Evidence that biological activity of NGF is mediated through a novel subclass of high affinity receptors

Neuron,

Vol. 6, 649-663,

April,

1991, Copyright

0 1991 by Cell Press

Evidence That Biological Activity of NGF Is Mediated through a Novel Subclass of High Affinity Rkeptors Gisela Weskamp

and Louis F. Reichardt

Department of Physiology and Howard Hughes Medical Institute ;Jniversity of California San Francisco San Francisco, California 94143-0724

Summary Trophic factors, such as NCF, regulate survival and differentiation of many classes of neurons by binding spe cific receptors. Two types of NGF receptors have been identified, which bind NGF with low and high affinity. The latter mediates the major biological actions of NCF. To determine the relationship between these two receptor types, polyclonal antibodies to the low affinity receptor have been prepared and used in ligand-binding, Iigand-cross-linking, and biological assays. These antibodies eliminated binding of NGF to low affinity receptors and to one class of high affinity receptors, but did not prevent binding to a second class of high affinity receptors. The same antibodies did not inhibit NGFstimulated neuronal survival or neurite outgrowth. Thus, a biologically important class of high affinity NGF receptors is antigenically distinct from the low affinity receptor and may be encoded by a novel gene. Introduction Neurotrophic factors ment of the nervous vival, differentiation,

regulate many steps in developsystem, including neuronal surand transmitter synthesis (see

Cowan et al., 1984; Barde, 1989). Nerve growth factor (NGF), the best characterized of these proteins, is required for survival and development of sensory and sympathetic neurons in the peripheral nervous system (see, for example, Levi-Montalcini, 1987; Johnson et al., 1980). NGF also regulates development of additional populations of neurons in the central nervous system, most notably cholinergic neurons located in the basal forebrain (Gnahn et al., 1983; Hefti, 1986; Mobley et al., 1986; Williams et al., 1986). In recent work, two new trophic factors that share extensive structural homologies with NGF have been identified (Leibrock et al., 1989; Hohn et al., 1990; Maisonpierre et al., 1990; Rosenthal et al., 1990; Ernfors et al., 1990; Jones and Reichardt, 1990). These two factors, brainderived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), appear to regulate survival and differentiation of distinct, but overlapping sets of neurons, including many neurons not responsive to NGF. Likeother proteinsthat regulatecell differentiation, NGF and other trophic factors exert their actions by binding to cell surface receptors. Binding studies using radiolabeled NGF or BDNF have revealed two types of receptors-high and low affinity-for each trophic factor (seeVale and Shooter, 1985; Rodriguez-

TCbar and Barde, 1988). High affinity receptors have an apparent dissociation constant (Kd) of ~10-” M and are present at a few thousand sites per cell, whereas low affinity receptors have a Kd of wIO-~ M and are expressed at much higher levels, as many as 150,000 sites per cell (Sutter et al., 1979; Landreth and Shooter, 1980; Schechter and Bothwell, 1981; Rodriguez-Tkbar and Barde, 1988; Hempstedt et al., 1989). The two receptor types can also be distinguished by their dissociation kinetics (slow versus fast), protease sensitivity, and detergent solubility (Landreth and Shooter, 1980; Schechter and Bothwell, 1981; Woodruff and Neet, 1986). Recent results indicate the two receptors also differ in ligand-binding specificity (Rodriguez-Tkbar et al., 1990).The lowaffinity NGF receptor is able to bind efficiently all known members of the NGF trophic factor family (Rodriguez-Tkbar et al., 1990; Ernfors et al., 1990). In contrast, as BDNF has been shown to bind very poorly to the high affinity NGF receptor (Rodriguez-TCbar et al., 1990), high affinity receptors appear to be specific for each trophic factor. Sequence analysis of cDNA clones encoding the low affinity NGF receptor reveals that each encodes a transmembrane glycoprotein, homologous in extracellular domains to the CD40 molecule (Stamenkovic et al., 1989) and the tumor necrosis factor receptor (Schall et al., 1990; Loetscher et al., 1990). Its cytoplasmic domain, however, lacks any obvious signaltransmitting motif, such as a protein kinase (Johnson et al., 1986; Radeke et al., 1987; Large et al., 1989). Low affinity NGF receptors are widely distributed among tissues (Raivich et al., 1985; Ernfors et al., 1988; Yan and Johnson, 1988). These receptors are found not only on neurons that are responsive to NGF, but also on neurons of other classes, such as embryonic motoneurons, upon which NGF has no detectable effects (Yan et al., 1988). Low, but not high affinity NGF receptors are also found on a variety of nonneuronal cells, including Schwann cells (Zimmerman and Sutter, 1983), which also do not appear to respond to NGF (see Yan and Johnson, 1988). These results suggest that expression of the low affinity receptor is not sufficient to confer responsiveness to NGF. In contrast to low affinity receptors, high affinity receptors exhibit appropriate specificities and distribution patterns to be implicated as mediators of trophic factor actions. The high affinity NGF and BDNF receptors discriminate between NGF and BDNF, and their distributions correlate closelywith biological responsiveness (Rodriguez-TCbar and Barde, 1988; Rodriguez-T&bar et al., 1990). High affinity NGF receptors have been found on neural crest-derived sensory neurons, sympathetic neurons, and central cholinergic neurons (Frazier et al., 1974; Sutter et al., 1979; Seiler and Schwab, 1984), as well as on NGF-responsive neuronal cell lines such as PC12 pheochromocy-

NeUrOtl 650

toma cells (see Vale and Shooter, 1985), but not on nonresponsive neurons or Schwann cells (Zimmerman and Sutter, 1983; Hosang and Shooter, 1985). The molecular differences between the low and high affinity receptors are not well understood. There is good evidence that low affinity receptors can be converted to high affinity receptors (Landreth and Shooter, 1980; Block and Bothwell, 1983; Hosang and Shooter, 1985,1987; Eveleth and Bradshaw, 1988; Buxser et al., 1990). For instance, compounds interfering with endocytosis have been shown to reduce the number of high affinity receptors dramatically, whereas agents that interfere with acidification of intracellular vesicles increasethe number of high affinity receptors (Eveleth and Bradshaw, 1988). These observations and kinetic analyses suggest that the low and high affinity receptors may reflect the same protein in different positions of the endocytotic cycle (Eveleth and Bradshaw, 1988; Buxser et al., 1990). Ligand-receptor cross-linking studies suggest that high affinity and low affinity receptor complexes with lz51-NCF differ in molecular weight (Hosang and Shooter, 1985). Using the cross-linker EDAC, only one major 1251-NGF-labeled species has been identified (Green and Greene, 1986) with the size (IOOK) expected for a complex between NGF and the low affinity NGF receptor. Since it has been seen under conditions where binding of lz51-NGF is targeted to either low or high affinity sites, the results suggest that the low affinity receptor is found in both low and high affinity binding sites. When similar experiments were performed using the more hydrophobic cross-linking agent HSAB, an additional, larger cross-linked (158K) species was detected; this species was proposed to represent binding of lz51-NGF to the low affinity receptor plus an additional 60K protein (Hosang and Shooter, 1985). Peptide mapping studies have also suggested that the two receptor types may be related (Kouchalakos and Bradshaw, 1986). Finally, transfection of a PC12 cell variant that does not express NGF receptor protein with the cDNA for the low affinity NGF receptor generates cell lines expressing both high and low affinity receptors (Hempstead et al., 1989). The cytoplasmic domain of the receptor must be expressed to generate high affinity receptors (Hempstead et al., 1990). These results also suggest that high affinity binding can be conferred by association of the low affinity receptor with an accessory protein. A prediction of the above model is that antibodies to the low affinity receptor should recognize both receptor classes and should inhibit the biological actions of NGF. In the present paper, we report the expression of the extracellular domains of the rat and chick low affinity receptors and the use of these recombinant proteins to generate polyclonal antibodies. Both antibodies eliminated binding of NGF to the low affinity receptor expressed in L cells. In studies using PC12cells, theanti-rat receptor antibodieswere shown to eliminate binding to the low affinity recep-

