Nerve growth factor-induced loss of cell-associated nerve growth factor receptor in human melanoma A875 cells

Neuroscience Letters, 136 (1992) 113-117 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

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Nerve growth factor-induced loss of cell-associated nerve growth factor receptor in human melanoma A875 cells Cornelia Hertel and Robert Schubenel Pharma Division, Preclinieal Research, E Hoffmann La Roche Ltd., Basel ( Switzerhmdj

(Received 23 September 1991; Revised version received 7 November 1991: Accepted 4 December 1991l Key words: Melanomacell; p75N~va; Receptor shedding; Immunoprecipitation

Human A875 melanoma cells are known to express the low-affinitynerve growth factor receptor p75NGj:~in a monomeric and a covalently linked probably dimericform. Kinetic analysisof the association of nerve growth factor (NGF) with its receptor revealed a rapid loss of binding sites at high ligand concentrations. Using cross-linkingand immunoprecipitationwith an anti-p75NG~Rantibody, this was found to be due to a decrease of lhe high molecular weight form of the receptor. Mechanisms for such a ligand-induced receptor loss are discussed.

Nerve growth factor ( N G F ) is a dimeric polypeptide with neurotrophic and neurotopic properties for some primary neural crest-derived sensory neurons, peripheral adrenergic and central cholinergic neurons [20]. Two binding sites for N G F have been described: a low-affinity or fast-dissociating receptor with a molecular weight of 75 k D a (p75 N~FR) and a high-affinity or slow-dissociating receptor [10]. The proto-oncogene p145 trk has recently been identified as a part of the high-affinity site [12]. p75 NGFR has been cloned from rat [15], chicken [9] and h u m a n [11] tissue. All share considerable homology including 4 cysteine-rich extracellular domains, a single membrane-spanning region, and an intracellular domain with no homology to any of the known second messenger generating enzymes [11]. Most functional responses to N G F , such as c-fos induction [2] and cell differentiation have been ascribed to the high-affinity receptor [6]. However, a large number of cells, such as Schwann cells express exclusively the low-affinity receptor [3]. The function of the N G F receptor in these cells still remains unclear. Schwann cells in rat sciatic nerve express transiently high levels of p75 N~FR after nerve lesion [18], and simultaneously shed a truncated form of p75 NCw [4, 23] suggesting that expression of these receptors is tightly controlled. It has been speculated that one function of Correspondence: C. Hertel, Pharma Division, Preclinical Research, F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland. Fax: (11) 141) 61 688 1720.

these receptors might be to bind secreted N G F and, thereby, create a localized source of N G F to guide the regenerating nerve [20]. H u m a n A875 melanoma cells express p75 N~k at a high level, but do not express p145 trk [12] thus providing a model to study the function of the low-affinity receptor [5] in the absence of p145 trk. Two forms of p75 NGFR have been identified under non-reducing conditions on SDSgels: a 75 k D a and a 160 k D a protein. The latter was suggested to be a S-S-linked receptor dimer [1]. Processing of the receptor in A875 cells has been described to occur by two mechanisms: truncation/shedding [23] and internalization [16]. In the present report we present a kinetic analysis of the interaction of N G F with its receptor in A875 cells. At low ligand concentrations receptor ligand binding is described by a second order isotherm, while at high ligand concentrations the association process becomes biphasic: the association is followed by a rapid loss of cell-bound ligand. These results suggest that N G F induces receptor downregulation. Mechanisms for such downregulation are discussed. Murine N G F was purified according to Longo et al. [13]. [12SI]NGF was purchased from Amersham, UK: RPMI-1640 from Gibco; fetal calf serum from Hyclone Lab Inc, USA; mouse monoclonal anti-human N G F R antibody 20.4 from Boehringer lngelheim, F R G ; l-ethyl3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) from Pierce, Rockford, USA. All other reagents used were reagent grade.

