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Proc. Nati. Acad. Sci. USA Vol. 74, No. 7, pp. 2785-2789, July 1977 Biochemistry Nerve receptors: Identification of distinct classes in plasma membranes and nuclei of embryonic dorsal root ( binding/nonionic detergent/retrograde axonal transport) ROGER Y. ANDRES, INGMING JENG*, AND RALPH A. BRADSHAW Department of Biological Chemistry, Washington University School of Medicine, St. Louis, MO 63110 Communicated by P. Roy Vagelos, May 2, 1977

ABSTRACT Two classes of receptors for '25I-labeled nerve sulin receptors (15), rather than from heterogene- growth factor in chick embryonic dorsal root neurons have been ity. observed. One type is associated with the plasma membrane (or Recently, it has also been demonstrated that NGF can be microsomal fraction) and can be completely solubilized by Triton X-100. These receptors display the nonsaturable binding taken up by the synaptic terminals of neurons of the superior isotherms and curvilinear Scatchard plots previously reported cervical ganglia after injection of 125I-NGF into the anterior for receptors in whole cells. The second chamber of the eye of the adult rat (16, 17). This internalized class of binding sites is located in the nucleus, firmly bound to NGF is carried to the perikarya by retrograde axoplasmic chromatin. These receptors are not solubilized by detergent, transport where it can also effect the synthesis of tyrosine hy- show saturable binding, and yield linear Scatchard plots of the type associated with a single class of binding sites of high af- droxylase and -f3-hydroxylase (18). It seems unlikely finity. The presence of the two receptor types suggests a bimodal that these effects could be mediated by the plasma membrane mechanism of action for nerve growth factor. receptors that direct neurite proliferation, suggesting more than one mechanism of action for NGF. This study provides direct Nerve growth factor (NGF) is an -like (1) that evidence for the existence of two classes of NGF receptors that induces the morphologic and metabolic differentiation of differ in their solubilization by Triton X-100, binding proper- sympathetic and sensory neurons in (2). However, ties, and subcellular localization. These two discrete NGF re- the responsiveness of these two tissues is quite different. Sensory ceptors could provide the separate loci for the expression of the neurons bind and are stimulated by NGF only during a rela- different NGF responses. tively narrow period of embryonic development (2-4). In contrast, the sympathetic cells retain sensitivity, even in adult MATERIALS AND METHODS (5), although the nature of the response changes during NGF was prepared from mouse submaxillary according different stages of development. to Bocchini and Angeletti (19). Subsequent references to NGF The types of cellular processes effected by NGF are also refer to this preparation, commonly identified as 2.5S NGF or variable. With low levels of hormone (about 1-10 ng), both NGF(AB). Oxidized NGF derivatives were prepared as de- sensory and sympathetic neurons produce the characteristic scribed by Frazier et al. (20). NGF was labeled with 1251 as neurite outgrowth with accompanying general stimulation of described previously (12). The specific activity of these prep- anabolic and neurotubule polymerization (2, 6). arations varied from 200 to 2000 cpm/fmol. Cytochrome c was At higher concentrations (about 1-10,tg), the specific induction labeled with 125I according to the procedure of Greenwood and of enzymes involved in nonadrenergic syn- Hunter (21), yielding preparations with specific activities of thesis is observed (7) as well as such effects as regeneration of 200-1000 cpm/fmol. Chick dorsal root ganglia (DRG) were adrenergic fibers in (8), inhibition of the of dissected from freshly decapitated chick embryos (8-day) and mucopolysaccharide in chrondrocytes (9), and stimulation of collected in an ice-cooled vessel by vacuum aspiration with a the temporal conversion of cell surface adhesive specificity in fine glass pipette. The inevitable crushing of the ganglia col- embryonic optic tectal cells (10). lected by this process did not alter their binding properties. The responses associated with the low concentrations of NGF Specific binding of 125I-NGF was measured according to appear to be mediated through receptors bound to cell surface Frazier et al. (12). Experiments were