EXPERIMENTAL

112,183-194

NEUROLOGY

(1991)

Rat NGF Receptor Is Recognized by the Tumor-Associated Antigen Monoclonal Antibody 217~ G. FERRARI, Fidia

Research

Laboratories,

M. FABRIS, P. POLATO, S. D. SKAPER, M. G. FIORI, AND Q. YAN**’ Via Ponte Washington

della Fabbrica University,

3/A, 35031 Abano School of Medicine,

The expression of the epitope recognized by the tumor-associated antigen monoclonal antibody 21’7~. increased on cultured rat Schwann cells with time. The same phenomenon has previously been reported for the rat nerve growth factor (NGF) receptor by using monoclonal antibody 192-IgG. The distribution of 217~ antibody immunoreactivity closely paralleled that observed for NGF receptors on NGF-primed pheochromocytoma (PC 12) cells in culture and a number of central neurons in vivo. Immunoprecipitation of affinity-labeled NGF receptors was used to examine whether these two antibodies shared common or unique antigens. Both the quantitative data and the electrophoretic mobility of the immunoprecipitated “‘1-NGF/receptor complex indicate that the antigen recognized by the 217~ mAb monoclonal antibody is, in fact, the NGF receptor. Furthermore, binding studies indicated that 192-&G and 2 17~ recognize different epitopes on the same molecule. The characterization of the 217~ antibody should provide a valuable new tool in the study of NGF receptors. 0 1991 Academic Press, Inc.

INTRODUCTION Nerve growth factor (NGF) is a well-defined protein which plays a critical role in supporting survival, neurite outgrowth, and expression of phenotypic traits of selected populations of neurons throughout the life of the organism (16, 25, 47). Although binding to specific receptors on the cell surface seems to be the initial step in NGF action, the primary mechanism by which the binding signal is transduced is still unknown. The presence and binding properties of NGF receptors have been extensively characterized on peripheral sympathetic neurons (4) and neural crest-derived sensory neurons (42). NGF receptors have also been localized to certain cholinergic neuronal populations in the central nervous system (CNS), in both the developing and the adult animals (22,31-33,35,40,46,48-50). NGF receptors have ’ Present CA 91320.

address:

Amgen,

Inc.,

Amgen

Center,

Thousand

Terme (PD), Italy; St. Louis, Missouri

and *Department 63110

of Pharmacology,

also been identified on Schwann cells, both in uivo (44) and in vitro (11, 52). Many tumor cell lines, such as clonal rat pheochromocytoma PC12 cells (5, 14, 20, 24, 37) and the A875 clonal line of human melanoma (29), also express NGF receptors. The protein species that constitutes the functional NGF receptor have not been fully characterized. NGFresponsive cells have two apparent binding sites. The high-affinity (Kd = 10-l’ M) or type I site is generally associated with the internalization of NGF and transduction of the biological message (5). The low-affinity (& = lo-’ M) or type II site also exists on responsive cells and is the only class found on nonresponsive cells (43). The primary kinetic difference between these two binding sites lies in their dissociation rates, with the high-affinity receptor having a much slower rate of NGF dissociation (37). A monoclonal antibody (192-IgG) presumably directed against the low-affinity form of the rat NGF receptor has been obtained (9). Both the 192-IgG antibody and NGF appear to bind to the NGF receptor in a noncompetitive fashion both in vitro and in vivo and are cotransported in a similar manner from axonal terminals to the cell body (21,45). The application of 192-IgG for immunoprecipitation and immunohistochemical procedures has proven useful in characterizing and localizing NGF receptor-bearing cells in the nervous system (46,48-50). The availability of other antibodies directed to this antigen would provide an additional tool for studying NGF receptors and the interaction between NGF and its receptors biochemically and pharmacologically. We report here that the monoclonal antibody 217~ (217~ mAb) (28), while first identified as an antibody against a tumor-associated antigen, also recognizes the NGF receptor. We demonstrate that 217~ mAb recognizes a different epitope on the NGF receptor than does 192-IgG. METHODS Reagents and Solutions NGF (2.5 S) was purified from male mouse submaxillary glands by the method of Bocchini and Angeletti (6).