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A875 cells were a gift from Dr. M. Hosang. Basel. Cells were grown in RPMI-1640 with L-glutamine containing I0% fetal calf serum in a humidified incubator containing 8% CO: at 37°C. Cells were subcultured 24 h prior to experiment at a cell density of 5 × 104 cells/era -~. [J25I]NGF binding was determined using the centrifugation assay described by Sutter et al. [17] Briefly, 1.2 x 105 cells were incubated in a final volume of 120 ¢tl with [~:5I]NGF at 4°C for the time indicated. To terminate the incubation 100 yl of the cell suspension were layered on top of 200 ¢tl 0.15 M sucrose and centrifuged 90 s at 10,000 x g. This method allowed separation within less than 15 s. Thereby most of the rapidly dissociating receptor ligand complex was captured. Viability was greater than 95% at the end of the experiment as determined by Dextran blue exclusion. For saturation binding experiments the specific radioactivity was kept constant at 191 Ci/mmol. Data were evaluated using a non-linear least square fitting program [8]. [t:SI]NGF was cross-linked to the receptor using EDC. Briefly, after incubation with [t25I]NGF EDC was added at a final concentration of 30 mM tbr 2 min at room temperature. This high concentration was chosen to shorten cross-linking time in the kinetic studies. TrisHCI pH 7.4 (final concentration 50 mM) was added to quench the reaction. Cells were centrifuged for 5 rain at 2500 rpm in a Beckman Microfuge 12. Cells were lysed in electrophoresis sample buffer (Gradipore) with or without reducing agent. Proteins were separated on precast 3 d 2 % gradient mini-gels (Gradipore, Pyrmont, Australia). Films were exposed for 3-5 days with intensifying screens (Kodak X-Omat AR diagnostic film). Supernatants and cell-containing pellets of the crosslinking experiment were treated separately. Cells were solubilized in assay buffer containing 1% nonident P40. The same concentration of solubilizer was added to the supernatant to reduce non-specific immunoprecipitation. Non-solubilized material was precipitated by centrifugation at 4°C for 10 rain at 12,000 x g. The latter supernatants were pre-cleared with agarose antimouse IgG, followed by overnight incubation with MAb 20.4 and precipitation with agarose-anti-mouse IgG. Immunoprecipitates were analysed on gradipore gradient minigels. Based on cross-linking studies at least two N G F binding proteins (80 kDa and 170 kDa) have been previously identified in A875 cells [1]. Here, [~25I]NGF was crosslinked to its binding protein(s) in intact cells and proteins were separated by SDS gel electrophoresis under reducing and non-reducing conditions (Fig. IA). Under reducing conditions there was one major band at 90 kDa representing the [12SI]NGF p75 N~;vR complex. Under non-

reducing conditions, the mobility of the complex was slightly reduced and a second band appeared at 160- 18(1 kDa. No additional band was identifed at 150 kDa, even alter prolonged exposure time (data not shown), suggesting that indeed p145 trk is absent in these cells [12]. Both bands were immunoprecipitated with a mouse antihuman-p75 N(;vR antibody (MAb 20.4) indicating that both bands contain p75 NGv~. (Fig. 1B). A high molecular weight form of the receptor has been shown to be stable during purification and is thought to be a receptor aggregate [14], most probably a disulfide bridge linked dimer [1]. In our experiments we always lbund a protein with a molecular weight of approximately t80 kDa, which would represent NGF-occupied receptor dimers, and occasionally an additional band with a molecular weight of 220 kDa representing higher aggregates. All further experiments were performed under non-reducing conditions to study if receptor aggregation is altered by ligand binding. The relative intensity of the labeling of the two proteins was found to depend on the NGF concentration (Fig. 1B). At low N G F concentrations (0.25 nM) the high-molecular weight band contained most of the label, while at high concentrations (26 nM) the label was distributed equally between the the low- and highmolecular weight band. Note, the specific radioactivity

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Fig. 1. Autoradiography of the cross-linked receptor ligand complex. A: autoradiogram of cross-linked proteins. Separation under reducing (200 mM 2-mercaptoethanol, fl-ME) or non-reducing conditions. Cells were incubated with 0.25 nM [~25I]NGF with or without 200 nM nonlabeled NGF, cross-linked, lysed and separated on gradient gels, B: immunoprecipitation of cross-linked proteins Cells were incubated for 30 min with either 0.25 nM [~25I]NGF (1.5 /.tCi/pmol) or 26 nM [~251]NGF (0.15 ,uCi/pmol) in the presence or absence of 200 nM nonlabeled NGF. Specific radioactivity was reduced to enable simultaneous exposure in the autoradiography. Receptors were crosslinked with EDC, cells were solubilized in 1% nonident P40 and receptors were immunoprecipitated with MAb 20.4. Gel separation was performed under non-reducing conditions. Autoradiogramms were exposed for 64 h (n=4).

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of N G F was decreased at the high ligand concentrations resulting in reduced overall labeling.