carried out in triplicate membranes. NGF covalently linked to Sepharose beads under and were corrected for the high-capacity, low-affinity binding conditions that prevent the release of soluble NGF produces a of NGF to the tube in the presence of incubation buffer stimulation of neurite outgrowth from embryonic dorsal root alone. sensory ganglia indistinguishable morphologically from that Triton X-100 extractions of particulate samples were per- of the unbound hormone (11). By using 125I-labeled NGF formed by incubating samples at room temperature for 10 min (125I-NGF), these receptors have been shown to possess an af- in Hanks' balanced salt solution (HBSS) (12) containing the finity of approximately 1010 liters/mol (12-14), corresponding appropriate concentration of detergent. The insoluble residue to the concentration range required for activity. However, the was collected by centrifugation (10 min, 3000 X g) and washed specific binding is a nonsaturable process with lower affinity twice with HBSS. DRG extracted with 1% (vol/vol) Triton are (about 2 X 107 liters/mol) of binding sites at higher concen- referred to as Triton-extracted DRG. trations of labeled NGF (12). The range of affinities observed Prior to the determination of specific binding to solubilized results from negatively cooperative interactions among a single components, 125I-NGF was purified by passage over a Sephadex class of receptor molecules, analogous to that observed for in- G-100 column previously equilibrated in 50 mM Tris, pH 7.4, Abbreviations: NGF, nerve growth factor; 125I-NGF, 125I-labeled NGF; containing 0.02% Triton and bovine albumin, 1 mg/ml. DRG, dorsal root ganglia; HBSS, Hanks' balanced salt solution. The peak eluting at a position corresponding to a molecular * Present address: Research Center, Department of Medicine, weight of 27,000 was used immediately in the binding assays. Washington University School of Medicine, St. Louis, MO 63110. Ganglia (usually 250 per experiment) were homogenized in 5 2785 Downloaded by guest on October 1, 2021 2786 Biochemistry: Andres et al. Proc. Nati. Acad. Sci. USA 74 (1977)

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2, 'O 60 -005 A 80 40 Zo2~~~~~~~ 1 2 20 125I-NGF, nM FIG. 2. Specific binding of 125I-NGF to nuclei (0) and micro- 02 OA Os 08 1 somes (0) from chick embryonic (8-day) DRG as a function of hor- Triton, % mone concentration. Each assay tube contained the material isolated from three ganglia in a final volume of 300,ul. The data are corrected FIG. 1. Specific binding of 125I-NGF to various subcellular for nonspecific binding (about 30% of specific binding). fractions of chick embryonic (8-day) DRG after extraction with dif- ferent concentrations of Triton X-100. Ganglia, microsomes, or nuclei of the specific binding was solubilized. These results clearly were incubated with the indicated concentrations of Triton (room binding sites, not temperature, 10 min). The nonsolubilized material was collected by suggest that DRG contain additional specific centrifugation and washed twice with ice cold HBSS containing bo- present in plasma membranes, that are exposed in detergent- vine serum albumin at 1 mg/ml. Specific binding assays were per- extracted homogenates and are not solubilized by this treat- formed on tubes containing three ganglia (0) or the equivalent ment. amount of microsomes (0) or nuclei (-) in a final volume of 300 M1. Support for this conclusion was provided by examination of the insoluble pellet, collected by centrifugation after Triton ml of HBSS and centrifuged (10 min, 3000 X g). The pellet was extraction of the DRG homogenates, which showed significant resuspended in 1 ml of 50 mM Tris, pH 7.4, containing 0.1% specific binding. Fractionation of this material by sucrose Triton. After 60 min at room temperature, the nonsolubilized density centrifugation revealed that virtually all of this binding material was removed by centrifugation (60 min, 48,000 X g). was located in the highest density fraction, containing pre- 125I-NGF was added to the supernatant (final concentration, dominately nuclei. Purification of nuclei from DRG (22) es- 30 pM) and, after incubation at room temperature for 60 min, tablished that these organelles indeed contained the binding the samples were chilled to 40 and chromatographed (at the sites for NGF that are not solubilized by Triton X-100 (Fig. 1, same temperature) on a column of Sephadex