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NERVE GROWTH FACTOR!

+972

Ralph A. Bradshaw Department of Biological Chemistry Washington University School of Medicine St. Louis, Missouri 63110

CONTENTS PERSPECTIVES AND SUMMARy ........................... ....... ..... ...... .............................

191

INTRODUCTION . . . . ............. ........ ............. ....... ...........................................................

193

MOLECULAR PROPERTIES . . ......... ............. ....... ....... ..............................................

Mouse Submaxillary Gland Snake Venom Other Tissues . .

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194

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198

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....... ............................... ..... ......

EVOLUTION

194

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200

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200

BIOSYNTHESIS .............................. .................................................... ..........................

203

BIOLOGIC PROPERTIES ................ ........... . ................ ................ ..............................

205

MECHANISM OF ACTION

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207

Plasma Membrane Receptors Internalization (Retrograde Axonal Transport) Nuclear Receptors . .. .. . ......... . ........... .. . .. .... A Mechanistic Model . . . . ... .. . .. ...... ... . . ......... ..... .... .... .... ......... ..............................................................................

207

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209

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210 210

PERSPECTIVES AND SUMMARY Since the discovery of nerve growth factor (NGp) some 30 years ago, there has been a constant interest in determining the precise nature of this sub­ stance and its true physiologic role in regulating growth, development, and maintenance in the nervous system. Although the potential importance of this understanding was clear from the outset, it has nonetheless remained an elusive go a l For the most part this has been en gendered by the problems .

lAbbreviations used: NGF, nerve growth factor; NSILA, nonsuppressible insu­ lin-like activity or insulin-like growth factor; EGF, epidermal growth factor. 191

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of working with neuronal tissue, which is difficult to obtain or culture in quantity, and by the problems of extrapolating in vitro results to in vivo systems. Thus, although considerable progress has been made in recent years, it is not possible at present to describe with certainty the biologically important species of NGF, its site(s) of synthesis and subsequent transport, the scope of responsive tissues, or its mechanism of action. Fortunately, this failure to resolve the fundamental features of the chem­ istry and biology of NGF have not dampened the collective spirits of the increasing number of researchers interested in this problem, and important inroads have been and are being made which are summarized in this review. Perhaps the most important of these has been the realization that NGF does not represent a unique entity, charged with the singular task of maintaining the viability of selectively responsive neurons, but rather is a member of a larger group of regulating peptides and proteins that possess the same responsibility albeit to other cell types located throughout the organism. Some of these substances, such as insulin, nonsuppressible insulin-like activ­ ity (NSILA), and relaxin, actually bear structural relatedness, presumably as the result of evolution from a common precursor, while others, such as epidermal growth factor (EGF), are apparently only functionally similar. However, all of these growth factors can justifiably be classed in one group, based on the nature and time course of the effects that they elicit in their individually responsive tissues. Furthermore, these factors can properly be thought of as "maintenance" or "secondary" hormones, in contrast to "primary" hormones. As detailed in the last section of this review, the distinction between primary and secondary hormones may well hinge on an extracellular as opposed to an intracellular site of action. Most of the knowledge about the properties of NGF are derived from studies with the male mouse submaxillary protein. However, it was first observed in the extracts of two mouse "sarcomas" and subsequently, in somewhat higher amounts, in the venoms of poisonous snakes, before the mouse submaxillary source was discovered. It has also been shown to be present, in very small amounts, in a variety of tissues, which generally correlate with sympathetic innervation. More recently this has been ex­ tended to a variety of cells in culture as well. The molecular properties of mouse submaxillary NGF have been studied in detail. The protein occurs as a noncovalent complex of three polypeptide chains, one of which, the ,B-subunit, possesses all of the nerve growth­ promoting activity. This subunit is composed of 2 identical polypeptide chains of 13,259 mol wt. The amino acid sequence of this unit has been determined. Partial sequence data for cobra (Naja naja) NGF has shown that venom NGFs also have a dimeric structure and are homologous to the mouse protein. Although studies on the biosynthesis of NGF have not

