytes expressing Science 252, 558-561.

D., Kaplan, D. R., Parada, L. F., and by NCF of meiotic maturation of the trk proto-oncogene product.

Norman, J. A., Hadjilambris, O., Baska, R., Shap, D. Y., and Kumar, R. (1992). Stableexpression, secretion, and characterization of active human renin in mammalian cells. Mol. Pharmacol. 41, 53-59. Pleasure, S. J., Reddy, U. R., Venkatakrishnan, G., Roy, A. K., Chen, J., Ross, A. H., Trojanowski, J. Q., Pleasure, D. E., and Lee, V. M.-Y. (1990). Introduction of nerve growth factor (NGF) receptors into a meduloblastoma cell line results in the expression of high- and low-affinity NGF receptors but not NGFmediated differentiation. Proc. Natl. Acad. Sci. USA 87, 84968500. Radeke, M. J., Misko, T. P., Hsu, C., Herzenberg, L. A., and Shooter, E. M. (1987). Gene transfer and molecular cloning of the rat nerve growth factor receptor. Nature 325, 593-597. Ragsdale, C., and Woodgett, tors. Nature 350, 660661.

J. (1991). Trking

neurotrophic

recep-

Redemann, N., Holzmann, B., von Ridden, T., Wagner, E. F., Schlessinger, J., and Ullrich, A. (1992). Anti-oncogenic activity of signalling-defective epidermal growth factor receptor mutants. Mol. Cell. Biol. 12, 491-498. Rodriguez-Tebar, A., Dechant, G., and Barde, Y.-A. (1990). Binding of brain-derived neurotrophic factor to the nerve growth factor receptor. Neuron 4, 487-492. Rodriguez-Tebar, A., Dechant, G., Cotz, R., and Barde, Y.-A. (1992). Binding of neurotrophin-3 to its neuronal receptors and interactions with nerve growth factor and brainderived neurotrophic factor. EMBO J. 11, 917-922. Schall, T. I., Lewis, M., Koller, K. J., Lee, A., Rice, G. C., Wong, G. H. W., Catanaga, T., Cranger, G. A., Lentz, R., Raab, H., Kohr, W. J., and Coeddel, D. V. (1990). Molecular cloning and expression of a receptor for human tumor necrosis factor. Cell 61,361370. Seed, B., and Aruffo, A. (1987). Molecular cloning of the CD2 antigen, theTcell erythrocyte receptor, bya rapid immunoselection procedure. Proc. Natl. Acad. Sci. USA 84, 3365-3369. Smith, C., Davis, T., Anderson, D., Solam, L., Beckmann, M. P., Jerzy, R., Dower, S. K., Cosman, D., and Goodwin, R. C. (1990). A receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins. Science 248, 1019-1023.

trk Homodimers 1079

Mediate

NCF Signal Transduction

Soppet, D., Escandon, E., Maragos, J., Middlemas, D. S., Reid, S. W., Blair, J., Burton, L. E., Stanton, B. R., Kaplan, D. R., Hunter, T., Nikolics, K., and Parada, L. F. (1991). The neurotrophic factors brainderived neurotrophic factor and neurotrophin-3 are ligands for the tr/cB tyrosine kinase receptor. Cell 65,895-903. Squinto, S. P., Stitt, T. N., Aldrich, T. H., Davis, S., Bianco, S. M., Radziejewski, C., Glass, D. J., Masiakowski, P., Furth, M. E., Valenzuela, D. M., DiStefano, P. S., and Yancopoulos, G. (1991). trkB encodes a functional receptor for brain-derived neurotrophicfactorand neurotrophin-3 but not nervegrowth factor. Cell 65, 885-893. Stamenkovic, I., Clark, E. A., and Seed, B. (1989). A B-lymphocyte activation molecule related to the nerve growth factor receptor and induced by cytokines in carcinomas. EMBO J. 8,1403-1410. Sutter, A., Riopelle, R. J., Harris-Warrick, R. M., and Shooter, E. M. (1979). Nerve growth factor receptors. J. Biol. Chem. 254, 5972-5982. Tapley, P., Lamballe, F., and Barbacid, tive inhibitor of the tyrosine protein family of oncogenes and neurotrophin 371-381.

M. (1992). K252a is a seleckinase activity of the trk receptors. Oncogene 7,

Tsujimoto, M., Yip, Y. K., and Vilcek, J. (1985). Tumor necrosis factor: specific binding and internalization in sensitive and resistant cells. Proc. Natl. Acad. Sci. USA 82, 7626-7630. Ueno, H., Colbert, H., Escobedo, J. A., and Williams, L. T. (1991). Inhibition of PDCF B receptor signal transduction by coexpression of a truncated receptor. Science 252, 844-848. Ullrich, A., and Schlessinger, J. (1990). Signal transduction receptors with tyrosine kinase activity. Cell 67, 203-212.

by

Weskamp, G., and Reichardt, L. F. (1991). Evidence that biological activity of NGF is mediated through a novel subclass of high affinity receptors. Neuron 6, 649-663. Wigler, M., Pellicer, A., Silverstein, S., and Axel, R. (1978). Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor, Cell 14, 725-731. Yan, H., Schlessinger, J., and Chao, M. V. (1991). Chimeric NCFEGF receptorsdefinedomains responsible for neuronal differentiation. Science 252, 561-563.