tor and to one class of high affinity receptors. Surprisingly, these antibodies did not prevent binding of NGF to a second class of high affinity receptors. In ligand cross-linking experiments, this antibody precipitated the complex of NGF with the low, but not the high affinity receptor. Furthermore, the anti-rat receptor antibodies did not inhibit NCF-dependent survival or neurite outgrowth by rat sensory neurons or PC12 cells. Similarily, the anti-chick receptor antibodies did not reduce NGF-dependent survival and neurite outgrowth by embryonic chick sensory neurons. In agreement with previous data, the results suggest that one class of high affinity receptors is derived from the low affinity receptor. However, the results also argue for the existence of a second, antigenically distinct class of high affinity receptors responsible for mediating the major biological actions of NGF. Results Expression of REX and ChEX in Baculovirus-Infected Insect Cells Since there is no natural source suitable for purification of largeamountsof NGF receptors, theextracellular domains of the rat NGF receptor (referred to as REX) and of the chick NCF receptor (named ChEX) were expressed in insect cells, using a baculovirus expression system. To characterize the recombinant rat protein (REX), infected Spodopterafrugiperda (Sf9) cells were metabolically labeled with [35S]cysteine. As shown in Figure IA (lane I), only two major proteins with estimated M,values of 60K and 47K were secreted into the supernatant. Both proteins were recognized by a monoclonal antibody specific for the rat NGF receptor, mc192 (Figure IA, lane 2), indicating that the two proteins represent different forms of the NGF receptor’s ectodomain. The rat NGF receptor contains potential extracellular N-linked glycosylation sites (Radeke et al., 1987). To determine whether the two secreted forms of the recombinant receptor were glycosylated, [35S]cysteine-labeled REX was isolated by immunoprecipitation and then digested with endoglycosidase F (endo F), an enzyme that cleaves both high mannose and complex glycans (Tarentino et al., 1985). Treatment of immunoprecipitated REX with endo F reduces the M, of both bands by IOK-15K, indicating that both forms contain N-linked carbohydrates (Figure IA, lane 3). When infected cells were treated with tunicamycin, an inhibitor of N-linked sugar addition, REX migrated as one band with an M, of about 35K on SDS-PAGE (data not shown), indicating that the two proteins differ in carbohydrate content and not in amino acid sequence. Clycoproteins from Sf9 cells are known to contain mainly high mannose branched glycans, since these cells do not express N-sialyltransferase (Butters et al., 1981). Therefore, in contrast to the naturally occurring NGF receptor on PC12 cells (Vale et al., 1985), the recombinant receptor is not expected to contain sialic acid residues. Consistent with this expectation, the NGF

iigh ‘151

Affinity

NGF Receptors

1

A

1

97.4

66.2

2

3

200.0

97.4

-

66.2

-

-

:

42.7

-

21.5

-

Figure

-

31.0

-

Figure

-

31.0

42.7

1. Expression

and

Purification

ectodomain Sepharose

of REX

does not bind to wheat (data not shown).

3

4

s

-

116.0

-

97.4

-

66.2

-

42.7

-

2. lmmunoblot

Analysis

Using

Anti-REX

IgG

Cell extracts of L cells (lane I), PCNA cells (lane 2), PC12 cells (lane 3), neonatal rat dorsal root ganglion neurons Bane 4), and nonneuronal cells from neonatal rat dorsal root ganglion (lane 5) were fractionated by SDS-PAGE and subjected to immunoblot analysis using anti-REX IgG as described in Experimental Procedures. Positions of molecular weight markers (in kilodalton) are shown.

(A) Autoradiography of YS-labeled proteins. Sf9 cells infected with recombinant baculovirus were metabolically labeled with [35S]cysteine as described in Experimental Procedures. Aliquots from culture supernatants were directly fractionated by SDSPAGE (lane 1) or immunoprecipitated with mc192 IgC and subsequentlyincubated without (lane2)orwith (lane3) endo F.Autoradiographs after 15 hr exposureare shown; positions of molecular weight markers (in kilodaltons) are indicated. (B) Purified REX (IO pg)from baculovirus-infected insectcells was analyzed on a 15% polyacrylamidegel and stained with Coomassie blue. Positions of molecular weight markers (in kilodaltons) are indicated.

receptor agglutinin

2

germ

Purification of the NGF Receptor Ectodomain Using supernatants from infected Sf9 cells, purification of REX was achieved by two chromatography steps. Culture supernatants were collected 3 days after infection and glycoproteins were enriched on lentil-lectin Sepharose. Glycoproteins were then further purified by Mono Q anion exchange chromatography. When purified REX was analyzed on a reducing SDS-polyacrylamide gel stained with Coomassie blue, two bands with M, values of 60K and 47K were detected (Figure 16). The yield of purified REX was typically between 2-3 mg per liter of culture supernatant. ChEX was purified using the same procedure (data not shown), yielding about 1 mg per liter of culture supernatant. Antibodies to REX Recognize the NGF Receptor on Mammalian Cells Using purified REX as an antigen, polyclonal anti-REX

antibodiesweregeneratedasdescribed in Experimental Procedures. Purified anti-REX IgG reacted strongly with REX on Western blots (data not shown). To determine whether anti-REX IgG also recognizes the NGF receptor expressed by mammalian cells, different celIularextractswerefractionated by SDS-PAGEandanalyzed in immunoblots. In L cells, a mouse fibroblast cell line that does not express the NGF receptor (Radeke et al., 1987), no antigen was recognized by antiREX IgG (Figure 2, lane 1). However, in PCNA cells, an L cell line stably transfected with the NGF receptor gene, the antibodies bound an antigen with an M, of 60K (Figure 2, lane 2). PC12 cells (Greene and Tischler, 1976), which express both high and low affinity NCF receptors, expressed a major antigen with an M, of 68K and a minor antigen with an M, of 200K. These values are in agreement with results from immunoprecipitations using the anti-rat NGF receptor monoclonal antibody, mc192 (Radeke et al., 1987). The lower molecularweight moiety is the low affinity NGF receptor, whereas the slower migrating band may represent aggregates of this receptor (Buxser et al., 1985). The difference in M, of the NGF receptor expressed in PCNA cells compared with that present in PC12 cells probably reflects incomplete glycosylation of the PCNA cell receptor. Anti-REX IgG also recognized a 68K antigen in extracts of cultured neonatal rat sensory neurons (Figure 2, lane 4). In extracts of nonneuronal cells derived from neonatal rat dorsal root ganglia, an antigen with the same M,was present, but was expressed at much lower levels (Figure 2, lane 5). The antigen in both cell types has the same M, as the low affinity NGF receptor, which has been shown to be expressed on both neurons and Schwann cells in these ganglia (Taniuchi et al., 1986). Anti-REX antibodies therefore appear to react both with the recombinant fragment of the NGF receptor expressed in insect

Neuron 652

A

F 5 .r P g I m N -

100

100

60

80

60

60

40

40

20

20

0 20

0

40

6” t

0

20

40

60

(min)

4 Figure 3. Effects of Anti-NCF Dissociation of ‘?NGF from

Receptor IgC on the Kinetics PCNA and ChNL Cells

of

PCNA cells (A) were incubated at 4°C for 60 min with either preimmune IgG (open circles) or anti-REX IgC (closed circles), both at 50 fig/ml. ChNL cells (6) were incubated at 4°C for 60 min with either preimmune IgC (open triangles) or antiChEX IgC (closed triangles), both at 50 Kg/ml. After addition of 2 nM ‘251-NCF, cells were incubated at 4’Y for 60 min. The dissociation reaction was initiated by theaddition of unlabeled NCF to a final concentration of 500 nM. At the times indicated, aliquots were removed to determine cell-bound radioactivity as described in Experimental Procedures. Results are expressed as percentage of 12VNGF bound at time 0 in the absence of antibody.

cells ferent

and

with

mammalian

NGF

receptors cell

expressed

in several

dif-

types.