Equilibrium binding to intact cells was performed at room temperature using a concentration range from 0.1 x Kd to 10 × Kd. A bell-shaped curve rather than the expected sigmoidal shaped curve was found (Fig. 2). At concentrations above 5 nM, the amount of bound N G F decreased drastically. Scatchard transformation resulted in downward concave plots. At 4°C the effect was less evident, while at 37°C it was even more pronounced (data not shown). Since the specific radioactivity (labeled ligand/non-labeled ligand) was kept constant over the total concentration range, this behavior cannot bc explained by different affinities ot" the receptor tk~r iodinated and non-iodinated ligand [19]. Possible mechanisms producing bell-shaped binding curves with an actual reduction in cell-associated N G F tire concentration-dependent dissociation of N G F from its receptor, dissociation of the receptor ligand complex from the cell, or proteolytic degradation of the binding site. To determine association kinetics cells were incubated at room temperature with N G F at 1 nM (linear range of equilibrium binding), 3 nM and 5 nM (maximal binding), and 25 nM (descending part of binding curve; Fig. 3). At I nM N G F the association kinetics can be described by a second order isotherm. However, at high concentrations the earliest accessible time point (1 rain) showed maximal binding with a rapid decrease in binding at the later time points. Association kinetics at 37°( " and 4°C were similar to those observed at room temperature (data not shown). Shedding of N G F receptors has been described by Zupan et al. [23]. We investigated whether the observed loss of binding to the high molecular weight receptor might be due to NGF-induced shedding of a truncated receptor. Cells were incubated at room temperature with 25 nM N G F for 1, 5 and 15 min (Fig. 4) followed by cross-linking with EDC for 2 rain. Cross-linked receptors were determined in cells and supernatant by immunoprecipitation with anti-p75 ~'~ R antibody. A slight time-dependent reduction of the labeled complex occurred in the cellular fraction (Fig. 4, left pannel), which was less than what would be expected from the loss observed in the association experiment. This might be explained by the increased incubation periods due to lhe necessary incubation with EDC for 2 min. In the supernatant no corresponding appearance of a specifically NGF-cross-linked protein was observed. When immunoprecipitation was performed with anti-NGF antibody results were similar (data not shown). Human A875 melanoma cells express approximately 10s low-affinity nerve growth titctor receptors/cell (p75 Nc;~R) in a monomeric and an aggregated probably dimeric form [1]. Previous kinetic and equilibrium analysis of N G F binding to this receptor had revealed a single

116

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Fig. 3. Association kinetics. A875 cells were incubated at room temperature with 1 nM [~25I]NGF (n=6), 3 nM [~251]NGF (n=10), 5 nM [t25I]NGF (n=2), 25 nM [12~I]NGF (n=5).

binding site with a Kd in the low nanomolar range and a very rapid dissociation rate [1]. Recently A875 cells have been shown to lack expression of p 145trk, the protein participating in the high affinity complex [12]. The present detailed analysis of the binding properties of p75 NGFR revealed an unexpected binding behavior: a time- and temperature-dependent reduction in binding at high concentrations of NGF. While association kinetics at low ligand concentrations were seemingly monophasic, they became biphasic at higher concentrations with a very rapid initial increase in binding followed by a fast decrease. Such kinetics are indicative of a subsequent ligand-induced process, possibly receptor downregulation. Receptor internalization, a mechanism involved in receptor downregulation [7] is very unlikely to be the underlying mechanism, because the process described here occurred at 4°C and in lysates, although to a lesser extent (data not shown). Another possible explanation for the rapid loss of cellassociated [~2SI]NGF is based on the observed receptor aggregation. At low ligand concentrations aggregation is likely to occur between an occupied and an unoccupied receptor, while at high NGF concentrations aggregation is likely to occur with two ligand-occupied receptors.

Ligand occupation of the receptors will probably lead to sterical hindrance of aggregation when the aggregating receptors are ligand occupied and thus formation ofmonomeric receptor ligand complexes would be favored at high ligand concentrations. This is observed in the experiments described here. Such a mechanism per se would not induce loss of cell-associated NGF. However. the monomeric receptor has been described to be tess stable to proteolytic processes than the high molecular weight form [14]. Therefore preferential formation of the monomeric form of the receptor would favor proteolytic degradation resulting in loss of binding sites, as is observed in our experiments. This loss of binding sites is probably not due to receptor truncation/shedding [23], since no complex of NGF with another protein fragment could be identified in the cell supernatant by immunoprccipitation with either anti-p75 NCvR antibody or antiNGF antibody. This is in agreement with a recent report by Zupan and Johnson [22], who report that receptor shedding is independent of NGF. We therefore suggest that as yet unidentified proteolytic processes cause ttw observed loss in NGF binding. The mechanismts) m volved need to be further investigated.

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Fig. 4. Immunoprecipitation of the cross-linked receptor ligand complex in cell m e m b r a n e s and cell supernatant. Cells were incubated lk~r the time indicated with 26 nM [':q]NGF. Receptors were cross-linked, then supernatant and pellet (containing the cell membranes) were separated, and the receptor was immunoprecipitated with M A b 20.4. The fourth and eighth lane show labeling in the prescncc of 200 nM nonlabeled N G t : 1/~ 3). Autoradiogramms of the pellets ~ere exposed l\~r 20 h. of the supernatants [\~r 96 h.