G-100 (0.8 X 22 squares). It should be noted that, because of the differences in cm) previously equilibrated in 50 mM Tris, pH 7.4, containing the specific binding-versus-125I-NGF concentration relation- 0.02% Triton and bovine serum albumin, 1 mg/ml. The eluate ships of the microsomal and nuclear binding (see below), the was monitored for radioactivity. proportion of the binding to the two populations of binding sites Nuclei were prepared according to Biessmann and Rajewsky varies with the 1251-NGF concentration used in the binding (22). This procedure involves preparation of a crude nuclear assay. fraction by low-speed centrifugation, purification of nuclei by Properties of the Receptors Not Solubilized by Triton. The sedimentation in 2.3 M sucrose/i mM sodium cacodylate/1 specific binding of 125I-NGF, localized in the nuclear fraction mM MgCl2 at pH 6.4, and washing with 1% Triton. These nu- of homogenates of chick embryonic DRG, displayed distinctly clei were resuspended in HBSS by homogenization in a tightly different binding properties from those observed in either fitting glass-Teflon homogenizer. Aliquots were taken for the dissociated ganglia or the microsomal fraction. As shown in Fig. determination of DNA and were processed immediately. The 2, in the concentration range of 125I-NGF in which the plasma amount of sample calculated to give an appropriate concen- membrane receptors were not saturated (open circles), the tration of DNA in the binding assay was diluted 1:1 with HBSS specific binding to the nuclear fraction reached a maximum containing bovine serum albumin at 20 mg/ml. The volume at 1 nM 125I-NGF and did not increase further (closed circles). was adjusted with HBSS containing bovine serum albumin at A Scatchard analysis of these data for nuclei (Fig. 3, open cir- 10 mg/ml. Based on DNA determinations, this procedure yields about 75% of the nuclei found in a homogenate of DRG. The protein/DNA ratio was approximately 5.3:1 (23). Chromatin was prepared from purified nuclei according to Spelsberg and Hnilica (24), microsomes were isolated as described by Banerjee 16 et al. (14), and DNA was determined with the diphenylamine method described by Burton (25). x 12 RESULTS Effect of Triton Extraction on NGF Receptors of DRG. In ~8 order to determine the proper conditions for solubilizing the 0 d membrane-bound receptor for NGF (11-14), homogenates of DRG were extracted with different concentrations of Triton. As shown in Fig. 1, at concentrations of detergent as low as 0.2%, 4 a major portion of the ability of DRG to specifically bind NGF was removed from the homogenates. However, increasing the concentration of the detergent to 1% did not significantly sol- 4 8 12 ubilize more of the binding remaining in the pellet. In contrast, Bound, fmol when microsomal preparations, containing primarily plasma FIG. 3. Scatchard analyses of specific 125I-NGF binding data for membranes, were treated in the same manner, more than 90% microsomes (0) and nuclei (0) from DRG. Details as in Fig. 2. Downloaded by guest on October 1, 2021 Biochemistry: Andres et al. Proc. Nati. Acad. Sci. USA 74 (1977) 2787 8 e Ii 6I'I 0 6 > ~~~~II '.4 4 I II x x I

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0 10 20 30 Fraction number 5 10 15 20 25 Fraction number FIG. 4. Elution profile of Sephadex G-100 chromatography of solubilized NGF receptors from chick embryonic DRG. Ganglia (250) FIG. 5. Second chromatography of the first peak (high-molecu- lar-weight NGF-binding material) on a column of Sephadex G-100. were homogenized and incubated in 0.1% Triton X-100. The partic- The sample (fractions 8-10 of Fig. 4) was applied directly to the col- ulate material was removed by centrifugation (60 min, 48,000 X g), umn which was as in 4. 