Anti-NGF Receptor Antibodies Abolish Binding to Cells Expressing Exclusively Low Affinity NCF Receptors

NCF

PCNA cells are transfected L cells that express high levels of the rat low affinity NGF receptor (Radeke et al., 1987). To examine the effects of anti-REX IgG on the binding properties of these receptors, PCNAcells were first preequilibrated with either preimmune IgG or anti-REX IgG, both at 50 ug/ml. These cells were then incubated with 1251-NGF. Subsequently, the kinetics of ligand-receptor dissociation were monitored in the presence of high concentrations of unlabeled NGF. As seen in Figure 3A, 1251-NGF binds to PCNA cells in the presence of preimmune IgC. Upon addition of unlabeled ligand, 1251-NGF dissociates within minutes from these receptors. After 30 min, no measurable 1251-NGF remained bound to these cells, corroborating the previous demonstration that these cells express only low affinity, fast-dissociating receptors (Radeke et al., 1987). Results in Figure 3A also show that preincubation of PCNA cells with anti-REX IgG completely prevented specific binding of lZSI-NGF to these cells. Thus, anti-REX IgG can completely inhibit binding of NGF to rat low affinity, fast-dissociating NGF receptors. The same results were obtained using anti-ChEX IgG and ChNL cells, a cell line that expresses high

001

01 antibody

1

1

10

concentration

100

1000

(pg/ml)

Figure4. Dose-Dependentlnhibitionof’%NGFBindingtoPC12 Cells by Anti-REX IgG PC12 cells were incubated at 37°C for 60 min with the indicated concentrations of preimmune IgG (open circles) or anti-REX IgG (closed circles). Binding was then initiated by the addition of 800 pM 9-NCF. After incubation for another 60 min at 37OC, the amount of cell-associated radioactivity was determined as described in Experimental Procedures. Results are expressed as percentage of ‘251-NGF binding in the absence of antibody.

levels

of chick

low

affinity

NGF

receptors.

The

kinetics

of dissociation of 1251-NGF from chick low affinity receptors were determined as described above for the rat receptors. As shown in Figure 3B, 1251-NGF specifically bound to ChNL cells in the presence of control IgG and dissociated from these cells within minutes upon addition of unlabeled ligand. After 5 min, no measurable 1251-NGF remained bound. Preincubation of cells with anti-ChEX IgG completely abolished specific binding of 1251-NGF to ChNL cells (Figure 3B). Thus, antibodies to the chick low affinity NGF receptor completely prevent binding of NGF to chick low affinity receptors.

Anti-REX IgG Reduces Binding of NCF to PC12 Cells Next, we determined whether anti-REX IgG inhibits the binding of NGF to PC12 cells, which express both low and high affinity NGF receptors. In initial experiments, we measured the effects of increasing concentrations of preimmune IgG or anti-REX IgG on jZ51-NGF binding to PC12 cells. As shown in Figure 4, ligand binding was inhibited in a dose-dependent manner. At 0.3 ug/ml anti-REX IgG, ligand binding was reduced to 50% of control values. At IgG concentrations of 1-2 ug/ml, 80% of 1251-NGF binding was inhibited. Even in the presence of 500 uglml anti-REX IgG, no further increase of inhibition beyond 80% was observed. Thus, PC12 cells express two distinct types of NGF receptor-one that can be inhibited by low antibody concentrations and another that cannot be inhibited by a 500-fold higher concentrations of IgG.

hi h Affinity 6’S!

Figure %NGF

NGF Receptors

& values of 44 pM and 2.9 nM, (Table 1). Scatchard analysis indicates that the cells contained approximately 6000 high affinity sites and 150,000 low affinity sites per cell. These values are similar to those previously obtained for PC12 cells (see Vale and Shooter, 1985). When binding of lz51-NGF was measured in the presence of anti-REX IgG, the Scatchard plot yielded a straight line (Figure 5). Analysis indicated that lz51-NGF bound only to one class of receptors with an apparent Kd of 42 pM. Approximately 2500 antibody-resistant sites were present on each PC12 cell (Table 1). Under the conditions used in these assays, therefore, two distinct populations of high affinity receptors can be distinguished on PC12 cells. One population (about 3500 sites per cell) is antigenically related to the low affinity receptor and may well represent the low affinity receptor at a particular stage in the endocytotic cycle (Eveleth and Bradshaw, 1988; Buxser et al., 1990). However, the second class, of approximately 2500 sites per cell, appears to be antigenically distinct.

5. Scatchard Analysis of Steady-State Association of with PC12 Cells in the Absence or Presence of Anti-REX

PC12 cells were incubated at 37OC for 60 min in the presence of either preimmune IgG (open circles) or anti-REX IgC (closed circles), both at 50 &ml. Radiolabeled ligand at various concentrations (5 pM to 2 nM) was allowed to bind. After incubation at 37OC for 60 min, cell-bound radioactivity was determined, and values werecalculated as described in Experimental Procedures.

Effects of Anti-REX IgG of NCF to PC12 Cells

on

Equilibrium

Effects of Anti-REX of NCF from PC12

To characterize further which receptor classes on PC12 cells are inhibited by anti-REX IgG, kinetic dissociation assays were performed. PC12 cells were first incubated at 37OC for 60 min with lz51-NGF in the presence of control IgG or anti-REX IgG. Binding of 1251-NGF was measured; excess unlabeled NGF was then added, and the kinetics of dissociation of 1251-NGF were monitored (Figure 6). Two concentrations of 1251-NGF were used (50 pM and 800 PM) such that most of the high affinity receptors would be bound at both concentrations. However, relatively few of the low affinity receptors would be occupied at the low NGF concentration (Landreth and Shooter, 1980). To measure kinetics of dissociation, 500 nM unlabeled NGF was then added and cells were incubated at either 37OC (Figures 6A and 6B) or 4OC (Figures 6C and 6D). Results in Figure 6 show that two populations of receptors were detected in each experiment performed in the presence of control IgG. As expected, the proportion of rapid versus slow dissociating lz51-NGF was dependent on the concentration of radiolabeled ligand. At low concentrations of lz51-NGF (50 PM), 65% of specifically bound NGF dissociated slowly (Figures 6A and 6C), whereas at 800 pM

Binding

To determine which types of NGF receptors were inhibited by anti-REX IgC, the effects of this antibody on ligand-receptor interactions were assayed in the sameequilibrium and kinetic binding assays used previousiytocharacterize NGF interactionswith high and low affinity receptors (see Vale and Shooter, 1985). To quantitate the effects of anti-REX IgG on equilibrium binding, specific association of NCF with the two receptor classes was measured at 37OC and binding data were analyzed by Scatchard analysis as described in Experimental Procedures. In agreementwith previous reports (Sutter et al., 1979; Schechter and Bothwell, 1981), equilibrium, as determined by measuring incubation time-dependent association of radiolabeled ligand, was reached in 35 min at 37OC (data not shown). In the presence of control IgG (Figure 5), the Scatchard plot yielded a curvilinear line, indicating that PC12 cells have two classes of NGF receptors with apparent

Table

1. Effects

of Anti-REX

IRG on Equilibrium

Binding

Control

4.4 (k2.5) 2.9 (tl.2)

of 1251-NGF to PC12 Cells

-___

Anti-REX-Treated # Receptors

Kd CM) x IO-” x 10m9

Data are derived ments * SEM.

(per Cell)

~6,000 ~150,000 from

binding

experiments

---__~ # Receptors (per Cell)

Kd CM) 4.2 (kO.9)

as described

IgG on Dissociation Cells

in Experimental

x IO-”

Procedures.