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definitively located the physiologically significant sites of production, other than the submaxillary gland of mice, they have established that the mole­ cule is first constructed as prohormone of 22,000 mol wt, which is subse­ quently processed by proteolytic modification to the 13,000 mol wt species. The significance of this cleavage in biologic terms is not known. The biologic effects of NGF, which have been studied in detail, are concentrated on the sympathetic and portions of the embryonic sensory nervous systems. NGF exerts a positive pleiotypic activation on responsive cells resulting in stimulation of a variety of catabolic and anabolic path­ ways. The polymerization of microtubules is thought to be a key step in the eventual proliferation of neurites, the morphological basis for the only reliable bioassay for the hormone. An additional important effect of the hormone is the maintenance of viability of responsive neurons in vivo and in vitro. NGF also exerts a number of effects on tissues of the central nervous systems (CNS) as well as some of nonneuronal origin. The impor­ tance of these effects, which are usually different in character from periph­ eral neuron responses, is not presently appreciated Receptors for NGF have been located in both the plasma membrane and the nucleus of peripheral neurons. The plasma membrane receptors show multiphasic specific binding of 125I_NGF, similar to the behavior of insulin receptors in lymphocytes. Kinetic analyses indicate that this apparent high and low affinity binding results from negatively cooperative interactions among a single type of receptor molecules. These molecules are also readily dissolved by nonionic detergents. In contrast, nuclear receptors show only high affinity binding sites and are not solubilized by detergents. Based on considerable evidence that NGF can be internalized specifically by nerve terminals and transported to the cell bodies by retrograde axonal flow, a three step mechanism for NGF involving complexation with external mem­ brane receptors, internalization and transport to the nucleus, and complexa­ tion with nuclear receptors to directly effect transcriptional events is proposed. In keeping with the structural and functional relationships of NGF to other maintenance hormones, such as insulin, NSILA, and relaxin, this mechanism may have broad applicability. .

INTRODUCTION

Nerve growth factor has been identified in a wide variety of vertebrate tissues (1-3) but with two exceptions, all contain very low concentrations and none, as yet, has been shown to be relevant to nervous system physi­ ology as a site of synthesis. The two sources of the factor from which milligram quantities can be obtained are adult male mouse submaxillary gland (4) and the venom of the three families of poisonous snakes, Crotali-

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dae, Viperidae, and Elapidae (5). It should be noted that both mouse submaxillary gland and snake venom gland represent true sites of synthesis in vivo and, among other sites, may even be important elements in the network of tissues that produce NGP for the maintenance of responsive neurons, primarily those found in the sympathetic nervous system. How­ ever, the greatly increased amounts found in these sources is considerably higher than the physiologically active levels of the hormone and it seems likely that these concentrations are a manifestation of other metabolic features of the gland, which results in the increased synthesis of many compounds in addition to NGP. Thus, these high concentrations are proba­ bly unrelated to the action of NGP on the nervous system and may even play an exocrine role, possibly of a vestigial nature, in these animals. The NGP of mouse submaxillary gland has been studied in greatest detail, due primarily to its ready availability. It seems reasonable to assume that the salient features, at least of the biologically active structure, will be common to all vertebrate NGP molecules, as judged by biologic and im­ munologic cross-reactivity. Thus, it may be viewed as representative of the class. However, as noted below, significant structural variations may exist, particularly at the quaternary level, between the NGF molecules isolated

from different species, and those isolated from different tissues within an organism, which may be of importance in regulating one or another aspect of the synthesis, transport, or mechanism of the hormone. This review deals primarily with mouse submaxillary gland NGF. It describes the molecular properties, as they are presently understood, and the evolutionary relationships of this hormone with other growth factors. It also summarizes the biologic properties at the organismic and cellular levels, which can be integrated into a single mechanism of action involving both extracellular and intracellular receptor interactions. Several reviews dealing more explicitly with various aspects of the chemical and biologic features of NGP are available (6- 13). A more general treatment of growth factors can be found in the article by Gospodarowicz & Moran (14). MOLECULAR PROPERTIES

Mouse Submaxillary Gland 7S NGF Mouse submaxillary NGF is isolated from gland homogenates as a high molecular weight complex containing three types of polypeptide chains, designated a, /3 and Y ( 1 5, 16). The stoichiometry of the complex is a2/3Y2 but since the /3 -subunit is actually a dimer of identical chains, (17, 1 8) at least a twofold axis of symmetry is maintained. The molecular weight