0, Untreated and 125I-NGF was added to the supernatant (final conc., 0.3 pM). developed Fig. sample; *, sample pretreated with an excess (1 uM) of unlabeled NGF. After 1 hr at room temperature, the sample was analyzed on a Sephadex G-100 column previously equilibrated in 50 mM Tris, pH 7.4, containing bovine serum albumin (1 mg/ml) and 0.02% Triton. structures. Cell nuclei, prepared by using ionic conditions and *, Sample containing only 1251-NGF; 0, sample containing 1251-NGF pH different from those of simple Triton extraction, were not plus an excess (1 AM) of unlabeled NGF. distinguishable from crude Triton-extracted DRG with respect to saturability and affinity, confirming this conclusion. Al- cles) yielded a linear plot consistent with a single class of re- though the purity of the nuclei, isolated as described in Mate- ceptor molecules. The binding constant was comparable to that rials and Methods, has been confirmed by biochemical and calculated for the high-affinity portion of the binding curve of electron microscopic standards (23), the presence of unidenti- the plasma membrane receptors, approximately 5 X 109 li- fied contaminants is difficult to exclude rigorously. In order to ters/mol (Fig. 3, closed circles). establish that the binding observed in the nuclear fraction was Properties of the Receptors Solubilized by Triton. The loss not due to the presence of microsomal elements, a homogenate of a major portion of the specific binding as the result of the of DRG was prepared and divided into two portions. One was Triton treatment may be due either to the solubilization of a supplemented with previously prepared microsomes. Nuclei plasma membrane receptor or to the irreversible inhibition of were isolated from both samples (DNA was adjusted to the same low-affinity receptor. Although the localization of the re- concentration in both samples to counteract any nonreprodu- maining high-affinity binding in the nuclear pellet argues cible loss during isolation) and the specific 125I-NGF binding against the latter explanation, the demonstration of receptors was determined. As indicated in Table 1, there was no differ- in the soluble fraction was deemed to be important. Accord- ence in the amount of specific binding observed in the sup- ingly, the supernatant obtained after centrifugation (48,000 X plemented and unsupplemented samples. In the absence of g, 1 hr) of chick embryonic DRG incubated with 0. 1% Triton Triton extraction, however, the specific binding of the nuclear X-100 for 1 hr was diluted with buffer, mixed with 125I-NGF, and microsomal fractions was additive. Thus, the nuclear and analyzed by gel filtration at 4°. In the elution profile (Fig. binding does not appear to be related to that found in the mi- 4), two peaks of radioactivity were observed (solid line) in crosomal fraction. contrast to the sample (dashed line) that also contained an excess However, these experiments do not eliminate the possibility of unlabeled NGF. The second peak, found in both samples that the nuclear binding is due to a cytoplasmic component that (around fraction 20), eluted close to the position of cytochrome has become associated with or entrapped in nuclear material. c and corresponds to native unbound 125I-NGF. To exclude this possibility, chromatin was prepared according When the initial peak of radioactivity found in the sample to Spelsberg and Hnilica (24). This preparation displayed mixed with extract supernatant was collected and reanalyzed binding characteristics for 125I-NGF identical to those of the on the same gel filtration column, the complex eluted in the nuclear preparations with respect to affinity and saturability same position with little dissociation to the low molecular weight (data not shown). The observed specific binding of NGF to form (Fig. 5, dashed line). However, when this high molecular nuclei appears, therefore, to be inherent to chromatin. weight fraction was incubated with unlabeled NGF at room temperature for 30 min before it was applied to the gel filtration Table 1. Effect of addition of microsomes on nuclear binding column, the majority of the radioactivity eluted in the position of 125I-NGF of unbound hormone (solid line), establishing both the speci- ficity and reversibility of the solubilized receptors. Sample Specific binding, %* To ascertain the relationship of the specific binding sites solubilized by the detergent and the receptors identified by A. Nuclei 100 + 5 binding assays with whole cell preparations (12), microsomal B. Nuclei and microsomes (extracted) 95 + 5 fractions were prepared and extracted with detergent. As shown C. Nuclei and microsomes (unextracted) 260 5 in Fig. 1, the receptor molecules present in this subcellular fraction were readily solubilized by Triton, showed nonsatu- Homogenates from 100 chick embryo (8-day) DRG were prepared, rable binding isotherms (Fig. 2), and showed apparent high- and sample B was supplemented with microsomes prepared from and low-affinity binding in Scatchard analysis (Fig. 3). another 100 DRG. Nuclei were isolated from both