Oaks,

183

0014-4886191

Copyright All

rights

0

1991 of reproduction

by

Academic in any

form

$3.00

Press, Inc. reserved.

FIG. 1, , Immu lnohistochemical staining of cultured rat Schwann cells for 217~ mAb and SlOO. (A, B) 217~ mAb staining of cultu ,188 land8 days after ’ plating, respectively. (C) SlOO staining of Day 8 cultures. Note the increased staining intensity of Schwann cells by 217~ :mP ib from Day 1 to 1Day 8 in vitro and the failure of fibroblasts to stain with either antibody (arrows). 184

185

A NOVEL RAT NGF RECEPTOR ANTIBODY ‘251-NGF was purchased from Amersham (Arlington Heights, IL) with a specific activity of 1800 cpm/fmol. Mouse anti-rat NGF receptor monoclonal antibody (192-IgG) (9) (hybridoma cells kindly provided by Dr. Eugene Johnson) and polyclonal antibodies against 192-IgG were affinity-purified as previously described (48). Monoclonal antibody 217~ hybridoma-conditioned medium, as described by Peng et al. (28), was a gift of Dr. J. de Vellis. Rabbit antiserum to bovine SlOO protein was obtained from DAK0 Laboratories. Anti-mouse IgG ABC-peroxidase kits were purchased from Vector Laboratories (Burlingame, CA). n-Octylglucoside was from Boehringer-Mannheim (Indianapolis, IN), and formalin-fixed Staphylococcus aureus (Pansorbin) from Calbiochem (La Jolla, CA). Cell Cultures Schwann cells were prepared from neonatal rat sciatic nerves according to the method of Brockes et al. (8). In brief, pooled nerves were incubated three times for 15 min with phosphate-buffered saline (PBS) containing 0.25% (w/v) trypsin and 0.1% (w/v) collagenase. The protease activities were stopped by addition of 2 ml of Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS). Cells were dissociated by trituration through a syringe with a 22-gauge needle, passed through a 20-pm Nytex sheet, centrifuged, resuspended in DMEM/lO% FCS, and plated on poly-L-lysine (0.1 mg/ml)-coated dishes at 2-3 X lo6 tells/35-mm dish. After 48 h, the cells were treated with 10e5 M cytosine arabinoside for 2 days. This treatment selectively destroys rapidly dividing fibroblasts, while having limited effects on the Schwann cell population. For longer term cultures, cells were grown in medium consisting of DMEM and Ham’s F12 (l:l, v/v) with 25 mM glucose, 2 n&f glutamine, 0.5 U/ml penicillin, 0.5 pg/ml streptomycin, and the following supplements, as described by Bottenstein and Sato (7): 5 pg/ml insulin, 10 pg/ml transferrin, 20 nM progesterone, 100 ph4 putrescine, 30 nM sodium selenite. After 8 days, the cultures were 8590% enriched in Schwann cells as determined by visual inspection (see Fig. 1). PC12 cells (a gift of Dr. Lloyd Greene) were grown as described (15) on collagen-coated tissue culture dishes in RPM1 1640 containing 5% FCS and 10% heat-inactivated horse serum. The cultures (5 X lo5 tells/60-mm dish) were supplemented with 50 rig/ml of NGF for 3 weeks to obtain cells with extensive neurite outgrowth. Immunohistochemistry Cells were rinsed three times with PBS, fixed with paraformaldehyde (4% in PBS) for 30 min at room temperature, and washed three times with PBS. For demonstrating the intracellular antigen SlOO, cells were per-