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of either the a- and y- subunits is approximately 26,000 ( 13, 19) while the combined molecular weight of the two polypeptides of the p-subunit is 26,5 18 (20). Thus, the complex has a molecular weight of approximately 130,000 , which yields a sedimentation coefficient of 7S ( 1 5). This value is commonly used to differentiate this high molecular weight complex from other forms of mouse NGF. The 7S complex also contains between 1 and 2 moles zinc ion per mole (2 1 ), apparently bound to the y-subunit. Removal of the zinc does not materially affect the stability of the complex (22). The three unique subunits of 78 NGF are associated weakly by noncon­ valent forces and can be dissociated above pH 8, below pH 5, or by simple dilution ( 1 6, 22, 23). In fact, at physiologically active concentrations, the complex is fully dissociated to its constituent subunits, suggesting that the 78 form of NGF is not important as an active entity (23). The weak association of the subunits of the 78 complex has also led Murphy et al (24) to suggest that it may not occur at all in the gland but is formed as an artifact of preparation. This conclusion is based on the observation that gland homogenates directly diluted by 104 and measured hydrodynamically show high molecular weight material, whereas 7S NGF under the same conditions is completely dissociated (23). However, it is unclear how such a complex would be dissociated to allow the formation of 78 NGF during the isolation procedures used and it seems rather more likely that these observations are due to a nonspecific association of the p (active)-subunit with other cellular elements after the dissociation of the naturally occurring 78 complex is induced by dilution. The three constituent polypeptide chains of 78 NGF differ sufficiently in their isoelectric point to allow their complete separation by ion-exchange chromatography. This can be accomplished after the 7S complex has been purified to homogeneity (25) or on partially purified samples (12). In the latter case, the homogenate is first fractionated on a column of Sephadex G-l00 and then chromatographed on a column of eM-cellulose eluted at pH 5.0. The p-subunit is tightly bound and is eluted at the end of the salt gradient in homogeneous form. The a-subunit, with an average pI of 4.3 passes through the column un retained and the y-subunit, with an average pI of 5.5 (26), is eluted in the initial phase of the gradient. Since both of these subunits are contaminated only by high molecular weight material, they are readily purified to homogeneity by additional gel filtration steps. The subunits obtained by either procedure can be reassociated, essentially quantitatively, to the 78 form ( 1 2, 1 6). Of the three SUbunits, only the p -moiety possesses nerve growth promot­ ing activity and it is solely responsible for the maintenance of adrenergic neurons in vitro. It is unclear, therefore, what role the a- and y-subunits

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play in either the associated or dissociated state. This is particularly true of the a-subunit that possesses no known biologic activity. The a-subunit, on the other hand, has been clearly established to be an arginine-specific estero-peptidase of the serine protease family (27). It is inhibited by diiso­ propyl-fluorophosphate and shows 35% sequence identities with bovine trypsin in the 86 of 229 residues determined (28). It is also remarkably similar to the binding protein found in the high molecular weight form of EGF, which is also derived from mouse submaxillary glands (29). Sequence analysis of limited segments of this protein have indicated about 75% identities with the NGF y-subunit (28) and they are indistinguishable with respect to catalytic and physical properties. However, they show an abso­ lute specificity for complex formation; i.e. the y-subunit does not bind EOF or vice versa (29). It is possible that this specificity indicates that these enzymes may play a role in processing precursors of the hormone (see below). 2.5S or {3-NGF The active subunit of the 7S complex can be prepared by several procedures that yield similar but unique products which result from limited proteolysis that occurs during the isolation (30, 31). The most commonly used preparation is that of Bocchini & Angeletti (32) in which the active subunit is isolated from partially purified 7S NGF. This material is denoted 2.5S NOF to distinguish it from the /3-NOF isolated from homogeneous 7S complex. The fundamental polypeptide unit contains 118 amino acids with amino terminal serine and carboxyl terminal arginine (20). The modifications that occur result from a foreshortening of the amino terminal portion by eight residues and the removal of the carboxyl terminal arginyl residue (20, 30). Thus, the maximally modified subunit contains only 109 amino acids. If polypeptides with and without the amino terminal octapeptide are designated A and B, respectively, and the superscripts R and T are used to denote carboxyl terminal arginine or threonine (the penultimate amino acid of the 118 residue subunit), then the ten dimeric combinations of the active subunit that are possible are: ARAR

ARAT

A TBR

A TBT BRBR BRBT BTBT

A TA T

ARBR

ARBT

Although preparations highly enriched in one or more forms have been obtained, it has not been possible to obtain a single dimer type in a homoge­ neous state. However, preparations containing all representations have been shown to be indistinguishable in terms of biologic and immunologic activ-