samples and ad- same was deter- Identification of Nuclear Structures as the Site of Triton- justed to the DNA concentration. Specific binding Resistant mined in six duplicate samples, and the results are expressed as per- Specific NGF Binding. Initial fractionation, by ul- centage of control (sample A). Sample C: membranes from 100 DRG tracentrifugation through 2.3 M sucrose, of the pellet remaining were added to nuclei from 100 DRG immediately prior to determi- after Triton extraction of DRG clearly suggested that the de- nation of specific binding. tergent-resistant receptors were associated with nuclear * Mean + SD. Downloaded by guest on October 1, 2021 2788 Biochemistry: Andres et al. Proc. Natt. Acad. Sci. USA 74 (1977)

Specificity of NGF Binding to Nuclei of DRG. In view of Table 2. Displacement of specific binding of 125I-NGF to chick the positively charged character of NGF [pI = 9.3 (26)] under DRG neurons by various the conditions of the assay, the effect of other basic proteins on 125I-NGF the specific nuclear binding of 125I-NGF was tested. As shown in Table 2, cytochrome c and lysozyme, at concentrations of Protein displaced, % 1 5 and 10% of the control gM, displaced only (native NGF) Native NGF (mouse) 100 binding. Insulin, which has limited structural relatedness to 87 contrast, NGF 1-ox. NGF (mouse)* NGF (1), displaced only 3%. In samples of that 2-ox. NGF (mouse)* 71 had been treated with N-bromosuccinimide to the extent that 3-ox. NGF (mouse)* 65 one, two, or three residues of per mol of NGF mo- Lysozyme (chick) 10 nomer (molecular weight 13,259) had been converted to the Cytochrome c (horse) 5 oxindole moiety showed significant, albeit progressively less Insulin (pig) 3 with increasing oxidation, ability to displace the radioactively labeled hormone from isolated nuclei. These values are con- In binding assay containing nuclei from three chick DRG per tube, sistent with those observed for the displacement of 125I-NGF the proteins were added to a concentration of 1 MtM. The radioactivity from the plasma membrane receptors by the same derivatives displaced was expressed as percentage of the native NGF control. The (M. W. Pulliam, N. C. Baglan, and R. A. Bradshaw, unpublished numbers shown are the mean of at least two experiments. * 1-ox., 2-ox., and 3-ox. NGF: mouse NGF oxidized to the extent of data). one, two, and three residues of tryptophan modified per subunit To test whether basic proteins in general show "specific (20). binding" to nuclear components, cytochrome c was labeled with 125I (21) and tested for direct binding, with the same procedures nuclei is not required for NGF binding. Rather, the binding as with 125I-NGF. Absolutely no "specific" binding was ob- seems to be a property of chromatin itself. Second, as judged served although, interestingly, the nonspecific binding to the by the behavior of cytochrome c, basic proteins do not, in tubes (see Materials and Methods) was similar to that observed general, specifically bind to chromatin. Third, cytochrome c for 125I-NGF. and lysozyme, both basic proteins with molecular weights Finally, if the presence of specific binding in DRG nuclei is similar to that of monomer NGF, and insulin, which is physiologically meaningful, it should be heavily enriched in structurally related to NGF (1), are able to displace only in- the responsive cells as opposed to biologically unresponsive cells. significant amounts of NGF from its nuclear binding sites. Of To test this hypothesis, the specific binding to the nuclei of the proteins tested, only native NGF and, to a lesser extent, its erthrocytes was measured and compared to that of oxidized derivatives, can do so. Finally, the nuclei receptors are DRG. These cells were selected because they do not display concentrated in normally responsive cells as compared to other specific plasma membrane binding of 125I-NGF, in contrast to tissues. other non-neuronal cell types (27). When calculated as specific During development of the peripheral , the binding (fmol) per Aig of DNA, erthrocyte chromatin showed receptors located in the plasma membrane provide the initial about 15% of the specific binding found in DRG chromatin. site of interaction for NGF with the responsive neurons of the Similar enrichment has been found for sympathetic neurons sensory and sympathetic nervous system. Complexation with as compared to brain and erthrocytes (R. Y. Andres and R. A. these