meabilized with methanol for 10 min at -20°C. After incubation with horse serum (2% in PBS) for 30 min at room temperature, primary antibodies at concentrations of 4 pg/ml for 192-IgG, 1:200 dilution for 217~ hybridoma-conditioned medium, or 1:400 dilution for anti-S100 antibodies were added with incubation overnight at 4°C. Primary antibodies were removed with three PBS rinses and then processed for immunoperoxidase reaction. For 192IgG and 217~ mAb staining, the cultures were incubated with rat-adsorbed biotinylated horse antimouse IgG for 1 h, washed, and incubated with avidinhorseradish peroxidase using the Vectastain ABC kit according to the manufacturer’s instructions. A solution containing 0.0066% hydrogen peroxide plus 0.5 mg/ml 3,3’-diaminobenzidine tetrahydrochloride in 0.1 M phosphate buffer (pH 7.4) was used as substrate for the immunolocalized peroxidase. For SlOO staining the peroxidase-antiperoxidase method (41) was used, with goat anti-rabbit at 1:80 and peroxidase-antiperoxidase (rabbit) at 1:200. Sprague-Dawley rats, 2-5 months old, were used throughout. Rats were perfused via intraaortic cannula with 100 ml of saline containing 0.1% heparin, followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. Brains were then postfixed in the same solution at 4°C for 4 h and kept overnight in 20% sucrose/0.1 M sodium phosphate buffer. Coronal sections (14 pm) were cut on a cryostat (Reichert-Jung, Mod 2700-Frigocut) and indirect immunohistochemistry was carried out using 192-IgG (4 pg/ml), 217~ mAb (1:200), or a mixture of 192-IgG (4 pg/ml) and 217~ mAb (1:200) as primary antibodies and followed by the avidin-biotin-peroxidase complex method as described above. Differential staining intensities with 192-IgG, 217~ mAb, or a mixture of the two were determined by microscopic examination of stained tissue sections processed side by side in exactly the same fashion. Each experiment was repeated at least four times with identical results. Immunoprecipitation

of NGF Receptors

A modification of a previously described protocol (46, 48) was used. Naive PC12 cells suspended in PBS, pH 7.2 (0.5 ml, lo7 cells/ml), were incubated with 0.5 ti ‘251-NGF in the presence or absence of 250 nM unlabeled NGF at 35°C for 60 min. The receptor-bound NGF was crosslinked by a 20-min exposure to 10 mM I-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The cells were then washed with 2 ml of Tris-HCl-buffered saline (50 n&f Tris-HCl/lBO mM NaCl, pH 7.6) twice and solubilized by adding 0.5 ml of 2% n-octylglucoside with 1 mM phenylmethylsulfonyl fluoride/0.5% bovine serum albumin (BSA)/PBS, pH 7.2, at room tempera-

186

FIG. neurite

FERRARI

2. NGF-primed PC12 cells stained network with either antibody.

with

192-IgG

ET

AL.

(A) and 217~ mAb.

ture for 60 min. The mixtures were centrifuged at 12,000g for 15 min and the supernatants were incubated ‘251-NGF afwith monoclonal antibodies to precipitate finity-labeled receptors. After 60 min, 5 ~1 of 10% Pan-

(B).

Note

the intense

immunoreactivity

of both

cell bodies

sorbin preadsorbed with anti-mouse IgG secondary bodies was added for 45 min. The mixtures were centrifuged and the pellets were resuspended in of solubilizing buffer. The suspension was layered

and

antithen 100 ~1 onto

--.--

-

_...

9

. w.

I

-

. -1

L

FIG. 3. Immunohistochemical staining of the medial septal nucleus and vertical (B), a 192-IgG/217c mAb mixture (C), or nonspecific staining with omission ofthe first the higher magnification of the indicated areas. 187

limb of the diagonal band with 217~ mAb (A), 192-IgG antibody(D). Scale bar represents 100 pm. Inserts show

188

FERRARI Absorption

%

Area

60

%

90

l------*-.----l ,”

60

192

217~

192i217c

Ab FIG. 4. Quantitation of diagonal band immunostaining with 192IgG, 217~ mAb, or a combination of the two. The diagonal band areas from Fig. 3 were analyzed by computer-assisted morphometry and microdensitometry (1) using an AT-IBAS system (Kontron). 192-IgG staining and 217~ staining were processed at the same time using adjacent sections. Histograms display either staining intensity (solid bars) or immunoreactive area (hatched bars).

a 250~~1 cushion of 10% sucrose/l.3% n-octylglucoside in PBS in 400-~1 polyethylene tubes and centrifuged at 12,000g for 30 s. The tubes were immediately frozen in a methanol/dry ice bath, and tips, with pellets, were cut off and their radioactivities were measured.

