Journal of Neuroscience Research 27520-632 (1990)

Reverse-Phase High-Performance Liquid Chromatography of Nerve Growth Factor Receptor-Like Proteins Identified With Monoclonal Antibodies D.-E. Shan, C.E. Beck, K. Werrbach-Perez, and J.R. Perez-Polo Department of Human Biological Chemistry and Genetics and the Marine Biomedical Institute, University of Texas Medical Branch, Galveston Human neuroblastoma SK-N-SH-SY5Y (SY5Y) and rat pheochromocytoma PC12 cells are model cell lines used in the study of nerve growth factor (NGF) effect. The effects of NGF are initiated by binding to cell surface receptors (NGFK). The amino acid sequence for NGFR has been deduced based on the identification of a single gene for NGFR. However, there are two kinds of NGF binding activities and several reported molecular weights of NGFR. We report here on the demonstration of NGFR-like proteins from PC12 and SY5Y cells by sequential lectin chromatography, reverse-phase HPLC, and SDS-PAGE analysis of immunoprecipitates obtained with NGFR-specific monoclonal antibodies. For both human and rodent NGFR, there was a tendency for the higher molecular-weight species of NGFR-like proteins to be eluted in more hydrophobic fractions. Also, the expression of different species of NGFR could be modified by treatment with retinoic acid (RA). These results are consistent with the hypothesis that the different molecular species of NGFR may result from the generation of a truncated form of NGFR, the presence of sugar residues on the NGFK protein, dimer formation between NGFR, or the association of NGFR with a receptor-associated protein. Key words: NGF, NGF receptor, reverse-phase HPLC, hydrophobicity, receptor-associated protein, purification INTRODUCTION Nerve growth factor (NGF) regulates neuronal cell death, neurite extension, and synapse formation during the development of sensory and sympathetic ganglia and is trophic to some neurons in the central nervous system (Thoenen and Barde, 1980; Levi-Montalcini, 1987; Whittemore and Seiger, 1987). The effects of NGF are initiated by its binding to cell surface receptors (NGFR). 0 1990 Wiley-Liss, Inc.

The rat pheochromocytoma PC12 cell line is a model cell line used to study the structure of NGFR and the effects of NGF (Greene and Tischler, 1976; Greene, 1984; Levi et al., 1988). Human neuroblastoma cell lines are another model for the study of NGFR structure and NGF effects (Perez-Polo et al., 1982; Perez-Polo and Haber, 1983). These cell lines are genetically stable, independent of NGF for cell survival, and reversibly responsive to NGF. Similar to the findings in PC12 cells, treatment of neuroblastoma cells with NGF induced neurite outgrowth (Perez-Polo et al., 1979; Sonnenfeld and Ishii, 1982), increased protein synthesis (Perez-Polo et al., 1982; Sonnenfeld and Ishii, 1982), and induced electrical excitability (Kuramoto et al., I98 I j . ‘The study of NGF effects on neuroblastoma cells offers unique opportunities given that they have properties that are not present in PC12 cells. First, only the high-affinity NGFR-I-type binding has been detected in neuroblastorna SYSY cells (Sonnenfeld and Ishii, 1982, 1985). Second, SY5Y cells are reported to have NGFR mRNA of similar size to that reported for the rat lowaffinity NGFR. When the SYSY NGFR gene is transfected into mouse fibroblast L cells or NGF-insensitive PC 12nnr cells, the receptor expressed is also the NGFRI1 type (Chao et a]. , 1986; Hempstead et al., 1989). This implies that in SYSY cells there may exist a specific cellular environment that is responsible for the converReceived September 6, 1990; revised September 24, 1990: accepted September 24, 1990. Address reprint reqnests to J . K . Perez-Polo, Gail Borden 436, UTMB, Galveston. Texas, USA. 77550. Abbreviations used: K,, equilibriuni dissociation constant; kDa, kilodalton: mAb 192, monoclonal antibody 192; mAb 20.4, monoclonal antibody ME20.4: NGF, nerve growth factor: NGFK, NGF receptor; NP-40. Nonidet P-40; PAGE, polyacrylamide gel electrophoresis; PBS, phosphatc-buffered saline; RA, retinoic acid: RPHPLC, reverse-phase HPLC; SDS, sodium dodecyl sulfdte; WGA. wheat germ aglutinin.

HPLC of Human NGF Receptor

sion of NGFR-I1 to NGFR-I. Thus, it would be of interest to know if SY5Y cells, which display only highaffinity NGFR, express mainly the high molecular weight species of NGFR. In addition, we wished to evaluatc the effects of retinoic acid (RA) on NGFR expression. RA is a morphogen known to affect cell differentiation (Chao et al., 1986; Giguere et al., 1989). RA increases the number of high- and low-affinity NGF binding in LA-N- 1 cells and I T 1 2 cells (Haskell et a]., 1987; Jackson et al., 1990a,b,c). It has been suggested that RA might induce receptor accumulation by increasing its synthesis (Oberg et al., 1988). However, as we report here, the pattern of NGFR-like antigen expression in RA-treated ncuroblastoma cells is qualitatively different from that in untreated cells. Two NGF-binding activities have been demonstrated for most neuronal tissues with equilibrium dissociation constants (Kd)of around 1OP1IM and 10P’M (Sutter et al., 1979; Stach and Perez-Polo, 1987). The former represents a high-affinity, low-capacity binding site (NGFR-I) that has a slow dissociation rate constant for ligand; the latter represents a low-affinity, high-capacity binding site (NGFR-11) that has a fast dissociation rate constant. It is generally believed that the NCFR-I is the physiologically relevant receptor present on neurons (Sonnenfeld and Ishii, 1985; Green et al., 1986) and it has been proposed that binding of NGF to the low-affinity receptor is required for conversion to the highaffinity form (Landreth and Shooter, 1980). Four molecular species for NGFR have been reported in different tissues (Kouchalakos and Bradshaw , 1986): class A (70-81 kDa), class B (87-105 kDa), class C (120-145 kDa), and class D (190-300 kDa). NGFR-I1 in PC12 cells, in human neuroblastoma LA-N-I cells, and in human melanoma A875 cells have been assigned to classes A and B (Puma et al., 1983; Hosang and Shooter, 1985; Marano et al., 1987; Marchetti and Perez-Polo, 1987). Binding sites in the class D category are thought to be dimers of class B because peptide maps of class B and D are similar and direct conversion from class D to class B undcr reducing conditions can be demonstrated (Buxser et al., 1985; Grob et al., 198.5; Marchetti and Perez-Polo, 1987). It has also been shown that a high molecular-weight dimer is present in the human melanoma A875 cell line, a cell line that does not internalize or respond to NGF (Buxser et al., 1983; Rubenstein et al., 1985). Alternatively, NGFR belonging to class C have also been labelled as NGFR-I. The class C receptor could be generated from the class B receptor by addition of a proposed and as-yet-unidentified 60 kDa receptor-associated protein (Hosang and Shooter, 1985). Although the predicted amino acid sequences for NGFR are homologous, antibodies to human and rodent NGFR