surface receptors is sufficient to elicit the neurite prolif- Bradshaw, unpublished data). eration characteristic of the NGF bioassay (11). However, it has been established that NGF can be taken up by these cells (28) DISCUSSION and, in one case, it has been shown that the internalized hor- The results presented indicate that chick embryonic DRG mone can elicit a biological effect-namely, increased synthesis contain at least two classes of binding sites for NGF. They can of dopamine-f-hydroxylase and (18). be distinguished by means of subcellular fractionation or by These changes were observed after the retrograde axonal their solubility in detergent. One type of receptor is associated transport of 125I-NGF, injected into the anterior chamber of with the plasma membrane fraction and, as generally expected the eye, to the neuronal cell bodies located in the superior cer- for such structures, is readily solubilized by detergent. As shown vical ganglion (16, 17). Electron microscopic autoradiography by the binding properties of the microsomal preparations, these has shown that the majority of the material so transported is receptors are the same as those identified and characterized in found in the perikarya in secondary lysosomes or vesicles fused whole-cell preparations of DRG (12). The second type of with the smooth endoplasmic reticulum (29). It is attractive to binding is associated with the nuclear fraction of DRG and is propose, therefore, that some of the latter hormone is released characterized by insolubility in solutions of Triton X-100. This into the nucleus where it can regulate gene class of receptors apparently escaped detection in earlier through complexation with the receptors described in these binding studies (12-14), either because of the nature of the studies. Indeed, subcellular fractionation of superior cervical material used or the manner in which it was prepared. ganglia 24 hr after injection of 125I-NGF into the eye shows Examination of the other subcellular fractions of DRG did significant levels of labeled hormone in the nucleus (ref. 30; R. not reveal any other significant binding, suggesting that these Y. Andres and E. M. Johnson, unpublished data). two classes of NGF receptors represent the two principal sites The postulate that NGF can specifically regulate the me- of activity of the hormone. This is supported by the observation tabolism of responsive neurons through interaction with nuclear that the amount of binding in the microsomal and nuclear receptors does not alter previous conclusions concerning the role fractions accounts for all of the binding in the homogenate (Fig. of plasma membrane receptors as mediators of neurite prolif- NGF 1). eration, the characteristic morphologic response of (11). Several observations suggest that the nuclear receptors are Rather it suggests a dual mechanism of action for NGF (31, 32) themselves physiologically significant. First, repeated washing in which the plasma membrane receptors provide the site for and homogenization of nuclei does not appreciably alter the the trophic stimulation of embryonic neurons during their specific binding, suggesting that morphological integrity of development and the nuclear receptors provide the means by Downloaded by guest on October 1, 2021 Biochemistry: Andres et al. Proc. Nati. Acad. Sci. USA 74 (1977) 2789

which mature neurons are maintained through NGF provided 9.,. Eisenbqrth, G. S., Drezner, M. K. & Lebowitz, H. E. (1975) J. by specific uptake at the synaptic termini and retrograde axonal Phiirmacol. Exp. Ther. 192, 630-634. transport. This mechanism also provides a specific means for 10. Merrell, R., Pulliam, M. W., Randono, L., Boyd, L. F., Bradshaw, communication between termini and cell bodies in NGF-re- R. A. & Glaser, L. (1975) Proc. Nati. Acad. Sci. USA 72,4270- 4274. sponsive neurons and suggests that, in these neurons, NGF may 11. Frazier, W. A., Boyd, L. F. & Bradshaw, R. A. (1973) Proc. Natl. be the chromatolytic messenger. Acad. Sci. USA 70,2931-2935. Although the importance of such a role may be restricted to 12. Frazier, W. A., Boyd, L. F. & Bradshaw, R. A. (1974) J. Biol. mature neurons, it is entirely plausible that the nuclear receptors Chem. 249, 5513-5519. function over a much wider range of development. In view of 13. Herrup, K. & Shooter, E. M. (1973) Proc. Natl. Acad. Sci. USA the fact that it has been demonstrated that embryonic