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ity, (12, 30, 31) which makes this heterogeneity of only secondary interest. However,the absence of the carboxyl terminal arginyl residues does prevent {.J-y interactions (30). Thus, {.J-NGF, treated with carboxypeptidase B to remove this residue, will not form 7S complexes, although it will still combine with the a-moiety (29). Some evidence has been obtained that partial Joss of this residue results in 6S complexes where only one y-subunit is combined with a f3-subunit that contains one AT/BT and one AR/BR unit (28); the interaction presumably occurs with the latter type. The amino terminal octapeptide has been synthesized by solid-phase techniques and has been shown to be devoid of NGF activity. It was also inactive in a number of pharmacologic assays (33). The physiologically significant form of the f3-subunit may in fact be the monomer. Young et al (34), have reported a binding constant, based on hydrodynamic measurements, of 107 M-t for the association of the two polypeptides of f3-NGF, which suggests that at the ng/ml concentrations required for optimal activity only monomers are present. Shooter and co-workers (13, 35), on the other hand, using entirely different methodol­ ogy, have reported values of 1010 to Wit M-i for the same constant. If correct, this value would imply that the dimeric form is the biologically active one. It has been shown that covalently cross-linked f3- or 2.5S NGF (36, 37) is fully active in the bioassay, while the results of Frazier et al (38), with an insolubilized NGF prepared in 6 M guanidine HC l, suggest that the monomer also can be active. Thus, the nature of the biologically active form of f3-NGF is presently unclear. The amino acid sequence of the constituent polypeptide chain of 2.5S NGF was determined to be (39, 40): NH2 -Ser-Ser-Thr-His-Pro-Val-Phe-His-Met-Gly-Glu-Phe-Ser-Val-Cys-Asp­ Ser-VaI-Ser-VaI-Trp-VaI-Gly-Asp-Lys-Thr-Thr-Ala-Thr-Asn-Ile-Lys-Gly­

Lys-Glu-Val-Thr-Val-Leu-Ala-Glu-Val-Asn-IIe-Asn-Asn-Ser -Val-Phe-Arg­ Gln-Tyr-Phe-Phe-Glu-Thr-Lys-Cys-Arg-Ala-Ser-Asn-Pro-Val-Glu-Ser-Gly­ Cys-Arg-Gly-Ile-Asp-Ser-Lys -His -Trp-Asn-Ser-Tyr -Cys -Thr-Thr-Thr-His­ Thr -Phe-Val-Lys-Ala -Leu -Thr-Thr-Asp-Glu-Lys-Gln-Ala -Ala-Tyr-Arg -Phe­

lIe-Arg- lIe-Asn-Thr-Ala-Cys-Val-Cys-Val-Leu-Ser-Arg- Lys-Ala-Thr -Arg­ COOH.

The six half-cystinyl residues are paired I-IV, II-V, and III-VI and link residues 15 and 80, 58 and 108, and 68 and 110 (40). The structure was independently determined by Mobley et al (41 ) for f3-NGF and was found to be identical to that reported by Angeletti & Bradshaw (20). C hemical modification experiments with NGF have revealed several in­ teresting features of the molecule but have failed to localize definitively the

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receptor binding site(s) or any residues that make up part of it. However, inactivation of the molecule has been achieved by conversion oftwo or three of the three tryptophan residues to the oxindole derivative by the action of N-bromosuccinimide (42, 43) and by complete modification of the arginine residues by 1,2-cyclohexanedione in borate buffers (44). Kinetic analyses of both reactions suggest that changes in conformation rather than conversion of specific receptor residues are the cause of the loss in biologic activity. In the case of the tryptophan modification, specific complexation of the modi­ fied hormone with dorsal root neuron receptors still occurs, albeit with a lowered affinity constant (44). Modification of lysine residues is apparently without effect on biologic activity or receptor interactions. NGF, treated with dimethylsuberimidate to effect covalent cross-linking between the polypeptide subunits of {3-NGF, was fully active biologically (36, 37). Although the number of cross-links introduced was undoubtedly considerably less, virtually all the lysine resi­ dues were at least mono-substituted. Acetylation, succinylation, and attach­ ment of NGF to C NBr-actived Sepharose beads all produced derivatives extensively or completely modified on the E-amino groups without loss of biologic activity (44). The two tyrosine residues ofNGF can also be modified without alteration in the biologic properties. C onversion of these residues to the 3-nitro­ derivative by the action of tetranitromethane (43) or iodination (45) yielded modified proteins that were fully competent in their interaction with recep­ tor molecules. Although some information is available concerning the three-dimen­ sional structure of NGF, particularly as it relates to the evolutionarily related family of insulins and proinsulins (see below), detailed knowledge is lacking. However, Wlodawer et al (46) have recently obtained crystals of 2.58 NGF suitable for single crystal x-ray diffraction analyses. The crystals are prepared by the hanging drop-vapor diffusion method, and have a hexagonal unit cell of P6}22 (or its enantiomorph P6s22). Appropriate heavy atom derivatives have been prepared and the determination of the complete three-dimensional structure is in progress (13).