SDS-PAGE

AL.

spectively) and mixed gently. After 60 min on ice, 200 ~1 of ‘251-NGF (1 ti) was added, and incubation continued for another 60 min on ice. A 500-fold excess of unlabeled NGF was included in parallel tubes for determination of nonspecific binding, which accounted for less than 10% of control binding in all cases. Cell-associated ligand was assayed by layering a 100~~1 sample in triplicate onto 175 ~1 of 10% sucrose/PBS/BSA in a microfuge tube and centrifuging for 2 min, followed by freezing in an ethanol/dry ice bath. Tips containing the cell pellet were cut from the frozen tubes and counted in a gamma counter to determine the amount of cell-bound ligand. The upper portion of the tube was counted to determine the corresponding amount of free 12’1-NGF. Antibody competition binding experiments were carried out as follows. The 217~ mAb was affinity-purified from hybridoma-conditioned medium by a protein G column. Iodination of 192-IgG was done using Na1251 (Amersham) and Iodo-gen reagent (Pierce) according to the manufacturer’s instructions. PC12 cells were washed with PBSIBSA, resuspended in PBSlBSA (5 X 10” cells/ml), and incubated with 2 nM ‘251-192-IgG (1100 cpm/fmol) at 37°C for 1 h in the presence of different amounts of nonradioactive 192-IgG or 217~ mAb in a total volume of 0.4 ml. Aliquots (100 ~1) of cell suspension in triplicate were layered onto 250 ~1 of 10% sucrose/PBS/BSA in a microfuge tube and centrifuged for 30 s. Cell pellets were counted as above. RESULTS

Autoradiography

After determining their radioactivities, pellets were resuspended in 100 ~1 of water. Half of each 100~~1 sample was mixed with 50 ~1 of either reducing (5% p-mercaptoethanol) or nonreducing SDS-PAGE sample buffer, heated in a boiling water bath for 5 min, and electrophoresed on 7% polyacrylamide gels (23). Gels were stained with Coomassie brilliant blue R, destained, and dried. Autoradiograms were made with Kodak XOmat AR film and a DuPont lightning-plus intensifying screen.

NGF and NGF Receptor Antibody

ET

Binding

Assays

The effects of 192-IgG and 217~ mAb on the binding of NGF to its receptor were performed according to Chandler et al. (9). In brief, PC12 cell monolayers were washed three times with 0.1% (w/v) BSA in PBS and mechanically dislodged. Cells were washed two additional times by centrifuging in PBS/BSA and resuspended in PBS/BSA at 4 X lo6 cells/ml. Cells and all reagents were cooled to 4°C before use. Aliquots (100 ~1) of cell suspension were added either to 100 ~1 of PBS/ BSA (control) or to 100 ~1 of 4-fold-concentrated antibody solutions (1:25 and 1:2.5 for 192-IgG and 217c, re-

Schwann cells, 1 and 8 days after plating, were stained with both the 217~ mAb and antibodies to SlOO (Fig. 1). At 1 day, the staining of Schwann cells with 217~ mAb was faint, but increased in intensity at 8 days. Staining was restricted to bipolar, spindle-shaped cells. In parallel cultures, cells with similar morphology were stained with anti-S100 antibodies (a generally accepted marker for Schwann cells). A similar time course of staining was obtained with Schwann cells from adult rat (not shown). In contrast, cells with fibroblast-like morphologies did not react with either the 217~ or SlOO antibodies. Also, no staining was evident on &day cultures following incubation with either mouse myeloma supernatant or normal rabbit serum (negative controls). This up-regulation of 217~ mAb antigen on Schwann cells in culture shares a similar pattern of staining with the rat NGF receptor monoclonal antibody 192-IgG (Ref. (ll), also our unpublished observations), suggesting that 217~ mAb recognizes the NGF receptor. The pattern of staining of 217~ mAb, in comparison to that of 192-IgG, was examined on NGF-primed PC12 cells and basal forebrain cholinergic neurons, two wellestablished NGF responsive cells. When NGF-primed PC12 cells were stained with either 192-IgG (Fig. 2A) or