DRG 70,3884-3888. neurons possess nuclear receptors and that these cells can take 14. Banerjee, S. P., Snyder, S. H., Cuatrecasas, P. & Greene, L. A. up NGF, it seems likely that these receptors also play a regu- (1973) Proc. Nati. Acad. Sci. USA 70,2519-2523. latory role prior to the formation of functioning synaptic 15. De Meyts, P., Bianco, A. R. & Roth, J. (1975) J. Biol. Chem. 251, junctions. other processes, could the 1877-1888. Among they regulate 16. Hendry, I. A., Stoeckel, K., Thoenen, H. & Iversen, L. L. (1974) synthesis of the plasma membrane receptors, thus providing Brain Res. 68, 103-121. an opportunity for the regulation of the number of these cell 17. Stoeckel, K., Paravicini, U. & Thoenen, H. (1974) Brain Res. 76, surface receptors, in analogy to the membrane receptor regu- 413-421. lation demonstrated in other hormone systems (33, 34). This last 18. Paravicini, U., Stoeckel, K. & Thoenen, H. (1975) Brain Res. 84, possibility raises the interesting question of whether other 279-291. hormone systems may also possess surface membrane and nu- 19. Bocchini, V. & Angeletti, P. U. (1969) Proc. Nati. Acad. Sci. USA clear receptors that work in concert to allow a greater latitude 64,787-794. of target cell regulation of the hormonal response. The recent 20. Frazier, W. A., Hogue-Angeletti, R. A., Sherman, R. & Bradshaw, finding of nuclear receptors for insulin (35), which show R. A. (1973) Biochemistry 12,3281-3293. 21. Greenwood, F. C. & Hunter, W. M. (1963) Biochem. J. 89, properties similar to those observed for NGF, is suggestive that 114-123. this may be the case. 22. Biessmann, H. & Rajewsky, M. F. (1975) J. Neurochem. 24, 387-393. The authors thank Drs. William A. Frazier, Luis Glaser, Martin 23. Austoker, J., Cox, D. & Mathias, A. P. (1972) Biochem. J. 129, Schwab, and Hans Thoenen for helpful suggestions and discussions. 1139-1155. This work was supported by Research Grant NS10229 from the Na- 24. Spelsberg, T. C. & Hnilica, L. S. (1971) Biochim. Biophys. Acta tional Institutes of Health. R.Y.A. is a fellow of the Swiss National 228,202-211. Foundation. 25. Burton, K. (1956) Biochem. J. 62,315-323. The costs of publication of this article were defrayed in part by the 26. Bocchini, V. (1970) Eur. J. Biochem. 15, 127-131. payment of page charges from funds made available to support the 27. Frazier, W. A., Boyd, L. F., Pulliam, M. W., Szutowicz, A. &- research which is the subject of the article. This article must therefore Bradshaw, R. A. (1974) J. Biol. Chem. 249, 5918-5923. be hereby marked "advertisement" in accordance with 18 U. S. C. 28. Norr, C. S. & Varon, S. (1975) Neurobiology 5, 101-118. §1734 solely to indicate this fact. 29. Schwab, M. & Thoenen, H. (1976) Brain Res., in press. 30. Stoeckel, K., Guroff, G., Schwab, M. & Thoenen, H. (1976) Brain 1. Frazier, W. A., Angeletti, R. H. & Bradshaw, R. A. (1972) Science Res. 109, 271-284. 176,482-488. 31. Bradshaw, R. A., Frazier, W. A., Pulliam, M. W., Szutowicz, A., 2. Levi-Montalcini, R. & Angeletti, P. U. (1968) Physiol. Rev. 48, Jeng, I., Hogue-Angeletti, R. A., Boyd, L. F. & Silverman, R. E. 534-569. (1975) "," Vol. 2, Proc. Int. Congr. Pharma- 3. Herrup, K. & Shooter, E. M. (1975) J. Cell Biol. 67, 118-125. col., 6th, 231-238. 4. Frazier, W. A., Boyd, L. F., Szutowicz, A., Pulliam, M. W. & 32. Bradshaw, R. A., Pulliam, M. W., Jeng, I. M., Andres, R. Y., Bradshaw, R. A. (1974) Biochem. Biophys. Res. Commun. 57, Szutowicz, A., Frazier, W. A., Hogue-Angeletti, R. A. & Silver- 1096-1103. , R. E. (1976) in Surface Membrane Receptors, eds. Brad- 5. Angeletti, P. U., Levi-Montalcini, R. & Caramia, F. (1971) J. shaw, R. A., Frazier, W. A., Merrell, R. C., Gottlieb, D. I. & Ultrastruct. Res. 36,24-36. Hogue-Angeletti, R. A. (Plenum Press, New York), pp. 227- 6. Angeletti, P. U., Gandini-Attardi, D., Toschi, G., Salvi, M. L. & 246. Levi-Montalcini, R. (1965) Biochim. Biophys. Acta 95, 111- 33. Gavin, J. R., III, Roth, J., Neville, D. M., Jr., De Meyts, P. & Buell, 120. D. N. (1974) Proc. Natl. Acad. Sci. USA 71, 84-88. 7. Thoenen, H., Angeletti, P. U., Levi-Montalcini, R. & Kettler, R. 34. Hinkle, P. M. & Tashijian, D. H., Jr. (1975) Biochemistry 14, (1971) Proc. Natl. Acad. Sci. USA 68, 1598-1602. 3845-3851. 8. Bjerre, B., Bjorklund, A. & Stenevi, U. (1973) Brain Res. 60, 35. Goldfine, I. D. & Smith, G. J. (1976) Proc. Nati. Acad. Sci. USA 161-176. 73, 1427-1431. Downloaded by guest on October 1, 2021