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do not cross-react (Chandler et al., 1984; Ross et al., 1984; Taniuchi et al., 1986). Based on the known NGFR DNA sequence, it has been determined that NGFR is synthesized as a precursor molecule with 425 (rat> or 427 (human) amino acids (Johnson et a]., 1986; Radeke et al., 1987; Large et al., 1989). After removal of the N-terminal signal peptide, the protein core consists of 396 or 399 amino acids, with an estimated molecular weight of 42 or 49 kDa. The protein core is subsequently glycosylated to yield a 75 kDa NGFR (Grob et a]., 1985; Johnson et al., 1986). In this report, reverse-phase HPLC (RP-HPLC) was used to separale different species of NGFR according to their relative hydrophobicities. RP-HPLC has been used to purify other membrane proteins (Hancock and Sparrow, 1983; Potter and Lewis, 1986). Problems frequently encountered during the purification of membrane proteins by RP-HPLC include insolubility, aggregation, and irreversible adsorption of the proteins to reversephase columns (Tarr and Crabb, 1983; Wehr et al., 1989). The detergents traditionally used to solubilize membrane proteins are not compatible with RP-HPLC columns because of high viscosity and immiscibility with organic solvents (Hcukeshoven and Dernick, 1985). Alternative compounds are a mixture of chloroform and methanol; 70% or 89% formic acid; and anhydrous trifluoroacetic acid (Blondin, 1979; Tarr and Crabb, 1983; Heukeshoven and Dernick, 1985; Sussman, 1988). Due to the acidic PI of NCFR (Grob et al., 1985; Marchetti and Perez-Polo, 1987), a column capable of performing at basic pH was required. Proteins thus isolated were then immunoprecipitated with NGFR-specific monoclonal antibodies and thus high molecular-weight NGFRlike species that contained the 192-recognizable epitope present in NGFR were recognized. This finding supports the current view that the high-affinity, high molecularweight NGFR is derived from its low-affinity, low molecular-weight receptor. Tn addition, the molecular weight differences among the high molecular weight species detected suggests that there may be other proteins participating in the formation of high molecular-weight NGFR complexes.

MATERIALS AND METHODS A flow-chart diagram of the analysis of the NGFR used in this study is given in Figure 1, with approximate estimates of the recovery of labelled proteins included.

Materials Lactoperoxidasc, hydrogen peroxide, Nonidet P-40 (NP-40), lentil-lectin Sepharose-4B, a-methyl-D-mannoside, I-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC), and deoxycholate were obtained

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Fig. 1 . A summary of the methods used. Approximate yields of radioactivity at several steps are included.

from Sigma Chemical, St. Louis, MO. Acrylamide, N1 N1 -methylene bis-acrylamide, ammonium persulfate, trizma base, sodium dodecyl sulfate (SDS), N,N,N 1 ,N1tetramethylethylenediamine (TEMED), dithiothreitol, glycine, and Coomassie brilliant blue R-250 were purchased from Bio-Rad Laboratories, Richmond, CA. Standards for SDS-PAGE were obtained from Bethesda Research Laboratories, Gaithersburg, MD. Sucrose was purchased from J.T. Baker Chemical, Phillipsburg, NJ. Methanol and sodium hydroxide were purchased from Fisher Scientific, Plano, TX. Protein G-bearing StuphyEococcus aureus (Omnisorb) was obtained from Calbiochem, San Diego, CA. Glacial acetic acid, HPLC-grade acetonitrile, and water were purchased from Baxter Healthcare Corporation, Houston, TX. Carrier-free Na'251 was purchased from Amersham, Arlington Heights, IL. Culture medium and antibiotics were purchased from GIBCO Laboratories, Grand Island, NY. Culture serum was purchased from lrvine Scientific, Santa Fe, CA. A hybridoma clone for mAb 192 to the rodent NGFR was kindly provided by Dr. E.M. Johnson, Jr. The mAb 192 is specific for the rat NGFR (Chandler et al., 1984; Taniuchi et al., 1986). Immunoglobulins from ascites prepared in pristine-primed BALB/c mice

were purified by chromatography with DEAE Affi-Gel Blue column (Bio-Rad). A hybridoma clone for mAb ME20.4 to the human NGFR was obtained from the American Type Culture Collection, Rockville, MD. Tmmunoglobulins from ascites were prepared as described above. The mAb ME20.4 is specific for the human NGFR and does not cross-react with the rat NGFR (Ross et al., 1984; Hempstead et al., 1989). Immunoglobulins from ascites prepared in pristine-primed BALBic mice were purified by affinity chromatography with DEAE Affi-Gel Blue column (Bio-Rad). The antibody was shown to be specific for the human NGFR (Ross et al., 1984; Hempstead et al., 1989).

Cell Culture PC12 cells were kindly provided by Dr. L A . Greene. Cells were maintained with RPMI 1640 medium supplemented with 5% fetal calf serum, 5% heat-inactivated horse serum, and a 1% PSN antibiotic mixture consisting of 5 mgiml penicillin and streptomycin and 10 mg/ml neomycin. When harvesting, PC 12 cells were shaken off, dissociated into single-cell suspensions by trituration through a siliconised glass Pasteur pipette, and counted with a hemocytometer. Harvested PC12 cells were centrifuged at 110 g for 5 min and washed three times in phosphate-buffered saline (PBS), pH 7.2, before iodination. A subclone of LA-N- 1 human neuroblastoma cells, 2L1 cells, was kindly provided by Dr. J.E. Bottenstein. Cells were maintained with a medium made up of equal amounts of Dulbecco's Modified Eagle Medium and F12 medium, supplemented with 1 mM L-glutamate and 15% fetal calf serum. The SY5Y subclone of the SK-N-SH cells was kindly provided by Dr. J . Biedler, and was maintained as described elsewhere (Perez-Polo et al., 1979). All-transretinoic acid was prepared as described elsewhere (Haskell et al., 1987). 2L1 cells were treated with 10-5M RA and incubated at 3792 for 4 days. When harvesting. 2L1 cells, SY5Y cells, or RAtreated 2Ll cells were shaken off, dissociated into single-cell suspensions by trituration through a siliconised glass Pasteur pipette, and counted with a hemocytometer. Harvested neuroblastoma cells were centrifuged at llOg for 5 min and washed three times in phosphatebuffered saline (PBS), pH 7.2, before iodination. Iodination of Cell Surface Membrane Proteins Intact cells were labelled with 125-iodine as described elsewhere (Marchetti and Perez-Polo, 1987). Briefly, 2 mCi 125-iodine, 2.3 IU lacto-peroxidase, and 50 ~1 3% H,O, were added to 2-3 x lo7 PC12 cells in 0.5 ml PBS, incubated for 15 min at room temperature, and frequently shaken. The reaction was stopped by thc addition of 100 ~ 1 0 . 4 % acetic acid. Membrane proteins were solubilized in 4 ml 0.5% NP-40/PBS at 4°C for 1

HPLC of Human NGF Receptor

hr. Greater than 90% of the radioactivity was acid-precipitable.