FIG. 5. Immunohistochemical staining of newborn (PO) rat cervical spinal cord with 192IgG (A), 217~ mAb (B), and a 192-IgG1217c mAb mixture (C). Nonspecific staining is shown in (D), as done in Fig. 3. (E) Staining of nucleus basalis magnocellularis with 217~ mAb; (F) a higher magnification of the indicated area in (El. 3V, third ventricle; ic, internal capsule. Scale bars represent 100 pm. 189

190

FERRARI

ET

AL. 350

300

s E z m

250

200

0’ E B Z s

150

100

50 7

no Ab

1:1000

‘1

1:lOO

1:lO

5pg

1924gG

217c dilution

~, 192lgo, + -‘+

217~ -

-

-

NO Ab II,

FIG. 6. Immunoprecipitation of NGF receptor from PC12 cells by 217~ mAb and 192-IgG. Experimental details are described under Methods. (A) Quantitation of NGF receptor precipitated. (B) Autoradiogram of ‘%I-NGF receptor immunoprecipitated by 192-IgG and 217~ mAb run on a 7% SDS-PAGE gel. Lanes with “+” indicate the presence of 1 PM unlabeled NGF in the initial Iz51-NGF binding step. The molecular weight standards are shown on the right. Some noncross-linked “‘1-NGF run on the dye-front. The iz61-NGF/receptor complex runs around 90 and 200 kDa.

0

1 CONTROL

192 IgG

217c

FIG. 7. Differential effect of 217~ and 192-IgG antibodies on the binding of ‘*‘I-NGF to PC12 cells. Cells were incubated with PBS/ BSA (control), 192-IgG, or 217~ for 60 min (4”(Z), followed by addition of iz51-NGF (0.5 nM) without or with a 500-fold excess of nonradioactive NGF. Samples were assayed for bound ‘251-NGF after 60 min. Nonspecific binding was subtracted from all points. Data are presented as the percentage of control NGF binding (no antibody present); 100% = 2230 + 148 cpm (1.24 + 0.083 fmol, triplicate determinations in each of three experiments).

217~ mAb (Fig. 2B), a similar distribution of immunoreactivity was observed. The 217~ mAb, like 192-IgG, stained intensely not only the cell bodies, but also the neurite network. The distribution of 192-IgG and 217~ mAb immunoreactive neurons in cryostat sections of the rat basal forebrain is shown in Figs. 3A-3D. In the diagonal band of Broca both antibodies showed similar patterns. of staining, with immunoreactivity distributed on the cell bodies and along dendritic processes, as well as varicose fibers, presumably the projecting axons of these neurons; nonspecific staining was considerably weaker (Fig. 3D). When a mixture of the two antibodies was utilized, a stronger staining of these neurons than with either 217~ mAb or 192-IgG was observed (Fig. 3C). This latter finding was a very reproducible phenomenon and was confirmed by direct quantitation of the immunostained tissue sections processed in exactly the same way. The staining intensity of the diagonal band area with 217~ mAb was less than that with 192-IgG (Fig. 4), as seen light microscopically. However, the immunoreactive area measured with either antibody or in combination was the same (Fig. 4), indicating that 217~ mAb and 192-IgG recognize the same cellular structures. Similar results were obtained for newborn rat cervical spinal cord (Figs. 5A and 5B), another area reported to be NGF receptor positive (49). Here also, the staining pattern with 217~ mAb closely followed that of 192-IgG

A NOVEL

u --t-

0.25

1 Non-labeled

10

RAT

NGF

192lgG 217~

100 antibody

1000 (nM)

FIG. 8. The 217~ mAb does not inhibit binding of ‘251-192-IgG to PC12 cells. Naive PC12 cells were incubated with 2 nM ‘251-192-IgG in the presence of different amounts of nonradioactive 192-IgG or affinity-purified 217~ mAb. After 60 min at 37°C cells were separated from unbound ‘x51-192-IgG and bound radioactivity was determined. Values are the mean + SD in triplicate. Two other experiments gave similar results.