-2,500 Values

represent

the mean

of 3 separate

experi-

Neuron 654

B

t

(min) D

C

m .c : I g I : c

100

100

80

80

60

60

40

20 Ed!:::_

0

20

40

60

0 t

20

40

60

(min)

Figure 6. Effect of Anti-REX IgG on the Kinetics of ‘WNGF from PC12 Cells

of Dissociation

PC12 cells were incubated at 37°C for 60 min with either preimune IgG (open circles) or anti-REX IgG (closed circles), both at 50 pg/ml. ‘251-NGF was then added to 50 pM (A and C) or 800 pM (6 and D). The dissociation reaction was initiated by the addition of unlabeled NGF to a final concentration of 500 nM. At the times indicated, aliquots were removed to determine cell-bound radioactivity as described in Experimental Procedures. Results are expressed as percentage of Y-NGF bound at time 0 in the absence of antibody. (A and B) Dissociation at 37°C. (C and D) Dissociation at 4OC.

only 20%-30% of initial binding was to the slow receptor (Figures 68 and 6D). When kinetics of dissociation under control conditions were compared at 37OC and 4OC, data in Figure 6 show that the rates of dissociation are slowed at the lower temperature. At 37OC, 12+NGF dissociated from the fast receptor with a half-time of seconds, whereas dissociation from the slower receptor occurred with a half-time of about 60 min (Figures 6A and 66). At 4’C, however, dissociation from the fast receptor occurred within minutes, but no detectable release of lz51-NGF from the remaining slow receptors was seen after 60 min (Figures 6C and 6D). These results agree with previous characterizations of NGF receptors on PC12 cells, in which NGF has been reported to dissociate from the fast receptor with half-times of 0.5 and 5 min at 37OC and 4OC, respectively (Landreth and Shooter,

1251-NGF,

1980; Schechter and Bothwell, 1981). The half-times of NCF dissociation from the slow receptor at the same two temperatures have been reported to be ~30 min and several hours (see Landreth and Shooter, 1980; Schechter and Bothwell, 1981). The two major classes of NGF receptors have also been shown to exhibit different kinetics of ligandreceptor dissociation, both on PC12 cells and on primary neurons (see Vale and Shooter, 1985). To determine which receptor classes were inhibited by anti-REX IgG, the same assays were performed in the presenceof anti-REX IgG. Results in Figure6showthat anti-REX IgG appears to inhibit completely binding of 1251-NGF to low affinity receptors, when assayed by dissociation either at 37OC (Figures 6A and 6B) or at 4OC (Figures 6C and 6D). The same antibody also appears to reduce binding to the high affinity receptors by 50%-70% in these assays. This inhibition did not depend upon the initial concentrations of 12+NGF. The presenceof anti-REX IgG did not, however, significantly alter the kinetics of dissociation of lZSI-NGF from the slow receptors. At 37OC, dissociation occurred with a half-time of 60 min (Figures 6A and 66). At 4”C, no release was detectable (Figures 6C and 6D). Thus, the anti-REX antibody prevented any detectable binding of NGF to the fast receptor and reduced binding of NGF to the slow receptor by 50%-70%. Kinetics of dissociation from the slow receptor were not altered by anti-REX IgG. In agreementwith results in the previous section, these data indicate that there are two distinct populations of high affinity receptors present on PC12 cells, only one of which is antigenitally related to the cloned low affinity NGF receptor.

Anti-REX Binds the NGF-Low Affinity, but Not the NCF-High Affinity Receptor Complex in Cross-linking Experiments Upon cross-linking with NGF, low and high affinity NGF receptors can be distinguished by their different electrophoretic mobility (Hosang and Shooter, 1985). To determine the effects of anti-REX IgG on the binding properties of both receptors and to determine which ligand-receptor complexes are bound by antiREX IgG, we performed ligand-cross-linking experiments. Cell suspensions of PC12 cells were preincubated with preimmune IgG (Figure 7, lanes 1, 3, and 4) or anti-REX IgG (lane 5). 1251-NGF was added and subsequently cross-linked with the water-soluble, membrane-impermeablecross-linkerbis(sulfosuccinimidyl)suberate (BS3). When extracts of these cells were immunoprecipitated with anti-NGF antibodies, two major cross-linked species with M, values of 95K and 150K were detected, as well as a minor band of about 200K-220K (Figure 7, lane 3). The size of the cross-linked NGF-binding proteins is in agreement with previous studies, which used HSAB as a crosslinking agent (Hosang and Shooter, 1985). The rapidly migrating species represents the NGF-low affinity receptor complex. The slower migrating moiety of 150K

Fiigh Affinity hi5

NGF Receptors

1

Unlabeled

200

-

116

-

97.4

-

66.2

-

42.7

-

+-

NGF

Preincubation

3

4

5

-

-

--

+ -

+ -

+

reaction was reprecipitated with anti-NGF antibodies, the high molecular weight entity of 150K was precipitated, as well as the remainder of the low molecular weight component (Figure 7, lane 6). As seen in Figure 7, lane 5, preincubation of PC12 cells with anti-REX IgG prevents cross-linking of lz51-NGF to the lower molecular weight species, but not to the higher mo: lecular weight 150K species. In this lane, the 150K species was precipitated by anti-NGF antibodies. The specificity of these cross-linking reactions was demonstrated by binding assays performed in the presence of an excess of unlabeled NGF (Figure 7, lane 1). In this case, no labeled products were seen. Similarly, no labeled proteins were detected after precipitation with control IgC (Figure 7, lane 2). Therefore, in ligand cross-linking experiments, anti-REX IgG binds to the NGF-low affinity, but not to the NGF-high affinity receptor complex, providing further evidence that these two receptor proteins are antigenically distinct.

6

with

preimmune anti-REX Precipitation

2

+-

-

supernatant from lane 4

with

preimmune

-

+

-

-

-

-

anti-NGF

+

-

+

-

+

+

anti-REX

+

-

-

+

-

-

Figure 7. Autoradiography to PC12 Cells

of Affinity

Cross-Linking

of ‘WNCF

4 cell suspension of PC12 cells was incubated at 4OC for 60 min with either preimmune IgC (lanes 1, 3, and 4) or anti-REX IgC (lane 5), both at 50 &ml. After addition of 1 nM ‘251-NGF, cells were incubated at 4°C for 60 min and cross-linked with BS3 as described in Experimental Procedures. Cell extracts were then precipitated with either preimmune IgG (lane 2), anti-NCF antibodies (lanes 1.3, and 5), or anti-REX IgG (lanes 1 and 4). Binding of ‘251-NGF was performed in the presenceof 10 vglml unlabeled NGF (lane 2). After precipitation with anti-REX IgC, supernatant from lane 4 was reprecipitated with anti-NGF antibodies (lane 6). An autoradiograph after 4 days exposure is shown; positions of molecular weight markers (in kilodaltons) are indicated. Note that anti-NGF antibodies precipitate both the 150K and the 95K species (lane 3), whereas anti-NGF receptor antibodies precipitateonlythe95Kspecies (lane4).The150Kmoiety is notdegraded by incubation with anti-receptor antibodies, since it can be precipitated by subsequent addition of anti-NGF antiserum (lane 6).

NGFcross-linked tothe high affinity receptor, whereas the 220K moitey has been shown to represent aggregates of NGF and its receptors (Buxser et al., 1985). In cell extracts precipitated with anti-REX IgC, the 95K entity was the major labeled antigen. A minor labeled protein was found with an M, of 190K (Figure 7, lane 4). When the supernatant from this

consistsof

Anti-NCF Receptor Antibodies Do Not inhibit Biological Responses to NGF High affinity NGF receptors appear to mediate the most important neuronal responses to NGF. To determine which population of high affinity NGF receptors mediates these responses, effects of NGF on neuronal survival and neurite outgrowth were examined in the presence of control IgG and anti-REX IgG, using PC12 cells and dissociated neonatal rat sensory neurons (see Experimental Procedures). As shown in Figure 8, no significant neurite outgrowth was observed when primed PC12 cells were grown in the absence of NGF for 4 days. Addition of NCF (5 nglml) promoted neurite outgrowth and induced neuronal phenotypic changes (see Greene, 1978). Neither preimmune nor anti-REX IgG had any effect on the NGF-mediated morphological changes on PC12 cells. Similar results were obtained using dissociated rat sensory neurons (Figure 8; Table 2). When these neurons were cultured in the absence of added NGF for 4 days, no significant survival of neurons was observed. At 50 rig/ml NGF, 67% of the neurons plated survived and exhibited extensive neurite outgrowth. Addition of preimmune IgG to the culture medium did not reduce either survival or neurite outgrowth (Table2). Similarly, anti-REX IgG did not reduce NGF-mediated survival or neurite extension of these neurons in vitro. As illustrated in Figure 8 and quantitated in Table 2, no significant difference in survival or morphology could beobserved. When added without NGF, anti-REX IgG did not enhance either survival or neurite outgrowth (Table 2). When antiChEX IgG was used in bioassays with dissociated chickembryonic sensory neurons, this antibody also failed to prevent responses to NGF. As quantitated in Table 2, no significant survival or neurite outgrowth was observed when these cells were cultured for 4 days without added NGF or with only anti-ChEX IgG. Addition of 50 nglml NGF resulted in survival of 52% of plated neurons, which showed an extensive neurite network. Addition of neither preim-