Lentil-Lectin Chromatography of NGFR Solubilized membrane proteins were centrifuged at 900g for 15 min. The supernatant was taken and chromatographed on a lentil-lectin Sepharose column (2 cm X 9 mm), which had been equilibrated with 0.5% deoxycholate in 0.02 M Tris-HC1, pH 8.0. The column was prepared according to a method described elsewhere (Takacs and Staehelin, 1981; Hedo, 1984). After the sample was loaded the column was extensively washcd with 0.5% deoxycholate in Tris-HC1. Glycoproteins were eluted with 10% (wtivol) a-methyl-D-mannoside in the same buffer. The eluted glycoproteins were dialyzed sequentially against 0.1% NP-40, 0.1% acetic acid, and distilled water, with a considerable amount of glycoprotein being precipitated during dialysis. HPLC Chromatography of NGFR HPLC chromatography was performed by using a Beckman gradient liquid chromatography system. The acid-precipitated proteins were washed twice with HPLC-grade water and solubilized with 400 p1 10% acetonitrile in 0.05M NaOH, pH 12.5. The samples were filtered through a Millipore Millex-HV,, filter (0.45 pm) before injection. An RP-HPLC column that could tolerate basic solvents (Dupont POLY F, 8 cm X 6.2 mm) was used with a POLY F packing guard column (2 cm X 2 mm). Injections were performed by means of a Beckman 210 sample injection valve with 1 ml loop. The mobile phase contained a mixture of solvent A and solvent B delivered by separate pumps. Solvent A consisted of 10% acetonitrile in 0.05M NaOH, pH 12.5, while solvent B contained 90% acetonitrile in 0.05M NaOH, pH 12.5. All solvents were degassed and filtered through a Rainin Nylon-66 filter (0.45 pm). The sample was applied to the column, and proteins were eluted with a linear gradient of solvents A and B (100% A for 10 min, 0-100% B in 40 min). The flow rate was 0.5 ml/min, and 1-min fractions (0.5 ml) were collected. Immunoprecipitation of NGFR The pH of the HPLC effluent was immediately adjusted with 0.7M phosphate buffer, pH 8.0, and the effluent was lyophilized with a SAVANT model RTlOOA system. After washing with water, the pellet was solubilized in 100 pl 1% NP-40 and incubated with 3 p g of anti-NGFR mAb 192 at 4°C overnight. For some experiments, 3 pg of anti-human NGFR mAb ME20.4 was used as a substitute for the mAb 192. As a control experiment, resolubilized proteins in the HPLC fractions were crosslinked with 3 pg mAb 192 by addition of 10 mM EDAC for 20 min followed by 50 mM Tris-HCI (pH

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7.6) as described elsewhere (Taniuchi et al., 1986). The antigen-antibody complex was precipitated by incubating with 20 p1 protein G-bearing S. aureus (10% wtivol) in 0.5% NP-40IPBS for at least 1 hr and centrifuged at 1,700g for 20 minutes. The pellet was washed with 10% sucrose, solubilized with 200 p1 2% SDS in boiling water for 2 min, and centrifuged at 1,700g for 15 min. Fractions yielding supernatant with significant radioactivity were mixed with a reducing Laemmli electrophoresis buffer containing 40 mM dithiothreitol and boiled for another 5 min.

SDS-Polyacrylamide Gel Electrophoresis (PAGE) One-dimensional electrophoresis was performed according to the method described by Laemmli ( 1 970). The samples were loaded onto 3% stacking gels, 1.5 mm thick, on top of 7 or 7.5% resolving gels, with a final sample volume of 260 pl for each fraction. Two 14cm-long slab gels were run at a constant current of 15 mA per gel until the bromphenol blue dye reached the bottom of the gel. After Coomassie blue staining and destaining, gels were dried by using a slab gel dryer, model SE540 (Hoefer Scientific Instruments). Autoradiography of SDS Gels Autoradiograms were prepared from dried gels by using a screen-intensified Kodak X-Omat-R film at -70°C for 1-3 weeks. RESULTS The determination of the extent of recovery of the NGFR-like proteins and their binding capacity was not measured here. However, it has been reported that approximately 40% of the binding capacity of NGFR can be lost during lectin chromatography (Lyons et al., 1983). The recovery of radioactivity in the glycoprotein pool was estimated to be 0.8% of the total radioactivity associated with the solubilized proteins present. The recovery of radioactivity following HPLC chromatography varied from 70% to loo%, depending on the amount of sample injected onto the column. Smaller sample loads were associated with an almost complete recovery of the radioactivity injected.

PC12 Cells The elution pattern of acid-precipitable glycoproteins present in PC12 cells was reproducible for different preparations (Fig. 2). There were five protein peaks occurring consistently and repeatedly at 26.4 -+ 0.4, 27.8 2 0.2, 29.2 2 0.3. 31.8 2 0.7, and 43.1 f 0.2 min (n24), which may serve as markers of the elution profile. Peaks at 33.5 and 35.5 min were occasionally seen, but were usually masked by protein eluted at 31.5 min

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Fig. 2. RP-HPLC of acid-precipitable glycoproteins from PC 12 cells. '251-labelled glycoproteins were dissolved with 400 p1 10% acetonitrile in 0.05M NaOH and applied to a Dupont POLY F column (6.2 mm X 8 cm; 20 pm particle size). Elution was performed with solvent A containing 10% acetonitrile in 0.05M NaOH, pH 12.5, and solvent B containing 90% acetonitrile in 0.05M NaOH, pH 12.5. The flow rate was 0.5 mlimin at room temperature (22°C). The chart speed was 3 mmimin. The dashed line represents the acetonitrile gradient. The solid line indicates the absorbance monitored at 254 nm. The solid line with filled circles indicates the radioactivity detected in each fraction. The fraction size was 0.5 ml and l-min fractions (0.5 ml) were collected. There are consistently five protein peaks occumng at 26.2, 27.8, 29.0, 31.5, and 43.0 min.

and, thus, were not used as markers. There was a l-min delay in the appearance of proteins in the effluent due to the dead space volume of the HPLC system. The pattern of radioactivity in the HPLC effluent did not reflect the pattern of protein detected by absorbance at 254 nm because of averaging of the radioactivity in the l-min fraction collected and because of the varying extent of iodination of different proteins. Most hydrophobic proteins on the cell membrane were readily accessible to iodination of whole cells while most hydrophilic proteins in the cytoplasm were not. Measurement of radioactivity in all fractions eluted by RP-HPLC (Fig. 2) showed that there were equal amounts of radioactivity associated with fractions 27, 30 and 35. lmmunoprecipitation of the PC12 proteins in RP-HPLC fractions was followed by SDS-PAGE analysis and autoradiography as described in the Materials and Methods. An autoradiogram of the SDS-PAGE gel showed a 40 kDa band (41 .0 k 0.6, n = 2) and a 61-55 kDaband(61.3 f 0 . 2 , n = 3 ; 5 7 . 2 1.2,n=3)present in fractions 6 and 26 through 30, a 74-76 kDa band (77.3 ? 1. I , n = 5 ) in fractions 27 through 3 1, a I 17 kDa +_

band (1 15.9 % 3.9, n = 5 ) in fractions 29 through 31, and 0.9, n = 5 ) in fractions 35 a 133 kDa band (133.2 through 36 (Fig. 3A). In general, the 40 and 61 kDa molecular species were eluted predominantly in fraction 27 of the HPLC chromatogram; the 76 kDa molecular species was eluted predominantly in fraction 29, the 1 17 kDa molecular species was eluted predominantly in fraction 30, and the 133 kDa molecular species was eluted predominantly in fraction 35. A few 40 and 61 kDa species were precipitated in fraction 6, which may not be retained in the RP-HPLC column. By comparison, an autoradiogram of the gel using mAb ME20.4, which does not cross-react with rodent NGFR on PC12, since it is specific for human NGFR (Ross et al., 1984), showed three minor bands in fractions 28, 30, and 33, corresponding to 38 kDa, SO kDa, and 71 kDa, respectively (Fig. 3B). This different elution profile had little radioactivity present in those frac-