RECEPTOR

191

ANTIBODY

covered (Fig. 6A). The SDS-PAGE/autoradiographic analysis of the 217~ mAb-immunoprecipitated 1251NGFlreceptor complex showed a major band at 90 kDa and a minor band at 200 kDa under reducing conditions, the same as those for 192-IgG (Fig. 6B). Under nonreducing conditions, the 1251-NGF/receptor complex precipitated by both 192-IgG and 217~ mAb displayed the same, slightly lower molecular weight, species reported previously (44) (data not shown). Because the 192-IgG antibody has been reported to increase NGF binding to PC12 cells at 4°C (9), the effect of 217~ mAb on this parameter was assessed.Figure 7 shows that 217~ mAb (used at a 1:lO dilution), unlike 192-IgG, did not increase NGF binding to PC12 cells at 4”C, suggesting that these two monoclonal antibodies recognize a different epitope on the NGF receptor. This possibility was directly tested by an antibody competition binding assay. The binding of ‘251-192-IgG to PC12 cells was inhibited by coincubation with nonradioactive 192-IgG, but not by the same amount of 217c mAb, even at a 500-fold excess (Fig. 8). As both antibodies immunoprecipitate the same ‘251-NGF cross-linked receptor, 192-IgG and 217~ mAb likely recognize different epitopes on the same molecule. DISCUSSION

and the combination of 192-IgG and 217~ mAb produced a more intense staining (Fig. 5C). The nucleus basalis magnocellularis stained with 217~ mAb (Figs. 5E and 5F) displayed a pattern similar to that already described for 192-IgG (22). This latter distribution was identical to that observed for choline acetyltransferase (3), suggesting that 217~ mAb recognizes basal forebrain cholinergic neurons bearing NGF receptors. Since the 217~ mAb immunohistochemical study revealed staining patterns identical to those of 192-IgG in a number of cell types, the possibility that these two antibodies recognize the same antigen was examined. An unequivocal test of the recognition of the NGF receptor by 217~ mAb would be immunoprecipitation of affinity-labeled NGF receptors. Naive PC12 cells in suspension were affinity-labeled with ‘251-NGF and then the NGF/receptor complex was covalently cross-linked. The solubilized 1251-NGF/receptor complex thus obtained was readily immunoprecipitated by both 192-IgG and 217~ mAb (Fig. 6). Nonspecific counts with excess unlabeled NGF in the initial binding step or with no primary antibody were quite similar, being less than 3% of the total counts immunoprecipitated by 192-IgG (Fig. 6A). The ability of 217~ mAb to immunoprecipitate NGF receptor was concentration dependent: using a 1:lO dilution of 217~ mAb hybridoma supernatant, about 80% of the 192-IgG precipitable counts were re-

The 217~ monoclonal antibody was first identified as an antibody against a tumor-associated surface antigen (28). Recent evidence, however, indicates that 217~ mAb also recognizes an antigen expressed by Schwann cells, as well as nonneuronal cells derived from newborn rat olfactory bulb in vitro (12). Expression of the antigen recognized by 217~ mAb is shown here to increase on Schwann cells with time in culture. Furthermore, the distribution of 217~ mAb immunoreactivity was identical to that observed for NGF receptors using the monoclonal antibody 192-IgG on NGF-primed PC12 cells and known NGF receptor-positive CNS neurons in tissue sections. The identical pattern of staining of 217~ mAb and 192-IgG may be explained in one of two ways: (a) the same cells express both the NGF receptor and an antigen specifically recognized by 217~ mAb; or (b) the 217~ antigen is, in fact, the NGF receptor. A similar question was raised previously, although not answered, using neural crest-derived cells (38). The present finding, that 217~ mAb immunoprecipitates an ‘251-NGF/receptor complex unequivocally supports the second possibility, that 217~ mAb recognizes the NGF receptor. The 217~ mAb, when used at the lowest dilution (1:lO of the hybridoma supernatant), yielded a recovery of about 80% of the 192-IgG immunoprecipitable counts of 1251-NGF/receptor complex. Possible explanations for this observation can be that: (i) since the amount of 217~