Neuron 656

PC12

Figure

8. Effects

Dissociated collagen-coated the absence with either 4 days. Bar,

of Anti-REX

IgG on Survival

and Neurite

DRG

Outgrowth

of PC12 Cells

and

Peripheral

Sensory

Neurons

sensory neurons derived from newborn rat dorsal root ganglia (DRG) and primed PC12 cells (PC12) were grown on tissue culture dishes as described in Experimental Procedures. PC12 cells and DRG neurons were incubated in either of NGF (control) or the presence of 5 or 50 rig/ml NGF, respectively. Before addition of NCF, medium was supplemented preimmune IgG (Preimmune) or anti-REX IgG (Anti-REX), both at 100 ug/ml. Phase-contrast micrographs were taken after 100 urn.

mune IgG nor anti-ChEX IgG altered NGF-induced survival and neurite outgrowth by these neurons. Therefore, NGFdependent neurite outgrowth by PC12 cells and NGF-dependent survival and neurite outgrowth of rat and chick spinal sensory neurons do not require the binding of NGF to the low affinity NGF receptor. Discussion The most important conclusion of our work is that there appear to be three types of NGF receptors, one low affinity and two distinct high affinity receptors. Binding of NGF to low affinity receptors, defined either by equilibrium binding, by dissociation kinetics, or by ligand-receptor cross-linking, is completely inhibited by antibodies specific for the extracellular domain of the cloned rat low affinity NGF receptor. This result indicates that, at least on PC12 cells, all of the low affinity receptors are products of this single gene.

Previous work has shown that expression of this single protein in transfected cells is sufficienttogenerate a low affinity receptor (Johnson et al., 1986; Radeke et al., 1987). Data in the present paper also show that the sameantibodies inhibited bindingof NGFtooneclass of high affinity receptors, again identified by both equilibrium binding and dissociation kinetics. Consequently, this major class of high affinity receptors seems to be related to the low affinity receptor. The difference between low and high affinity receptors of this class could be defined by the position of the receptor in the endocytotic cycle (Eveleth and Bradshaw, 1988; Buxser et al., 1990). Alternatively, this receptor class may represent the low affinity receptor as a subunit of a multi-protein complex (Landreth and Shooter, 1980; Hosang and Shooter, 1985). In these models, ligand affinitywould bedetermined by receptor aggregation or association with accessory proteins (Vale and Shooter, 1982, 1983; Hosang and Shooter, 1985). However, the same anti-low affinity NGF recep-

Iiigh (S7

Affinity

NGF Receptors

rable 2. Effects of Anti-Receptor Yeurite Outgrowth of Cultured

IgC on Survival Sensory Neurons % of Surviving Neurons

and

% of Neurite-Bearing Cells

Newborn Rat No NGF 4nti-REX IgC ‘\lGF UCF + preimmune IgG NGF + anti-REX IgC

1.2 0.6 100.0 93.7 98.8

* * k * f

0.4 0.3 8.3 5.2 7.4

0 0 100.0 f 5.9 105.9 + 9.9 97.3 * 8.6

Embryonic Dav 8 Chick No NCF Anti-ChEX IgC NCF NCF + preimmune IgG NGF + anti-ChEX IgG

1.5 1.2 100.0 103.5 112.0

* f f * *

0.7 0.4 8.2 5.0 9.9

0 0 100.0 107.6 103.4

f 7.8 f 4.9 it 5.2

Dissociated sensory neurons obtained from newborn rats and embryonic day 8 chick were grown as described in Experimental Procedures. Cells were incubated either without NGF (No NCF) or with NGF (50 nglml) in the presence of either preimmune IgC or anti-receptor IgC (Anti-REX and Anti-ChEX), both at 100 pglml. Cells were cultured for 3-4 days and scored for survival and neurite outgrowth. Values for newborn rat represent the average of 8 experiments; those for embryonic chick are the average of 3 experiments. Values are expressed as a percentage of control (cells grown with 50 nglml NGF).

tor antibodies did not inhibit binding of NCF to a newly defined class of high affinity receptors, even when antibodies were used at concentrations up to 500-fold higher than that required to eliminate binding to low affinity receptors. The specificity of the antibodies for a subset of NGF-binding sites was also demonstrated in ligand cross-linking experiments, in which the antibodies were shown to bind to the 95K species that corresponds to a complex of ‘*+NGF and the low affinity NCF receptor. The antibodies did not recognize the 150K species, previously shown to correlate with binding of lz51-NGF to high affinity sites (Hosang and Shooter, 1985). Thus, a second high affinity receptor that is antigenically distinct from the low affinity receptor exists on the surface of PC12 cells. While the results are not absolutely definitive, they suggest that this second high affinity receptor is unlikely to contain the low affinity receptor as a subunit and therefore might be the product of a distinct gene. A second major conclusion is that this novel high affinity receptor is sufficient to mediate the major biological actions of NGF. Neither NCF-dependent survival of sensory neurons nor NGF-induced neurite outgrowth by sensory neurons or PC12 cells was detectably inhibited by antibodies to the low affinity receptor (Table 2). Similarly, at least one rapid response to NGF, tyrosine phosphorylation of phospholipase C-y, was also not inhibited by these same antibodies (Vetter et al., unpublished data). When added in the absence of NGF, antibodies to the low affinity receptor did not detectably activate receptor function, as assayed by survival or neurite outgrowth (Table 2). In the biological assays, essentially identical results were

obtained using two antibodies, one of which was prepared to the ectodomain of the chick receptor protein, which has diverged substantially (36%) from the primary sequence of mammalian receptors (Large et al., 1989). These results argue that the low affinity receptor and the high affinity receptors derived from it are not required for neuronal survival or neurite outgrowth. Our results do not address other possible functions, such as the regulation of neurotransmitter synthesis, that may be mediated by the low affinity and related high affinity receptors. Thefailureof the low affinity receptor-specificantibodies to prevent binding to one class of high affinity receptors is not likely to reflect inefficient binding to a single receptor class. First, the antibodies clearly inhibit binding to one class of high affinity receptors (Figure5). As shown in a dose-response measurement (Figure 4), anti-REX IgG prevents binding of NGF to one class of high affinity binding sites at 1 pg/ml, but does not detectably inhibit binding to the second class of high affinity binding sites at 500-fold higher antibody concentrations, even though both classes bind NGF with approximately the same affinity. Second,thefailureofantibodiestointerferewith binding to this second receptor class is not likely to reflect replenishment of surface NGF receptors from an internal pool. In all binding assays, PC12 cells were preequilibrated with anti-REX IgG for60 min priortoaddition of NGF. The NGF receptor has been shown to internalize and recycle to the cell surface within this time span (Eveleth and Bradshaw, 1988). If recycling of surface NGF receptors accounted for binding of lz51-NGF at 37OC, higher concentrations of anti-REX IgG should bind agreater proportion of the high affinity receptor as it appeared on the cell surface, resulting in a further reduction of NGF binding over a 500-fold range of antibody concentration. Instead, a partial but constant tnhibition of 80% was observed. Moreover, in the presence of anti-REX IgG, there are specific binding sites for 1251-NGF on PC12 cells incubated at 4°C in the absence of glucose and the presence of NaN,(G. Weskamp, unpublished data), conditions that prevent membrane internalization and recycling (Haigler et al., 1979). Results in the present paper indicate that there are striking biochemical and antigenic differences between the two classes of high affinity receptors. In ligand cross-linking experiments using 1251-NGF, PC12 cells, and thewater-soluble, membrane-impermeable cross-linking agent BS3, two receptor-ligand complexes shown previously to represent low and high affinity receptor complexes were identified (Hosang and Shooter, 1985). One conclusion from this work is thatthe high affinity receptor contains a novel protein constituent with an extracellular domain. It cannot be formed solely by association with additional intracellular proteins. A second conclusion is that the polyclonal anti-REX IgG distinguishes between these two complexes. Anti-REX IgG can inhibit formation of the low, but not the high molecular weight ligand-recep-