Snake Venom Nerve growth f actor has been found to be present in the venom of the three families of poisonous snakes. Nine species of Crotalidae, six species of Viperidae, and five species of Elapidae have been examined to varying degrees, although homogeneous samples of NGF have only been obtained from one species of each family (1 1). Nonetheless, based on both structural and immunologic data, true homology exists between the various forms of snake NGF and the mouse {3-NGF (47, 48).

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The most detailed information is available for the NGF from cobra (Naja naja) venom. The homogeneous preparation of NGF, which was achieved by gel filtration, CM-cellulose column chromatography, and preparative disc gel electrophoresis, has a native molecular weight of 28,000, which was reduced by 6 M guanidine HCI to about 13,000 (49). Thus, it possesses a dimeric structure analagous to the mouse 2.5S or {3-NGF. No evidence of a higher molecular weight form was obtained. Partial sequence analysis indicated that the constituent polypeptide chains are very similar or identi­ cal and are composed of 1 16 amino acids. A total of 73 residues were directly identified of which 6 1 % are identical to the mouse protein. If the compositions of the undetermined regions were maximally optimized, 64% of the residues would be the same in both proteins. Although the disulfide bonds were not determined, the identical position of the half-cystinyl resi­ dues in both proteins suggests a similar pattern in the venom protein. Cobra NGF has some properties that are distinctly different from the

mouse protein. First, it is much less basic in character, possessing a pI of 6.75 as compared to 9. 1 for mouse 2.5S NGF (50). Second, it does not have a carboxyl-terminal arginine residue and does not interact to form a com­ plex with either the 0.- or y-subunit of the mouse 7S complex. Finally, although it shows the same dose response curve as the mouse protein, it is only about 50% as effective in the bioassay (using embryonic chick ganglia) and it will displace only 80% of the mouse 125I-NGF bound to such prepa­ rations. In marked contrast to the results obtained for cobra NGF, which estab­ lish a clear structural relationship between the snake and mouse proteins, Pearce et al (51) have reported the isolation of a viperid NGF (Vipera russelli) that differs significantly from both the mouse and cobra NGFs. In particular, this NGF preparation gave molecular weight values of about 35,000 and contained 20% (w/w) carbohydrate in the form of fucose, mannose, galactose, N-acetylglucosamine, and N-acetylneuraminic acid. No evidence was provided that the molecule contained more than one polypeptide chain. Interestingly, these workers (48, 52) have also reported that the NGF from the two crotalids, B. atrox and A. rhodostoma, and another viper, V. ammodytes ammodytes. are glycoproteins as well, al­ though in these cases the preparations were heterogeneous. It should be emphasized that it has been clearly established that the well characterized proteins from mouse and cobra are completely devoid of carbohydrate moieties. The NGF from a number of other crotalid, viperid, and elapid venoms has been isolated in partially purified form (11). In general, these forms of NGF appear to be of about 25,000 mol wt and show some degree of immunologic cross-reactivity with antisera raised against venom or mouse

200

BRADSHAW

NGFs. This includes one study (47) in which it was shown that the NGF of V. russel/i venom cross reacts with anticobra (N. naja) NGF. Thus, although the presence of carboydrate in some venom NGF preparations adds a confusing and somewhat perplexing feature to the understanding of venom NGFs in general, it seems clear that venom NGFs are homologous proteins to themselves and bear the same relationship with the NGFs of higher vertebrates.