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Fig. 3. A: Autoradiogram of the immunoprecipitated '251-labelled NGFR from PC12 cells following SDS-PAGE of the HPLC fractions (Fig. 2). All HPLC fractions were immediately neutralized to pH 8 and concentrated by speed vacuum centrifugation. The proteins were washed with water, solubilized with 1% NP-40, and precipitated with 3 pg mAb 192 and 20 pl protein G-bearing Staphylococcus uureus (10% wiv). Precipitated proteins were washed with 10% sucrose, solubilized with 2% SDS and sample buffer containing dithiothreitol, and run on a 7 or 7.5% SDS polyacrylamide gel. The gel was dried and autoradiographed on screen-intensified Kodak X-Omat-R film at -70°C for 1-3 weeks. This autoradiogram represents the combination of two gels prepared at the same time. Short arrows on the left side indicate the positions of molecular weight standards: myosin, 200 kDa; phosphorylase B, 97.4 kDa; bovine serum albumin, 68 kDa; ovalbumin, 43 kDa; and carbonic anhydrase, 29 kDa. Long arrows on the right side correspond to a 40 kDa band and a 6 1-55 kDa band in fractions 6, 26-30; a 74-76 kDa band in fractions 27-31; a 117 kDa band in fractions 29-31; and a 133 kDa band in fractions 35-36. B: Autoradiogram of a control SDS polyacrylamide gel prepared as described in Figure 3A, with the exception that mAb ME20.4 (3 pg) replaced mAb 192. The dried gel was autoradiographed on a screen-intensified Kodak X-Omat-R film for the same period of time as in Figure 3A. The results indicated the presence of three minor bands in fractions 28, 30, and 33, corresponding to 38 kDa, 50 kDa, and 71 kDa, respectively. C: Autoradiogram of a control SDS polyacrylamide gel prepared as described in Figure 3A, with the exception that mAb 192 (3 pg) was crosslinked to the proteins in the HPLC fractions before immunoprecipitation. The dried gel was autoradiographed on a screen-intensified Kodak X-Omat-R film for the same period of time as in Figure 3A. Arrow on the right side corresponds to a single 95 kDa band in fractions 29 through 38, a 65 kDa band in fraction 28, and a 50 kDa band in fraction 26.

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tions where a high molecular-weight band had been recognized by mAb 192 in PC12. This would suggest that the molecular species, particularly the high molecularweight species, detected by mAb 192, were specific to the mAb 192 antibody and likely to be NGFR. We could not rule out the possibility that minor bands detected by mAb 192 were carried through all the purification steps. Another autoradiogram of an experiment where mAb 192 was crosslinked to PC 12 proteins present in the different HPLC fractions showed a single 95 kDa band to be present in fractions 29 through 38 (Fig. 3C). A 65 kDa band was also present in fraction 28 and there was a 50 kDa band in fraction 26. These last two protein bands may reflect the recognition of truncated forms of NGFR by the mAb 192 antibody.

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Human Neuroblastoma Fig. 4. RP-HPLC of acid-precipitableglycoproteins from neuThe HPLC chromatogram of acid-precipitable gly- roblastoma 2L1 cells. 1251-labelledglycoproteins were discoproteins from human neuroblastoma 2L1 cells (Fig. 4) solved with 400 pl 10% acetonitrile in 0.05M NaOH and inwas very similar to that from PC12 cells with eluted jected into a 20-km Dupont Poly F column (6.2 mm X 8 cm). protein peaks at 26.2, 27.8, 29.0, 31.5, and 43.0 min; Elution was performed with solvent A containing 10% acetothere were three blunted radioactivity peaks in fractions nitrile in 0.05M NaOH and solvent B containing 90% acelo27, 30, and 35. The autoradiographic pattern of the im- nitrile in 0.05M NaOH, pH 12.5. The flow rate was 0.5 mli min at room temperature. The chart speed was 3 rnmimin. The munoprecipitated NGFR-like proteins from 2L 1 cells usdashed line represents the acetonitrile gradient. The solid line ing the monoclonal antibody mAb ME20.4 specific for indicates the absorbance monitored at 254 nm, 0.02 AUFS. human NGFR (Fig. 5 ) showed a few differences from the The solid line with filled circles indicates the radioactivity results obtained with PC12 cells. The predominant mo- detected in each fraction. The fraction size was 0.5 rnl collecular species in 2L1 cells was a 93 kDa band in frac- lected per minute. Consistent with the findings in PC12 cells, tions 27 and 29 through 37. Broad radioactivity extended there are five protein peaks occurring at 26.2, 27.8, 29.0, from the 93 kDa species upward and downward in frac- 31.5, and 43.0 min in 2L1 cells. tions 30 and 31. There were three small molecular weight species in fractions 26 and 27, with molecular weights of 34 kDa, 42 kDa, and 59 kDa, respectively. In a sharp protein peak at 26.7 min. The radioactivity curve contrast to the results with PC12 cells, the predominant of the HPLC effluent from RA-treated 2L1 cells was high molecular-weight species eluted in fractions 34 distinctively different from that of PC12 cells or 2L1 through 37 in experiments with 2L1 cells was a 148 kDa cells. The sharp radioactivity peak that usually appeared band. In addition to the 93 kDa band, there was also a 76 in fraction 30 was replaced by a corxave curve from kDa band present in fractions 35 through 37. fractions 28 through 30 in RA-treated cells. The major difference between the autoradiogram of The autoradiogram of immunoprecipitated NGFR the immunoprecipitated NGFR from SY5Y cells (Figs. from RA-treated 2L1 cells (Fig. 9) was also different 6, 7) as compared to that from 2L1 cells was an almost from that of untreated 2L1 cells. Only two molecular complete absence of low molecular weight species in weight species could be identified in RA-treated 2LI fraction 26 of SYSY cells. A few bands with molecular cells, a 35 kDa band in fractions 26 and 27 and a 93 kDa weights around 42 kDa and 59 kDa were present in frac- band in fractions 29 through 31. In fractions 35 and 36, tions 27 through 30 and 35 through 37. Similar to what both the 35 kDa band and the 93 kDa band were present. was found in 2L1 cells, the predominant species in fractions 27 through 3 1 of SY5Y cells was a 91 kDa band. In addition, a 114 kDa band could be identified in fraction 30 in the experiment with SY5Y cells. Another broad DISCUSSION Different species of NGFR-like proteins have been band of radioactivity was present in fractions 35 and 36 of SY5Y cells, with the center of this broad band corre- characterized from rodent and human NGF-responsive cells by lentil-lectin chromatography, RP-HPLC, immusponding to a molecular weight of about 135 kDa. The HPLC chromatogram of the acid-precipitable noprecipitation, and SDS-PAGE by using monoclonal glycoproteins from RA-treated 2L1 cells (Fig. 8) showed antibodies to the rodent and human NGFR protein. In all