Neuron 658

tor complex. Similarly, it can immunoprecipitate the low, but not the high molecular weight species. The antibodies used in the present paper are polyclonal, prepared using the entire extracellular domains of either the rat or the chick receptor as antigens. One antigen, the chick receptor ectodomain, exhibits substantial divergence from the primary sequences of mammalian receptors (Large et al., 1989). The rat receptorantibodiesalsoalmostcertainlyrecognizemultiple epitopes, since they can inhibit binding of NGF and precipitate NGF-receptor complexes. Thus, the results argue that the two receptor-ligand complexes share no detectable antigenic epitopes. It is unlikely, that the antigenic differences could reflect associations of the low affinity receptor with other proteins that shield all such epitopes. Finally, the antibodies were not able to inhibit ligand-receptor interaction in assays lasting hours to days, times much longer than those required for receptor recycling (Eveleth and Bradshaw, 1988). If the novel class of receptors was formed by conversion of low affinity receptor subunits, the antibodies should have been able to inhibit their function, given sufficient time to act. Hosang and Shooter (1985) showed that the ratio of the high and low molecular weight 1251-NGF-crosslinked species did not vary over a 5000-fold concentration range of cross-linking agent, which also argues that the high molecular weight entity is not a ternary complex. They also showed that the high molecular weight species could be seen after chases with unlabeled NGFor after protease treatments that abolished subsequent cross-linking of prebound 1251-NGF to the lower molecular weight complex. This suggests that high affinity binding sites do not depend on the presence of the low affinity receptor, detected as a ternary complex with lz51-NGF. Recent work by others is also consistent with our observations. In a recent paper, published after completion of this work, two monoclonal antibodies and one anti-peptide antibody specific for the rat low affinity NGF receptor also were shown to precipitate the low, but not the high molecular weight ligand-receptor complex (Meakin and Shooter, 1991). Furthermore, Radeke and Feinstein (personal communication) have also detected formation of both ligand-receptor complexes using a hydrophilic cross-linking agent, in agreement with our results. These investigators have used reducible cross-linking agent to obtain more direct evidence suggesting that the two ligand-receptor complexes represent association of lz51-NGF with different glycoproteins. After purification, iodination, and reduction, the 135K and 85K species were detected in the high and low molecular weight NGF-NGF receptor complexes, respectively. While our results indicate that there are two classes of high affinity NGF receptors, the data do not differentiate between their binding properties. Results of equilibrium binding studies indicate that each class binds NGF with the same approximate affinity (Figure 5). Measurements of kinetics of dissociation of

1251-NGF-NGF receptor complexes also suggest that the two receptor classes have similar kinetic properties (Figure 6). The assays used in the present paper, however, were limited in their resolution. Consequently, there may be significant differences between these two receptor classes that were not detected in these assays. The proposal that two antigenically distinct classes of NCF receptors exist on PC12 cells and sensory neurons is compatible with most prior characterizations of NGF-binding sites. First, it provides a comparatively straightforward explanation for the differences in ligand-binding specificities detected between low and high affinity receptors. Both BDNF and NT-3 appear to bind the lowaffinity NGF receptorwith approximately the same efficiency as NGF (Rodriguez-TCbar et al., 1990; Ernfors et al., 1990). High affinity BDNF and NGF receptors are more specific, with each binding only one trophic factor with high affinity (Rodriguez-Tkbar et al., 1990). However, it has yet to be determined whether BDNF and/or NT-3 will bind to the subclass of high affinity NGF receptors recognized by the antiREX antibody. Our results can be most easily explained by postulating the existence of additional components that modify or replace the low affinity receptor, altering ligand-binding specificity as well as affinity. Results in the present paper suggest that these modifying components directly interact with NGF. As a precedent, interleukin-2 has a receptor with two subunits, each of which is able to bind ligand independently (see Teshigawara et al., 1987). Our results support the proposal that multiple classes of NGF binding are present on the surface of responsive cells. Schechter and Bothwell (1981) first proposed that NCF binds to more than one type of NGF receptors on PC12 cells, each of which exists independentlyof ligandoccupancyor internalization. Their results and our data derived from cross-linking experiments provide strong evidence for a population of high affinity NGF receptors that bind ligand directly and are not formed by conversion of ligand-low affinity receptor complexes. In contrast to previous studies (Hosang and Shooter, 1985), we detected both low and high molecular weight ligand-receptor complexes using a membrane-impermeable cross-linking agent, suggesting that cytoplasmic factors are not required for generating this high affinity receptor complex. Substantial evidence also indicates that high and low affinity binding sites are at least partially interconvertible. First, cross-linking agents such as the lectin wheat germ agglutinin and anti-NCF IgG convert more than 90% of fast-dissociating (low affinity) receptors to slow-dissociating (high affinity) receptors, suggesting that clustering of NGF-NGF receptor complexes may account for high affinity binding (Vale and Shooter, 1982, 1983). Second, preincubation of cells with low concentrations of NGF appears to convert low affinity receptor to high affinity receptors (Landreth and Shooter, 1980; Block and Bothwell, 1983). Finally, agents that interfere with endocytosis or in-

Iiigh Affinity t #59

NGF Receptors

nibit acidification of cytoplasmic vesicles reduce and Increase, respectively, the number of high affinity binding sites (Eveleth and Bradshaw, 1988). Our results demonstrating antigenic cross-reactivity between the low affinity and one class of high affinity receptors provide direct support for models postulating interconversion of the two receptor types. These findings suggest that this high affinity receptor class forms the same 12SI-NGF-cross-linked species as the low affinity receptor in ourcross-linkingexperiments.Our results do not support models that attribute all high affinity binding solely to stages in the internalization of the low affinity receptor (Buxser et al., 1990). With these models, it is also difficult to understand why high affinity binding sites for NGF have been detected in membrane preparations and on cells maintained at 4OC (Frazier et al., 1974; Schechter and Bothwell, 1981; Vale and Shooter, 1982; Hempstead et al., 1989). In recentwork to determine the molecular relationship between low and high affinity NGF receptors, cDNAs encoding the low affinity receptor have been transfected into NR18 cells, a variant of PC12 cells selected for the absence of 1251-NGF-binding sites (Bothwell et al., 1980; Hempstead et al., 1989, 1990). Cell lines expressing the product of the transfected cDNAs were isolated and shown to express both low and high affinity binding sites for NGF. Results also suggest that the cells may express the same two distinct lz51-NGF-receptor complexes identified by Hosang and Shooter (1985). High affinity receptors and the larger 1251-NGF-associated product of cross-linking were not seen in cells expressing truncations of the receptor lacking the cytoplasmic domain (Hempstead et al., 1990). However, substantially lower amounts of 1251-NCF-cross-linked complexes were seen in cells expressing the two receptor truncations. In NR18 cells expressing wild-type receptors, NGF application induced fos mRNA (Hempstead et al., 1989). fos responses were not determined in cell lines expressing truncated forms of the receptor. In similar experiments using the human neuroblastomacell line, HTLA 230, a small percentage of cells exhibited a limited neurite outgrowth response on a complex extracellular matrix in the presence of very high concentrations of NGF (0.5 pglml) (Matsushima and Bogenmann, 1990). Recently it was shown that expression of NGF receptors in a medulloblastoma cell line led to low and high affinity NGF binding, but did not result in morphological changes (Pleasure et al., 1990). All sets of experiments indicate that a high affinity receptor and a limited biological response can be reconstituted in appropriatecell lines by expression of the low affinity receptor. The experiments described above do not eliminate the possibility that there is a second NGF-binding entity that mediates the major biological responses of neurons to NGF. First, the nature of the mutation(s) in the NR18 cell line is not known (Bothwell et al., 1980). These cells were screened after intense chemical mutagenesis for the absence of specific lz51-NGF binding,