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Other Tissues Nerve growth factor has been identified in a wide variety of normal and diseased tissues by both biologic and immunologic assay (1-3, 8). These include heart, kidney, thymus, diaphragm, uterus, vas deferens, spleen, liver, and granuloma. The adrenal medulla, in organ culture, produces NGF (53) but there is disagreement on the amount (3, 54). Young and co-workers (55-59) have reported that a variety of cells in culture, including L cells, 3T3 cells, SV40 3T3 cells, primary fibroblasts, neuroblastoma cells, melanoma cells, myoblasts, glioma cells, glioblastoma cells, and primary synovial fibroblasts all produce NGF. Of particular interest is the L-cell NGF (60). Molecular weight determinations indicate a value for this form of NGF of 1 60,000. However, unlike the 7S complex of mouse submaxillary gland, it is not dissociated in dilute solution. It does, however, contain a subunit similar in size and electrophoretic behavior to the polypeptide chain unit of {1-NGF. The same form appears to be present both intracellularly and as an exported protein in the medium. These results clearly suggest an association of the fJ-NGF of these cells with a different protein(s) than the a- and y-subunit of the submaxillary gland. However, the significance of these observations is obscured by the fact that the protein was analyzed only in crude extracts or unfractionated media. It will be necessary to obtain homogeneous samples of this form of NGF before its relationship to sub­ maxillary gland NGF and its physiologic significance can be assessed. EVOLUTION

As has come to be appreciated for most proteins, nerve growth factor is a member of a larger family that owes its structural relatedness to evolution from a common precursor. It is interesting that, in this case, these relation­ ships have also been instrumental in developing and defining the family itself. The first step in the development of the concept that growth factors constitute a protein family was the observation that NGF and proinsulin share common regions of sequence relatedness (61 ). When aligned at the

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amino termini, the regions of the proinsulin molecule that yield the B and A chains of insulin, following proteolytic activation, show 30% and 52% identities, respectively, with the corresponding regions of NGF. Further­ more, these regions are appropriately spaced to accommodate the connect­ ing C-bridge region of proinsulin even though no significant similarity is found there. The alignment is further strengthened by the fact that the disulfide bond, NGF I-IV, corresponding to the B -19-A-20 pair in insulin, is also conserved, which adds a potential element of three-dimensional similarity as well. The greater length of the NGF chain (118 amino acids) as compared to that of the longest proinsulin (86 amino acids) suggests a possible additional step in the evolutionary development of NGF following the divergence created by the original gene duplication. A contiguous reduplication, which produced an internal repeat of the B region following the A chain-like region in NGF, would explain the longer length of the NGF molecule. It may also have generated repeated C and A regions as well, which were then subsequently lost by further genetic events. Alternatively, this region may be still expressed in the present structural gene product but be lost by proteolysis, perhaps by the catalytic action of the y-subunit at some post­ ribosomal stage. As discussed below, the latter possibility seems most likely. Evaluation of the three-dimensional structure ofNGF by physical chemi­ cal measurements and solution topographical mapping indicates that the two molecules could have similar conformations, at least in the structurally conserved regions. Generally, both molecules contain low proportions of a-helical structures (62,63). More specific measurements (43) indicate that the two tyrosine residues of NGF and tryptophan residues 21 and 76 are in similar environments, with respect to solvent availability, as the corre­ sponding residues in insulin as deduced from the three-di�ensional struc­ ture determined by X-ray analysis (64). In contrast, Argos (65), using three-dimensional prediction methodology, has concluded that the regions of NGF most likely to be in a-helical conformations do not coincide well with the known insulin structure, which suggests that the three-dimensional structure of NGF will not, in fact, resemble that of the insulins. Resolution of this problem must await results from the present crystal studies on NGF. Recent elucidation of the primary structure of porcine relaxin (66) and the amino terminal sequence of human NSILA (67) has established the structural relatedness of these two factors to insulin as well. Relaxin is a hormone-like substance synthesized by the granulosa cells of corpora lutea which acts on tissues of the pubic symphisis to aid in the delivery of the foetus. It may play a more general role in tissue remodeling in the nonpreg­ nant state. As isolated relaxin is a two chain structure joined by disulfide