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Fig. 6. RP-HPLC of acid-precipitableglycoproteins from neuroblastoma SYSY cells, carried out as in Figure 1. The arrow indicates the disappearance of the protein peak at 26.2 min. instances there was a tendency for the higher molecularweight species to be eluted in the more hydrophobic fractions. The method reported here proves useful for separating different species of NGFR as a prerequisite for the characterization of each species. Since all rodent molecular species were precipitated by mAb 192, this suggests that these molecular species may share the same epitope. There is other evidence indicating that NGFR-I and NGFR-I1 may share a subunit: 1) binding of wheat germ agglutinin (WGA) or NGF converts NCFR-I1 to

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gel. The gel was dried and autoradiographcd on a screen-intensified Kodak X-omat-R film at -70°C for 1-3 weeks. This autoradiogram is the combination of two gels prepared at the same time. Short arrows on the left side indicate the positions of the standards: myosin, 200 kDa; phosphorylasc B, 97.4 kDa; bovine serum albumin, 68 kDa; ovalbumin, 43 kDa; and carbonic anhydrase, 29 kDa. Long arrows on the right side correspond to (from bottom upward) 34 kDa, 42 kDa, 5'3 kDa, 76 kDa, 94 kDa. 148 kDa.

NGFR-I (Landreth and Shooter, 1980; Grob and Bothwell, 1983; Vale and Shooter, 1982, 1983); 2) NGFR-I in human neuroblastoma SYSY cells and NGFR-I1 in human melanoma A875 cells are recognized by the same antibody ME20.4, while NGFR-I in PC12 cells and NGFR-II in PC12""r5 cells can be precipitated by mAb 192 (Green and Greene, 1986; Buck et al., 1987); 3) only a single gene has been identified for both types of NGFR (Chao et al., 1986: Johnson et al., 1986; Radeke et al., 1987; Large et al., 1989); 4) NGFR mRNA in SYSY cells that display only NGFR-I is of the same size as that for NGFR-I1 (Chao et a]. , 1986); 5 ) neuraminidase cleaves equivalent amounts of sialic acid from both forms of NGFR (Hosang and Shooter, 1985); 6) the omission of reducing agents prior to SDS-PAGE gives a similar shift in mobility of NGFR-I and NGFR-I1 (Buxser et al., 1985; Green and Greene, 1986; Taniuchi et a]., 1986); 7) a single 103 kDa NGF-NGFR complex has been proposed to be the NGFR-I and NGFR-11 (Green and Greene, 1986); and 8) mAb 192 is generated against NGFR-I1 but can bind, be internalized, and retrogradely transported with NCFR that is thought to be NGFR-I (Johnson et al., 1987). Indeed, our control experiment (Fig. 3C) indicates that both the 117 and 133 kDa species are complexes with one of their components belonging to the same mAb 192-recognizable subunit. Among the molecular species isolated here by RPHPLC and precipitated by mAb 192, the most likely candidate for NGFR is the 76 kDa species, similar to the

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Fig. 7. Autoradiogram of the immunoprecipitated NGFR after SDS-PAGE from collected fractions, Figure 6. The procedure was carried out as in Figure 5 . The arrows on the right side correspond to (from bottom upward) 42 kDa, 59 kDa, 75 kDa, 9 1 kDa, 114 kDa, and 135 kDa. P 102

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Fig. 8. KP-HPLC of acid-precipitable glycvproteins from 2L1 cells treated with 10p5M RA for 4 days, carried out as in Figure 4. The arrow indicates a sharp protein peak at 26.7 min.

75 kDa NGFR reported for A875 cells and the 80 kDa NGFR in PC12 cells, Schwann cells, and rat brain (Ross et al., 1984; Grob et al., 1985; Taniuchi et al., 1986; Marano et al., 1987; DiStefano and Johnson, 1988), which has been shown capable of binding NGF and could represent the low-affinity NCFR-11. The 133 kDa protein in the more hydrophobic fraction 35 may correspond to the 158 kDa crosslinked NGFNGFR complex reported as NGFR-I (Massague et al., 1982; Hosang and Shooter, 1985). If the 133 kDa protein represents the high-affinity receptor, the crosslinking of a NGF dimer to this dimeric receptor could add 26 kDa to the molecule and result in a 158 kDa ligand-receptor

complex being reported. The difference in the molecular weights of the 75-80 kDa and 133 kDa species would suggest that the receptor-associated protein may be a 53-58 kDa protein similar to the 60 kDa NGFR-associated protein suggested by others (Hosang and Shooter, 1985; Green and Greene, 1986). The existence of a 5358 kDa receptor-associated protein is consistent with our identification of a 93 kDa and a 148 kDa species by the same technique in human neuroblastoma. The 148 kDa species could result from a complex that is made up of a 93 kDa NGFR-like protein and a putative 55 kDa receptor-associated protein. The 61 kDa species may represent an N-glycosylated form of NGFR, since a 62 kDa N-glycosylated form of NGFR has been reported by pulse-chase labelling in A875 cells (Grob et al., 1985). However, we do not exclude the possibility that this 61 kDa species may be artificially produced by the loss of 0-linked oligosaccharide from the 75 kDa mature form of NGFR at basic pHs. The fact that there are several molecular weight values reported for NGFR and that both NGFR-I and NGFR-I1 are recognized by the same monoclonal antibody would suggest that most, if not all, of the molecular species identified here may be derived from the mature form of the 75-80 kDa rodent N G m and its 93 kDa human counterpart. Since the difference in the deduced molecular weights of mature forms of NGFR-I1 in PC12 and A875 cells (75-80 kDa as compared to 93 kDa) and NGFR-I (133 kDa as compared to 148 kDa) suggest an NGFR-associated protein of 53-58 kDa or 55 kDa, the NGFR-associated protein may have common features in these two cell lines. Thus, tissue or species-specific dif-

HPLC of Human NGF Receptor

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Fig. 9. Autoradiogram of the imniunoprecipitated NGFR after SDS-PAGE from collected fractions, Figure 8. The procedure was carried out as in Figure 5. The arrows on the right side correspond to (from bottom upward) 35 kDa and 93 kDa. ferences in the reported molecular weights for NGFR may result from differences in the degree of expression of a receptor-associated protein. These results are in agreemcnt with other reports of receptor-associated proteins that rely on different techniques (Hosang and Shooter, 1985; Green and Greene, 1986; Marchetti and Perez-Polo, 1987). The nature of the interactions between the components of putative NGFR dimers or between NGFR and receptor-associated protein is not clear, and we can not explain why these high molecular-weight species can survive in boiling SDS. A few reports have mentioned the possible involvement of disulfide linkages in NGFR dimers, but the molecule weights are not the 5amc as in the present study (Grob et al., 1985; Taniuchi et al., 1986). Although for a molecule with an even number of -SH groups in the extracellular domain, such as NGFR, there is rarely a free -SH group available for intermolecular disulfide linkage (Thornton, 198 1). Intermolecular disulfide linkage can still occur through thiol-disulfide exchange reaction between this NGFR and another NGFR or with NGF as long as a free -SH group is present, as occurs between insulin and its receptor or between [D-Ala2,Leu5,Cys6]Enkephalin and &opiate receptor (Clark and Harrison, 1982; Bowen et al., 1987). In addition, disulfide linkages may also occur during homogenization of tissue, as in the case of the low-density lipoprotein receptor (van Driel et a]., 1987). It is conceivable that, for those high molecular-weight NGFR complexes, disulfide bond formation may be artificially produced in a similar way during in vitro experiments.