so that cell lines expressing normal levels of this postulated second binding entity would very likely have been discarded. Possibly for this reason, the NR18 cell line lacks several additional proteins expressed in normal PC12 cells. In addition to NGF receptors, epidermal growth factor receptors and tyrosine hydroxylase are also virtually absent (Bothwell, personal communication). The reconstitution of a limited NGFregulated response implies that the receptors expressed by these cells have at least some biological activity. In previous work, the fos response to NGF has been shown to saturate at very high NGF concentrations, suggesting involvement of low affinity receptors (Milbrandt, 1986). As discussed earlier, our results do not include any evidence arguing against a role in signal transduction for low affinity and related high affinity receptors. Our results indicate only that they are not required for major responses to NGF. It will be interesting to study the antigenic properties of the high affinity receptors present in thetransfected NR18 cells. In conclusion, results in this paper present evidence for the existence of three types of NGF receptors-a low affinity receptor, a high affinity receptor related to the low affinity receptor, and a newly defined high affinity receptor that is antigenically distinct. Existence of a distinct NGF binding protein would explain notonlythese results, but alsothe work of others demonstrating dramatic differences in binding specificity between low and high affinity receptors (Rodriguez-TCbar et al., 1990). A number of experiments can now be done to test predictions of this and other models. Experimental

Procedures

Materials and Solutions Sprague Dawley rats were purchased from Simenson Laboratories Glroy, CA). Restriction enzymes,T4 DNA ligase, and other enzymes were obtained from Boehringer Mannhein Diagnostics (Houston, TX). Bacterial strains E. coli CJ 236 and MV 1190, T4 DNA polymerase and other reagents used for site-directed mutagenesis, Affiprep-protein A column, and MAPS I I solutions were from Bio-Rad (Richmond, CA). Grace’s supplemented insect medium wasobtained from CIBCO (Grand Island, NY); Excel1 insect medium was purchased from JR Scientific (Woodland, CA). Dulbecco’s modified Eagle’s medium (DMEM), horse serum, and fetal calf serum (FCS) were from the UCSF Cell Culture Facility. Lentil-lectin Sepharose and a Mono Q anion exchange column were from Pharmacia (Piscataway, NJ). Nitrocellulose membranes were from Schleicher & Schuell (Keene, NH). Endo F was obtained from Calbiochem (San Diego, CA). Alkaline phosphatase-conjugated goat anti-rabbit antibodies were from Promega (Madison, WI). lodogen and BS3 was from Pierce (Rockford, IL). Anti-NGF antibodies were from Collaborative Research (Bedford, MA), and dioctyl phthalate was from Eastman Kodak (Rochester, NY). L-[%]cysteineand Na-‘251 were purchased from Amersham (Arlington Heights, IL). All other chemicals were obtained from Sigma (St. Louis, MO). Dulbecco’s phosphate buffered saline (PBS) is 15 mM NaZPOI, 1.5 mM KH2P04, 140 mM NaCI, 2 mM KCI, 1 mM CaC&, 0.5 mM MgClz. Baculovirus Expression of the Rat and Chick Ectodomain of the NCF Receptor For construction of the baculovirus NGF receptor expression vectors, either a 1.7 kb Ncol fragment encoding the full-length

Neuron 660

rat NCF receptor (Radeke et al., 1987) or a 2.1 kb Ncol fragment encoding the chick NGF receptor (Large et al., 1989) was used. Thefirsttransmembranecodonofeachfragment(basepairs868870 of the rat cDNA and basepairs 780-782 of the chick cDNA) was mutated to a stop codon by oligonucleotide-directed mutagenesis (Kunkel, 1985) using synthetic primers 5’ACCACCGAC AACTAGATTCCTCTCTAT-3’ and 5’-ACCGCCGATAACTACATCCCTGTCTAC-J, respectively. Each fragment was then cloned into a new Ncol site generated by linker insertion at the BamHl cloning site of the Autographa California nuclear polyhedrosis virus transfer vector pVL941 (Luckow and Summers, 1989). This vector expresses exogenous proteins not fused to a leader peptide. Each baculovirus transfer vector and genomic viral DNA were cotransfected into Sf9 insects cells, and recombinant baculoviruses containing foreign NGF receptor DNA were isolated and plaque purified as outlined in detail elsewhere (Summers and Smith, 1987). Purification of the NCF Receptor Ectodomain One liter suspension cultures of Sf9 cells (about 3 x IO9 cells) were infected with the recombinant baculovirus and subsequently grown in serum-free and chemically defined medium (ExcelI). Culture supernatants were collected on day 3 postinfection, and cells were pelleted at 500 x g. The supernatants were adjusted to 0.5 M NaCl and passed over a lentil-lectin Sepharose column. Bound glycoproteins were eluted with 0.5 M a-methylmannoside in PBS. For further purification, the eluted glycoproteins were then applied to a Mono Q column equilibrated in 20 mM Tris (pH 8.0), and fractionated with a salt gradient from 140500 mM NaCl in the same buffer. The rat NGF receptor ectodomain elutes as a single peak (OD 280 nm) at 310-350 mM NaCl and was named REX. Purification of the chick NCF receptor ectodomain was performed in the same manner, and the purified protein was given the name ChEX. Endo F Digestion Infected cells were grown in cysteinefree growth medium supplemented with 0.5 mCi of [‘*S]cysteine for 3 hr on day 2 postinfection. REX was immunoprecipitated from culture supernatants using 10 ug of a monoclonal antibody recognizing the rat low affinity NGF receptor (mc192; see below) and rabbit anti-mouse IgG-Sepharose. The washed precipitates were then suspended in 1% 2-mercaptoethanol and 1% SDS and boiled for 5 min to release immunecomplexes. Samples were then incubated for 16 hr at 37OC in 0.1 M sodium acetate (pH 5.5), 50 mM EDTA, 0.5% Nonidet P-40,1 % 2-mercaptoethanol in either the absence or the presence of 0.5 U of endo F. Digests were then separated under reducing conditions on 10% SDS-polyacrylamide gels (Laemmli, 1970). Enhanced gels were dried, and autoradiography was performed according to standard procedures. Antibodies Antibodies to the rat and chick NCF receptor ectodomain (antiREX and anti-ChEX, respectively) were obtained from rabbits immunized initially with 180 Pg of purified protein in Freund’s complete adjuvant. Several subsequent booster injections contained 50 ug of each antigen in Freund’s incomplete adjuvant. Antiserum was collected 4 weeks after the initial injection and then at biweekly intervals. IgG was purified on a Affiprep-protein A column according to the manufacturer’s instructions. Preimmuneserumwascollectedfromthesame rabbitsbeforeimmunization, and IgG was purified in the same manner. Hybridoma cells secreting the monoclonal antibody mc192 were grown as described (Chandler et al., 1984). mc192 recognizes an epitope on the rat NGF receptor (Chandler et al., 1984), but does not prevent binding of NGF to PC12 cells. mc192 IgC was purified from ascites fluid on an Affiprep-protein A column using the MAPS II system according to the recommendations of the manufacturer (Bio-Rad). lmmunoblot Analysis For antigen blots, cells (see below) were washed PBS and extracted with PBS containing 1% Triton were reduced with 2-mercaptoethanol, separated