15

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We would like to thank Drs. W. Haefely, D. Hartman and R Schoch for valuable discussion. 18 1 Buxser, S.E.. Puma, P. and Johnson. G i . , Properties of the nerve growth factor receptor, J. Biol. Chem., 260 (1985) 1917 1926. 2 Curran, T. and Morgan. J.l., Superinduction of c-fos b> nerxe growth Factor in the presence of peripherally actix.e benzodiazepincs, Science, 229 (1985) 1265 1268. 3 DiStefano. P.S. and Johnson Jr., E.M., Nerve growth factor receptors oncultured rat Schwann cells. J. Neurosci.. 8 (19881 231 241. 4 DiSteFano, P.S. and Johnson Jr., E.M., Identification of a truncated form of thc ncrvc growth factor receptor, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 270 274. 5 Fabricant. R.N.. DeLarco, J.[:,. and Todaro, G.J., Ner\c grov, th factor receptors on h u m a n melanoma cells m culture. Proc. Natl. Acad. Sci. U.S.A., 74 {1977) 565 569. 6 Greene, L.A. and Tischler, A.S., PC I2 pheochromocytoma cuhures in neurobiological research, Adv. (Tell. Neurobiol., 3 (19821 373 414. 7 Hertd, C. and Perkins, J.P., Receptor-specific mechanisms of de-

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sensitization of fl-adrenergic receptor Functions, Mol. Cell. Endocrinol., 37 119841 245 256, Hertel, {2".. Muellcr. R, Portenier, M. and Staehelin. m., Determination of the desensitization of fl-adrenergic receptors by [~H]CGP12177, Biochem J.. 2]6 (1983) 669 674. Heuer, J.G., Fatemie-Namie, S.. Whcllcr, E.F. and Bothwell, M., Structure and developmental expression of the chicken N G F receptor. [)ex. Biol.. 137 (1990) 287 304. Hosang, M. and Shooter. E.M., Molecular characteristics ol'nervc growth factor receptors on PC I2 cells. J. Biol. Chem.. 260 (1985) 655 6~)2. ,lohnson, D.. Lanahan, A., Buck, C.R.. Schgal, A., Morgan, C., Mcrccr, t!.. Both,acll. M. and Chao. M., Expression and structure of the h a m a n N G F receptor, Cell. 47 (1986) 545 554. Kaplan. I).R., Hempstead, B.L.. Martin-Zanca, D.. Chao, M.V. and Parada, [,.F.. The Irk proto-oncogene product: a signal transducing receptor lk~r ner\e growth Factor, Science, 252 (1991) 554 558. Longo, F.M., Woo, J.E. and Mobley, W.C,, Purification of nerve growth factor. In R.A. Rush (Ed.), Nerve Growth Factors. Wiley, Chichestcr, 1989. pp. 3 30. Puma. P., Buxser. S.E., Watson, L., Kelleher, D.J. and Johnson, G.L.. Purilication of the receptor for nerve growth Factor from A875 mclanoma cells by affinity chromatography. J. Biol. Chem., 258 (1983) 3370 3375. Radeke. M.J.. Misko. T.P.. Hsu. T.P.. Herzenberg, L.A., and Shooter, V.M.. Gene transfer and molecular cloning of the rat nerve growth factor receptor, Nature. 325(1987)593 597. Rako\~ic/-Szulczynska. E.M.. Lilmenbach, J.A. and Koprowski, tt., Intracellular receptor binding and nuclear transport of ner,,e growth Factor in intact cells aim ccll-l'rcc system, Mol. Carcinog., 2 (1989) 47 58. Seller. A., Riopelle, R.J., Harris-Warrick. R.M. and Shooter, E.M., Nerve growth factor receptor: characterization of two distinct classes of binding sites on chick embr_'~o sensory ganglia cells, J. Biol. Chem., 254 11979) 5972 5982. Taniuchi. M.. ('lark. H.B. and Johnson Jr., E.M., Induction of nerve growth factor receptor in Schx~.ann cells after axotomy, Proc. Natl. Acad. Sci. Li.S.A., 83 (1986)41)94 4098 Tayhn'. S.I., Binding of hormones to receptors. An alternative explanation of nonlmcar Scatchard plots. Biochemistry. 14 (1975) 2357 2361. Thoenen, H. and Barde, Y.-A., Physiology of nerve growth factor, Physiol. Rex., 60{19801 1284 1335. Thoenen, H., The changing scene ol" ncurotrophic lectors. Trends Neurosci.. 1411991) 165 170. Zupan, A.A. and Johnson Jr.. ['~.M., F,xidencc for endocytosis-dcpendent proteolysis in the gcncration o1" soluble truncated ncrve gro,.~,th factor receptor by A875 human melanoma cells, J. BiolI ('hem., 26611991)15384 15390. Zupan, A.A.. Osborne. P.A., Smith. C.E.. Siegel. N.R., Leimgrubcr, R.M. and Johnson ,It-.. E.M.. Identification. purification, and characterization of truncated l\~rms o1"the h u m a n nerve growth factor receptor, J. Biol. Chcm..26411989) 11714 117211.