The increased hydrophobicity provided by the binding of a receptor-associated protein to the NGFR may be required for NGF action. Such binding may facilitate hydrophobic interactions between NGFR and other proteins, such as an adaptor complex, or may provide a hydrophobic environment for the activation of protein kinase activity (Snoek et at., 1988). It has been demonstrated that NGF stimulates hydrolysis of phosphoinositides within 15 sec after addition; that the activity of protein kinase is affected by NGF binding; and that protein kinase C may be responsible for the NGF-induced phosphorylation of Nsp 100 kinase and tyrosine hydroxylase (Cremins et al., 1986; Hama et al., 1986; Contreras and Guroff, 1987). A third possibility is that the increased hydrophobicity of NGFR is required for binding to nuclear receptors in a similar fashion to what has been described for activatcd steroid receptors (Densmore et al., 1988). In these experiments, protease inhibitors were omitted by design. Stach et al. (1986) showed that a protease inhibitor, PMSF, inhibited NGF binding to the high-affinity NGFR. Here, we hoped to increase the appearance of high-affinity NGFR forms by omitting protease inhibitors. Indeed, this may explain why we found an abundance of 133 kDa NGFR-like species corresponding to the 158 kDa high-affinity NGF-NGFR complexes reported by Hosang and Shooter (1985, 1987). In control experiments with cells lacking NGF receptors there was no evidence for degradation of ‘2sI-labclled proteins. It is not feasible to compare the amount of recovered NGFR among different preparations because

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of variable yields of the radiolabelled NGFR during preparation and immunoprecipitation. The intensity of each species on the autoradiogram does not necessarily reflect the amount of each species in the native state. It has been demonstrated that contact with NGF-Sepharose during receptor purification increases the proportion of 200 kDa NGFR (Buxser et al., 1985). In the present experiments, the procedures of lectin chromatography and precipitation may alter the properties of the different molecular species reported. In contrast to our results with PC12 cells, the predominant molecular species of NGFR-like proteins found in human neuroblastoma cells was a 93 kDa species. This is consistent with our previous report of a 92.5 kDa NGFR in neuroblastoma LA-N-1 cells (Marchetti and Perez-Polo, 1987). It has been estimated that RA treatment in LA-N-1 cells increases the number of both the high- and low-affinity NGFRs (Haskell et al., 1987). It is interesting that the autoradiographic pattern of immunoprecipitated NGFRs in 2Ll cells is simplified by treatment with RA, in support of the concept of NGF receptor expression plasticity. Although direct conversion of high molecular-weight NGFR complex into two smaller molecular weight species has not been shown in our reports, we have demonstrated that for RA-treated 2L1 cells there is co-existence of a 93 kDa band and a 35 kDa band in fraction 35, where the high molecularweight species are usually found. This suggests that both molecular species are components of the high molecularweight NGFR complex. Another distinctive change found in RA-treated 2Ll cells is the disappearance of the 55 and/or 61 kDa band. These two molecular species of NGFR have been reported to represent the naked protein core and the Nglycosylated NGFR, respectively, in human melanoma A875 cells (Grob et al., 1985). The absence of these two molecular species in RA-treated cells suggests that either these species have been transformed to the 35 kDa truncated form of NGFR or that they are kept fully glycosylated. It has been reported that RA can enhance the glycosylation of a surface glycoprotein on mouse S91-C2 melanoma cells by increasing the activities of sialyltransferase and galactosyltransferase (Lotan and Irimura, 1987). Similar enhancement of glycosylation of NGFR may also take place in RA-treated 2L1 cells, which results in the disappearance of the 55-61 kDa species and the appearance of a 93 kDa mature NGFR. These results are consistent with the finding that RA does not have uniform effects on both high and low NGF-binding activity (Haskell et al., 1987; Jackson et al., 1990a,b,c). It would appear that RA effects on NGF are but one of many effects. In addition, even in NGF-responsive cells, RA does not elicit the same responses as NGF. R A neither

induces nor potentiates the NGF-induced increase of choline acetyltransferase activity in rat septa1 cultures (Knusel and Hefti, 1988). RA results in a reduced expression of N-myc oncogene prior to differentiation of neuroblastoma cells while the opposite is observed when PC12 are treated with NGF (Greenberg et al., 1985; Thiele et al., 1985). Also, RA does enhance prostaglandin E-stimulated accumulation of cyclic AMP in neuroblastoma cells, while NGF does not (Yu et al., 1988). Thus, even if the modification of the expression of NGFR were a significant factor, it might not account for all of the effects of RA. In conclusion, this HPLC study of NGFR-like proteins using two non-cross-reacting monoclonal antibodies to NGFR demonstrates that the NGFR species present in PC12 are also present in human neuroblastoma cells. These results are also consistent with the interpretation that the tissue-specific difference in the expression of one molecular species as compared to another among these cells may result in part from the differential expression of a receptor-associated protein. Lastly, the expression of different NGFR-like species can be modified by RA treatment.

ACKNOWLEDGMENTS This project was supported by NINDS grant NS18708. The authors are grateful to S.D. Pernia for technical assistance; to R.A. Bradshaw, M.V. Chao, and P.J. Foreman for helpful discussions; to E.M. Johnson, Jr., and L.W. Thorpe for preparing monoclonal antibodies to NGFR, and to D. Masters for manuscript preparation.