three times in X-100. Extracts on 7.5% poly-

acrylamide gels (Laemmli, 1970), and transferred to 0.2 urn nitrocellulose. After blocking in 5% dry milk dissolved in PBS, blots were reacted with 10 Fglml anti-REX IgC. Following washes and an additional incubation with alkaline phosphatase-conjugated anti-rabbit antibody, NGF receptor was visualized with 5-bromo, Cchloro, 3-indolyl phosphate and Nitro Blue Tetrazolium. Cell Culture and Bioassays Mouse L cell fibroblasts were maintained in DMEM containing 10% FCS and penicillin and streptomycin at 180 U/ml each. PCNA cells (L cells transfected with the rat NGF receptor gene) were grown in the same medium, but supplemented with 1% HAT (Radeke et al., 1987). ChNL cells were generated by transfection of mouse L cells with the chick NGF receptor gene. Briefly, an EcoRl fragment encoding the full-length chick NGF receptor was cloned into the expression vector pHBAPr-I-neo. This vector utilizes the human B-actin promotor to drive expression of foreign genes and contains the bacterial neo gene linked to the SV48 or; for selection of transfected clones (Gunning et al., 1987). ChNL cells were grown in DMEM supplemented with 10% FCS and 50 ug/ml C418. PC12 cells were grown in DMEM containing 0.5% glucose, 10% horse serum, and 5% FCS (Greene and Tischler, 1976). For bioassays, PC12 cells were primed with NGF for 2 weeks as described (Greene, 1978). Primed PC12 cells were then plated on collagen-coated plates in DMEM with 0.5% glucose, 1% horse serum, 5 rig/ml 2.5s NGF in the presence of either preimmune IgC or anti-REX IgC, both at 100 pg/ml. Primary cultures from dorsal root ganglia of newborn rats and embryonic day 8 chick were obtained as described (Fields et al., 1978; Davies, 1989). Briefly, ganglia were digested with trypsin and then dissociated mechanically. Dispersed cells were preplated twice to reduce number of nonneuronal cells, and 1 x IO4 cells per cm2 were plated on collagen-coated plastic chamber slides (Nunc, Naperville, IL). Cells were grown for 2 hr in the absenceof NCF, but in growth medium containing either preimmune IgG or anti-receptor IgC, both at 180 uglml. Then, completegrowth medium (DMEMcontaining0.5% glucose, 10% FCS, 100 U/ml penicillin and streptomycin, and 10 mM deoxyfluoruridine and 10 mM uridine as mitotic inhibitors) was added to the cells. When indicated, growth medium was supplemented with either preimmune IgC or anti-receptor IgC at 100 uglml and with or without 50 nglml 2.5s NGF. Cells were grown for 4 days, after which time phase-bright, neuritebearing cell bodies were counted. For immunoblot analysis, neuronal cells were grown for 24 hr in NGF-containing growth medium and processed as described above. Nonneuronal cells as obtained from preplating procedure were grown in NGF-free growth medium without mitotic inhibitors, and cell extracts were prepared in the same manner. Binding Studies 2.5s NGF was purified from male mouse submaxillary glands (Mobley et al., 1976) and labeled with Na-rZ51 using lodogen according to the manufacturer’s instructions. The specific activity was between 60 and 100 cpm per pg of NGF, and 95% of total counts were precipitable with trichloroacetic acid. SDS-denatured lZ51-NGF migrated as a single band on SDS-PAGE with an M, of ml3K. Binding assays were performed using conditions summarized by Vale and Shooter (1985), but the procedure for separating bound from free ligand was modified. Briefly, attached cells were washed twice with binding buffer (PBS containing glucose and bovine serum albumin at 1 mg/ml). Cells were then detached by gentle trituration, and the cell density was adjusted to 5 x IO5 cells per ml. Transfected L cells were washed twice in Ca2+- and Mg*+-free PBS and incubated for 15 min in the same buffer containing 1 mM EDTA. After dislodging cells from tissue culture dishes, PCNA and ChNL cells were centrifuged at 500 x g for 5 min and resuspended in binding buffer at a density of 5 x IO5 cells per ml. Prior to binding, cells were incubated with either preimmune IgG or anti-receptor IgG at the indicated concentrations, temperature, and time. Binding was initiated by incubating cells at either 4OC or 37OC with the indicated concentrations of rZSI-NGF. At various time points, 100 PI of cell suspension was layered onto 200 td of phthalate cushion

High Affinity 1161

NGF Receptors

(dibuturyl phthalate and dioctyl phthalate in a 3:2 ratio) in 400 ~1 microfuge tubes (Robbins Scientific, Sunnyvale, CA). Following centrifugation for 5 s in a Beckman microfuge, the tip of the tube containing cell-associated counts was cut off. Free ligand (on top of the phthalate cushion) and bound counts were counted separately in a Beckman gamma counter at 60% counting efficiency. This separation method (Segal and Hurwitz, 19m was compared with the sucrose gradient centrifugation (seeVale and Shooter, 1985) and gave similar results. For each experiment, specific binding was determined as the difference in binding of ‘?NGF in the presence and absence of 10 pg/ml unlabeled NGF. Nonspecific binding ranged from 5% to 30% of total binding. All determinations were done in triplicate, and dataare presented as mean f SEM. For Scatchard analysis, untransformed data were fitted using a nonlinear least-squarescurve-fitting program (Murlas et al., 1982). Cross-Linking of NCF to PC12 Cells For cross-linking experiments, PC12 cells were prepared as described for binding assays and resuspended at 3 x ‘lo6 cells per ml of binding buffer. When indicated, cells were preincubated with either preimmune IgC or anti-REX IgG, both at 50 pg/ml for 60 min at 4OC. Nonspecific binding was performed in the presence of 10 pglml unlabeled NCF. ‘*+NCF was then added to 1 nM, and after an additional 60 min, the pH was adjusted to 8.5. Cross-linking was achieved by addition of BS3 to a final concentration of 0.4 mM. After 30 min on ice, the reaction was stopped by the addition of glycine to 2 mM. Cells were then washed three times with binding buffer and subsequently extracted with PBS containing 1% Triton X-100 for 30 min at 4OC. Supernatants were immunoprecipitated with 20 pglml of either anti-NCF antibodies, preimmune IgC, or anti-Rex IgG and protein A-Sepharose. The washed precipitates were resuspended in reducing sample buffer and boiled for 5 min. Supernatants were then fractionated on 7.5% SDS-polyacrylamide gels (Laemmli, 1970). Fixed and dried gels wereautoradiographed for4days according to standard procedures. Acknowledgments The authors would like to thank Dr. Eric Shooter for providing hybridoma cells secreting the monoclonal antibody mc192, Drs. Monte Radekeand Stuart Feinstein for providing PCNAcells, Dr. David Schubert for providing PC12 cells, and Dr. Thomas Large for his help in generating the ChNL cells. We also wish to thank Dr. Max Summers for the generous contribution of Sf9 insect cells, plasmid pVL941, and baculovirus Autographacalifornia nuclear polyhedrosisvirus (AcNPV strain E2) and Dr. Dave Morgan and Mario Chamorro for their assistance with the baculovirus expression system. Wearegrateful to Dr. James Robertsand Kirk Riemer for help with the Scatchard analysis and Drs. Michael Ignatius, William Mobley, and Robert Edwards for helpful discussions. Drs. Douglas Clary, Zach Hall, Kevin Jones, Frances Leftort, Gene Napolitano, and Karla Neugebauer and Mrs. Monica Vetter gave comments on the manuscript. The secretarial sup port of Marion Meyerson was also greatly appreciated. G. W. was in part supported by Deutscher Akademischer Auslandsdienst, Centechnologie Programm. This work was supported by National Institutesof Health grant NS21824and the Howard Hughes Medical Institute. L. F. R. isan investigatorof theHoward Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

January

28, 1991; revised

February

20, 1991.

Block,

Trophic

T., and Bothwell,

factors

and neuronal

M. (1983). The nerve

growth

survival. factor

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Note

Added

in Proof

In recent work, the trk protein (135K) has been shown by two groups to bind NGF and to be a major constituent of the higher molecular weight (ISOK) ligand-receptor complex detected in affinity cross-linking experiments fD. R. Kaplan, B. Hempstead, D. Martin-Zanca, M. Chao, and L. Parada, personal communication; R. Klein, S. Jing, V. Nanduri, E. Q’Rourke, and M. Barbacid, personal communication).