192

FERRARI

ET AL.

mAb protein in the hybridoma supernatant was ungene copy (30) represents the major binding component known, increasing the amount of 217~ mAb might result for both the high- and low-affinity forms of the receptor in the same amount of NGF receptor precipitated, this (14, 17). It has been proposed that the high-affinity is consistent with the 217~ mAb dose-response curve; NGF receptor is composed of the low-affinity protein (ii) because the secondary antibodies used were raised associated with an additional protein (17, 18, 20). Reagainst 19%IgG and affinity-purified by a 192-IgG col- cent data indicating that the low-affinity NGF receptor umn, their affinity was higher for 192-IgG than for 217~ is also a low-affinity brain-derived neurotrophic factor mAb. Under optimal conditions, 217~ mAb might thus (BDNF) receptor (35) suggest that this receptor might immunoprecipitate the same amount of NGF receptors be a binding component used by a variety of ligands, as 192-IgG. NGF and BDNF being two of them (35). Furthermore, The use of SDS-PAGE autoradiography and immuthe mechanism that leads to high-affinity binding also noprecipitation clearly demonstrated the identity of the markedly increases the ability of the receptor to discrimantigen for 217~ mAb. In addition, our data indicate inate between the two ligands (35). Because 217~ mAb that 217~ mAb recognizes a different epitope on the appears to recognize a uniquely different epitope of the NGF receptor than does 192-IgG, because (i) a combinaNGF receptor, cross-linking experiments using labeled tion of 192-IgG and 217~ mAb stained more strongly the BDNF and immunoprecipitation with this antibody NGF receptor-positive neurons than did either antibody should prove useful in better characterizing the BDNF alone under identical conditions; (ii) the two monocloreceptor and, perhaps, other related members of the nal antibodies differed in their capacity to increase NGF NGF-BDNF gene family (19, 27). binding to PC12 cells at 4’C; and (iii) most importantly, The results described here also raise another issue of 217~ mAb did not compete with ‘251-192-IgG binding to some biological relevance. NGF receptors are expressed PC12 cells. Interestingly, preliminary experiments indiby many tissues and cell types that lack any apparent cate that 217~ mAb, unlike 192-IgG (9), does not block physiological responsiveness to NGF (32, 36, 49). The neurite outgrowth induced by NGF on PC12 cells (G. levels of NGF receptor are also highly regulated during Ferrari, unpublished observations). This provides yet development (23,48,49). It is interesting that many tufurther evidence that 217~ mAb identifies a different mor cell lines, in addition to PC12 cells and A875 cells, epitope of the NGF receptor than is identified by express an antigen (12,28) which appears to be identical 192-IgG. to the NGF receptor. Thus, the NGF receptor itself, beThe present findings describe the second monoclonal sides its traditional role in mediating neuronal actions antibody against the rat NGF receptor, whose determiof NGF, may possesssome specialized or as yet unidentinant is unique from that of 192-IgG. Thus, the 217~ fied role(s) in the regulation of nonneuronal cell growth mAb will be a very important tool for NGF receptor and differentiation, e.g., in developmental or transforstudy. For example, using 192-IgG and 217~ mAb, a sen- mation processes, as is now coming to be recognized for sitive and quantitative two-site enzyme immunoassay NGF action in the immune system (2). for NGF receptors can be developed, which will greatly In conclusion, we have shown that the antigen recogsimplify the existing lz51-NGF cross-linking immunonized by the monoclonal antibody 217~ is the NGF reprecipitation assay (44,48). This antibody may be use- ceptor. The results of immunohistochemistry, immunoful in exploring NGF receptors in neuropathological precipitation, and competition binding studies indicate states. As a result of lesioning or the aging process, NGF that 217~ recognizes an epitope on the NGF receptor receptor immunoreactivity in rat basal forebrain is di- different than that recognized by 192-IgG, the wellminished (13,39). It may be possible to obtain, by using characterized and widely used monoclonal antibody to a combination of 192-IgG and 217~ mAb, a clearer de- the rat NGF receptor. The identification of 217~ as an tection of NGF receptor-positive neurons (in comparianti-NGF receptor antibody should provide a valuable son to 192-IgG) in normal and experimental disease new tool for NGF receptor study and has important imconditions. The reported binding of 217~ mAb to a hu- plications for interpretation of previous studies using man glioma cell line (28) also encourages a determinathe 217~ antibody. tion of human NGF receptor recognition by this antibody. It is important to emphasize that the binding under ACKNOWLEDGMENTS consideration in this study may be predominantly to the We thank Dr. Jean de Vellis for the gift of 217~ monoclonal antilow-affinity form of the NGF receptor. At present, the body, Dr. Eugene Johnson for many reagents, and Drs. Maria Grazia significance of the low-affinity receptor, and its relation Nunzi and Gino Toffano for helpful discussions in the course of this to the high-affinity NGF receptor, remains a subject of work. We also thank Dr. Diego Guidolin for performing the quantitasome controversy. Molecular cloning of the NGF recep- tive image analysis and Antonia Bedeschi for preparation of the mantor indicates that the protein encoded by this single uscript.