REFERENCES Blondin GA (1979): Analysis of the protein subunit structure of the oligomycin sensitive ATPase:proteolipid fraction by reverse phase high pressure liquid chromatography. Biochem Biophys Res Commun 90:355-361. Bowen WD, Hellewell SB, Kelemen M, Huey R, Stewart D (1987): Affinity labelling of &-opiate receptors using [D-AlaZ,LeuS, Cys6lEnkephalin. J Biol Chem 262:23434-23439. Buck CR. Martinez HJ, Black IB, Chao MV (1987): Developmentally regulated expression of the nerve growth factor receptor gene in the periphery and brain. Proc Natl Acad Sci USA 84:30603063. Buxscr SE, Watson L, Johnson GL (1983): A comparison of binding properties and sttucture of NGF receptor on PC12 pheochromocytoma and A875 melanoma cells. J Cell Biochem 22:219233. Buxser S , Puma P, Johnson GL (1985): Properties of the nerve growth factor receptor. J Biol Chem 260: 1917-1926. Chandler CE, Parsons LM, Hosang M, Shooter EM (1984): A rnonoclonal antibody modulates the interaction of nerve growth factor with PC12 cells. J Biol Chem 259:2882-2889. Chao MV, Bothwell MA, Ross AH, Korprowski H, Lanahan AA,

HPLC of Human NGF Receptor Buck CR, Sehgal A (1986): Gene transfer and molecular cloning of the human NGF rcceptor. Science 2325 18-52 I . Clark S, llarrison LC (1982): Insulin binding leads to the formation of covalent (-S-S-) hormone receptor complexes. J Biol Chem 257: 12239-12244. Contreras ML, Guroff G (1987): Calcium-dependent nerve growth factor-stimulated hydrolysis of phosphoinositides in PC 12 cells. J Neurochem 48:1466- 1472. Cremins J. Wagner JA, Halegoua S (1986): Nerve growth factor action is mediated by cyclic AMP- and Ca+ 2/phospholipid-dependent protein kinases. J Cell Biol 1035387-893. Densmore CL, Chou YC, Luttge WG (1988): Activation of glucocorticoid-type I1 receptor complexcs in brain cytosol leads to an increase in surface hydrophobicity as dctcrmined by hydrophobic interaction chromatography. J Neurochcm SO: 1263-1271. DiStefano PS, Johnson EM J r (1988): Identification of a truncated form of the nerve growth factor receptor. Proc Natl Acad Sci USA 85:270-274. Giguere V, Ong ES, Evans KM, Tabin CJ (1989): Spatial and temporal expression of the retinoic acid reccptor in the regenerating amphibian limb. Nature 337566-569. Green SH, Greene LA (1986): A single Mr= 103,000 1251-B-nerve growth factor-affinity-labeled species represents both the low and high affinity forms of thc nervc growth factor rcceptor. J Biol Chem 261 : 15316-15326. Green SH, Rydel RE, Connolly JL, Greene LA (1986): PCl2 cell mutants that possess low- hut no high-affinity nerve growth factor receptors ncither respond to nor internalize nerve growth factor. J Cell Biol 102:830-843. Greenherg ME, Greene LA, Ziff EB (1985): Nerve growth factor and epidermal growth factor induce rapid transient changes in proto-oncogene transcription in PC 12 cells. J Biol Chem 260: 14101-141 lo. tireene LA, Tischler AS (1976): Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci USA 73: 2424-2428. Greene L (1984): The importance of both early and delayed responses in the biological actions of nerve growth factor. Trcnds Neurosci 7:91-94. Grob PM, Bothwell MA (1983): Modification of nerve growth factor receptor properties by wheat germ agglutinin. J Biol Chem 258: 14136-14 143. Grob PM, Ross AH. Koprowski H, Bothwell M il9SS): Characterization of the human melanoma nerve growth factor receptor. J Biol Chem 260:80U-8049. Hama T, Huang KP, Guroff G ( I 986): Protein kinase C as a component of a nerve growth factor-sensitive phosphorylation system in PC12 cells. Proc Natl Acad Sci USA 83:2352-2357. Hancock WS, Sparrow JT (1983): The separation of proteins by reversed-phase high-performance liquid chromatography. In Horvath C (ed): “High-Performance Liquid Chromatography.” London: Academic Press, tnc., pp 49-85. Haskell BE, Stach RW, Werrbach-Perez K, Perez-Polo JR (1987): Effect of retinoic acid on nerve growth factor receptors. Cell Tissue Res 247:67-73. Hedo JA (1984): Lectins as tools for the purification of menibrane receptors. In Ventner JC, Harrison LC (eds): “Receptor Purification Procedurcs.” New York: Alan R. Liss. Inc.. pp 45-60. Hempstead BL, Schleifer LS, Chao MV (1989): Expression of functional nerve growth factor receptors after gene transfer. Science 243:373-375. Heukeshoven J. Dernick R (1985): Characterization of a solvent system for separation of water-insoluble poliovirus proteins by

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reversed-phase high-pcrfonnance liquid chromatography. J Chromatogr 326:91-101. Hosang M, Shooter EM (1985): Molecular characteristics of nerve growth factor rcceptors on PC12 cells. J Bin1 Chem 26R65.5662. Hosang M, Shooter EM (1987): The internalization of nerve growth factor by high-affinity receptors of pheochromocytoma PC12 cells. EMBO J 6: 1197-1202. Jackson GR, Apffel L. Werrbach-Perez K , Perez-Polo JR (1990a): Role of nerve growth factor in oxidant-antioxidant balance and neuronal injury. 1. Stimulation of hydrogen peroxide resistance. J Neurosci Res 25:360-368. Jackson GR, Werrbach-Perez K, Perez-Polo JR (1990h): Role of nerve growth factor in oxidant-antioxidant balance and neuronal injury. 11. A conditioning Icsion paradigm. J Neurosci Kcs 25: 369-374. Jackson GR, Morgan BC, Werrbach-Perez K, Perez-Polo JR (1990~): Antioxidant effect of retinoic acid on PC 12 rat pheochromocytoma. Int J Devclop Neurosc (in press). Johnson D, Lanahan A , Buck CR, Sehgal A, Morgan C, Mercer E, Bothwell M , Chao M (1986): Expression and structure of the human NGF receptor. Cell 47545-554. Johnson EM Jr. Taniuchi M, Clark HR, Springer JE, Koh S, Tayrien MW, Loy R ( 1987): Demonstration of the retrograde transport of nerve growth factor receptor in the peripheral and ccntral nervous system. J Neurosci 7:923-929. Knusel B. Hefti F (1988): Development of cholinergic pcdunculopontine ncurons in vitro: comparison with cholinergic septa1 cells and response to nerve growth factor, ciliary ncuronotrophic factor, and retinoic acid. J Neurosci Res 21:365-375. Kouchalakos RN. Bradshaw R A (1986): Nerve growth factor receptor from rabbit sympathetic ganglia membranes. J Biol Cheni 261: 16054-16059. Kuramoto T, Werrbach-Perez K, Perez-Polo JK, Haber R (1981): Membrane propcrtics of a human neuroblastoma. 11: ECfects of differentiation. J Neurosci Res 6:44 1-449. Laemmli UK (1970): Cleavage of structural proteins during the asseinbly of the head of bacteriophage T4. Nature 227:680-685. Landreth GE, Shootcr EM (1980): Nerve growth factor receptors on PC 12 cells: Ligand-induced conversion from low- to high-affinity states. Proc Natl Acad Sci USA 77:4751-4755. Landrcth GE. Williams LK, McCutchen C (1985): Wheat germ agglutinin blocks the biological effects of nerve growth factor. J Cell Biol l0l:1690-1694. Large TH. Weskamp G. Helder JC, Kadcke MJ, Misko TP, Shooter EM, Reicha LF (1989): Structure and developmental expression of the nerve growth factor receptor in the chicken central nervous system. Neuron 2:l 123-1134. Levi A , Riocca S , Cattaneo A. Calissano P (1988): The mode of action of nervc growth factor in PC12 cells. Mol Neurobiol 2:20 1-226. Levi-Montalcini R (1987): The nerve growth factor 35 years later. Science 237:1154-1162. Lotan R, Irimura T (1987): Enhanced glycosylation of a melanoma cell surface glycoprotcin by rctinoic acid: carbohydrate chain analysis by lectin binding. Cancer Biochem Biophys 9:211221. Lyons CR, Stach RW, Perez-Polo JR (1983): Binding constants of isolated NGF-rcccptors from different spccics. Biochem Biophys Res Commun 115568-374. Marano N, Dietzschold B; Earley JJ, Jr, Schatteman G. Thompson S , Grob P, Ross AH, Bothwell M (1987): Purification and amino terminal sequencing of human melanoma nerve growth factor receptor. J Neurochem 48:225-232.

632

Shan et al.

Marchetti D, Perez-Polo JR (1987): Nerve growth factor receptors in human neuroblastoma cells. J Neurochem 49:475-486. Massague J , Buxser S, Johnson GL, Czech MP (1982): Affinity labelling of a nerve growth factor receptor component on rat pheochromocytoma (PC12) cells. Biochim Biophys Acta 6Y3: 205-212. Oberg KC, Soderquist AM, Carpenter G (1988): Accumulation of epidermal growth factor receptors in retinoic acid-treated fetal rat lung cells is due to enhanced receptor synthesis Mol Endocrinol 2:959-965. Perez-Polo JR, Haber B (1983): Neuronotrophic interactions. In Rosenbcrg RN, Willis Jr WD (eds): “The Clinical Neuroscience.” New York: Churchill Livingstone, Vol V, pp 37-51. Perez-Polo JR, Reynolds CP, Tiffany-Castiglioni E, Ziegler M, Schulze I , Werrbach-Perez K (1982): NGF effects on human neuroblastoma lines: A model system. In Haber B, Perez-Polo JR, Coulter J (eds): “Proteins in the Nervous System: Structure and Function.” New York: Alan R. Liss, Inc., pp 285-299. Perez-Polo JR, Werrbach-Perez K, Tiffany-Castiglioni E ( 1979): A human clonal cell line model of differentiating neurons. Dev Biol 71:341-355. Potter RL, Lewis RV (1986): Reversed-phase chromatography of proteins and nucleic acids: practical considerations. In Horvath C (ed): “High-Performance Liquid Chromatography.” London: Academic Press, Inc., pp 1-44. Puma P, Buxser SE, Watson L, Kelleher DJ. Johnson GL (1983): Purification of the receptor for nerve growth factor from A875 melanoma cells by affinity chromatography. J Biol Chem 258: 3370-3375. Radeke MJ, Misko TP, Hsu C, Herzenberg LA, Shooter EM (1987): Gene transfer and molecular cloning of the rat nerve growth factor receptor. Nature 325593-597. Ross AH, Grob P, Bothwell M, Elder DE, Ernst CS, Marano N, Christ BFD, Slemp CC (1984): Characterization of nerve growth factor receptor in neural crest tumors using monoclonal antibodies. Proc Natl Acad Sci USA 81:6681-6685. Rubenstein JL, Vale RD, Shooter EM (1985): Response to nerve growth factor of a human melanoma cell line. Clin Res 33:70A. Snoek GT, Feijen A, Hage WJ, Van Rotterdam W. De Laat SW (1988): The role of hydrophobic interactions in the phospholipid-dependent activation of protein kinase C. Biochem J 255: 629-637. Sonnenfeld KH, Ishii DN (1982): Ncrve growth factor effects and receptors in cultured human neuroblastoma cell lines. J Neurosci Res 8:375-391. Sonnenfeld KH, Ishii DN (1985): Fast and slow nerve growth factor binding sites in human ncuroblastoma and rat pheochromocytoma cell lines: relationship of sites to each other and to neurite formation. J Neurosci 5: 1717-1728.

Stach RW, Perez-Polo JR (1987): Binding of nerve growth factor to its receptor. J Neurosci Res 17: 1-10. Stach RW, Stach BM. Ennulat DJ (1986): Phenylmethyl-sulfonyl fluoride (PMSF) inhibits nerve growth factor binding to the high affinity (type I) nerve growth factor receptor. Biochem Biophy Res Commun 134:1000-1005. Sussman MR (1 988): Purification of integral plasma membrane proteins by reverse-phase high performance liquid chromatography. Anal Biochcin 169:395-399. Sutter A, Riopelle RJ, Harris-Warrick RM, Shooter EM (1979): Nerve growth factor receptors. Characterization of two distinct classes of binding sites on chick embryo sensory ganglia cells. J Biol Chem 2545972-5982. Takacs BJ, Staehelin T (1981): Biochemical characterization of ccll surface antigens using monoclonal antibodies. In Lefkovits I, Pernis B (eds): “Immunological Methods.” New York: Academic Press, Inc., pp 27-56. Taniuchi M. Schweitzer JB. Johnson EM Jr (1986): Nerve growth factor receptor molecules in rat brain. Proc Natl Acad Sci USA 83:1950-1954. Tarr GE, Crabb JW ( 1983): Reverse-phase high-performance liquid chromatography of hydrophobic proteins and fragments thereof. Anal Biochem 131:99-107. Thiele CJ, Reynolds CP, Israel MA (1985): Decreased expression of N-myc precedes reginoic acid-induced morphological differentiation of human neuroblastoma. Nature 313:404-406. Thoenen H, Barde YA (1980): Physiology of nerve growth factor. Physiol Rev 60:1284-1335. Thornton JM (1981): Disulphide bridges in globular proteins. J Mol Bid 151:261-287. Vale RD, Shooter EM (1982): Alteration of binding properties and cytoskeletal attachment of nerve growth factor receptors in PC12 cells by wheat germ agglutinin. J Cell Biol94:710-717. Vale RD, Shooter EM (1983): Conversion of nerve growth factorreceptor complexes to a slowly dissociating, Triton X- 100 insoluble state by anti nerve growth factor antibodies. Biochemistry 22:5022-5028. van Driel IR, Davis CG, Goldstein JL, Brown MS (1987): Self-association of the low density lipoprotein receptor mediated by the cytoplasmic domain. J Biol Chem 262: 16127-1 6 134. Wehr CT, Lundgard RP, Nugent KD (1980): Hydrophobic proteins: a challenge for reversed-phase HPLC. LC-GC 7:32-37. Whittemore SR, Seiger A (1987): The expression, localization and functional significance of p-nerve growth factor in the central nervous system. Brain Res Rev 12439-464. Yu VC, Hochhaus G, Chang FH, Richards ML, Bourne HR, Sadee W ( 1988): Differentiation of human neuroblastoma cells: marked potentiation of prostaglandin E-simulated accumulation of cyclic AMP by retinoic acid. J Ncurochem 51: 1892-1899.

Reverse-phase high-performance liquid chromatography of nerve growth factor receptor-like proteins identified with monoclonal antibodies.

Human neuroblastoma SK-N-SH-SY5Y (SY5Y) and rat pheochromocytoma PC12 cells are model cell lines used in the study of nerve growth factor (NGF) effect...
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