A NOVEL

RAT

NGF

REFERENCES 1.

AGNATI, L. F., K. FUXE, F. BENFENATI, I. ZINI, M. ZOLI, L. FABBRI, AND A. HARFSTRAND. 1984. Computer assisted morphometry and microdensitometry of transmitter identified neurons with special reference to the mesostriatal dopamine pathway. I. Methodological aspects. Acta Physiol. Stand. (S’uppl.) 532: 5-32.

2.

ALOE, L. 1988. The effect of nerve growth on mast cells in vivo. J. Neuroimmurwl.

3.

ARMSTRONG, D. M., C. B. SAPER, A. I. LEVEY, B. H. WAINER, AND R. D. TREEY. 1983. Distribution of cholinergic neurons in rat brain: Demonstrated by the immunocytochemical localization of choline acetyltransferase. J. Comp. Neurol. 216: 53-68.

4.

BANERJEE, S. P., S. H. SNYDER, P. CUATRECASAS, AND L. A. GREENE. 1973. Binding of nerve growth factor in sympathetic ganglia. Proc. Natl. Acad. Sci. USA 78: 7535-7539. BERND, P., AND L. A. GREENE. 1984. Association of iz51-nerve growth factor with PC12 pheochromocytoma cells: Evidence for internalization via high-affinity receptors only and for long term regulation by nerve growth factor of both high and low affinity receptors. J. BioZ. Chem. 259: 15,509-15,514. BOCCHINI, V., AND P. V. ANGELETTI. 1969. The nerve growth factor purification as a 30000 molecular weight protein. Proc. Natl. Acad. Sci. USA 64: 787-794.

5.

6.

7.

8.

9.

10.

11.

12.

factor

and its antibody

RECEPTOR 18.

HEUER, J. G., S. FATEMIE-NAINIE, E. F. WHEELER, AND M. BOTHWELL. 1990. Structure and developmental expression of the chicken NGF receptor. Dev. Biol. 137: 287304.

19.

HOHN, A., J. LEIBROCK, K. BAILEY, AND Y.-A. BARDE. 1990. Identification and characterization of a novel member of the nerve growth factor/brain-derived neurotrophic factor family. Nature (London) 344: 339-341.

20.

HOSANG, M., AND E. M. SHOOTER. 1985. Molecular tics of nerve growth factor receptors on PC12 Chem. 260: 655-662.

21.

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