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linkages, although preliminary evidence suggests the existence of a single chain prorelaxin molecule (68). NSILA has been recognized for some time as a serum factor with biologic properties similar to those of insulin. How­ ever, it is not blocked, or suppressed, by insulin antibodies. The soluble form exists as two closely related "isohormones," designated NSILA I and II (69). It contains only a single polypeptide chain of some 75 amino acids. As shown in Figure 1, the B chain of insulin and relaxin align with the amino termini of NSILA and NGF indicating the homologous nature of the four molecules. Of particular interest is the conserved cystine residue (posi­ tion B20 in insulin) found in all four molecules, and the close similarities in the region immediately following it. This region can be subdivided into two categories where insulin and NSILA show marked similarities but differ from NGF and relaxin, which share the Ser-Val-Ser-X-Trp sequence. The importance of this region as a putative portion of the receptor binding site of insulin (70) adds interest to this conservation. Similar relatedness is also found in the A chain of insulin and relaxin and the corresponding region of NGF. The NSILA sequence is not complete in this region at present. It should also be noted that several other growth factors, such as the somatomedins, A, B, and C, multiplication stimulating factor, and fib ro­ blast growth factor may also occupy positions in this family (71), although SER

NGF

RELAXIN

B BTHR

PRO--

-VAL-PHE

B

B

MET-CLy-

peA _ S3R - THR -ASN -ASP -PHE -ILE - LYS -ALA

PHE

INSULIN

-

VAL-ASN-GLN

NSILA II

ALA � TYR �ARG - PRO

NGF

PUE -SER-

RELAXIN

uu

INSULIN

LEU

TYR - LEU-

NSILA II

LEU

GLN



B

ARG-

GLU

LEU-CYS-GLY

GLU - Tlm--JLEU�CYS-GLY

- PHE -

----'

ALA-

ASP -TIIR-

L-___

SER-VAL-SER

VAL

SER-VAl! -SEn.

T HR

A �

VAL-GLY-ASP



GL Y - A RG

PUE-TYR -THR-PRO

'-------'

+

LYS -THR

I

TYR - PRE-SER -ARG-PRO­

Figure 1 Sequence comparison of the amino terminal portions of mouse nerve growth factor (NGF) (20) and human nonsuppressible insulin-like activity (NSILA II) (67) with the B chains of porcine relaxin (66) and human insulin (82). Residues identical in two or more of the proteins are enclosed in boxes. Dashed spaces indicate deletions arbitrarily introduced to increase the similarity. The half cystine marked with an asterisk is bonded to the same half-cystine residue, located elsewhere

in the molecule, in all four proteins.

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at present insufficient structural data is available to substantiate this. None­ theless, there exists the marked possibility that these factors share a com­ mon mechanism of action, as reflected in their ancestral development, on their respective target cells.

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BIOSYNTHESIS

One of the more obscure features of the physiology of NGF is the exact in vivo site(s) of synthesis and the subsequent method of delivery to target tissues. As might be expected, studies to establish the tissue of origin have focused on the submaxillary gland of adult male mice, the richest known source of the hormone. In new born mice of either sex, the levels of hormone are low and remain so until puberty. At that time the levels in male submax­ illary glands rise markedly. Such a rise can be induced in females by testost­ erone injections, or prevented in the male by castration (2, 72, 73). Furthermore, it has now been reasonably established that these changes are due to newly synthesized protein in situ and are not the result of increased synthesis elsewhere in the organism followed by specific uptake by the submaxillary gland (74-76). However, no other species, of either sex, con­ tains the high level of hormone found in the male mouse nor can it be induced in them by sex hormones or by other means, neoplastic situations notwithstanding. Furthermore, there is compelling evidence to suggest that this storehouse of NGF is not utilized by the mouse, at least not to regulate the neuronal processes presently attributed to the factor. Does this mean that all mouse submaxillary gland NGF is biologically irrelevant to the mouse? The answer is probably no, although this question has not been definitively resolved. The low concentrations of hormone in the prepubes­ cent male and the female most likely represent the "normal" concentration in that tissue. These levels are comparable to those found in other tissues that receive sympathetic innervation. Thus the submaxillary gland, as part of the network of sympathetic end organs, may be thought of as a physio­ logically relevant site of synthesis, because it is probably this group of organs as a whole that provides NGF to the target tissue, that is, the sympathetic nervous system. Germane to this question are the experiments of Hendry & Iversen (77) on the effects of removal of the submaxillary gland on the circulating levels of the hormone. These authors observed that sialoadenectomy resulted in a drop in the systemic levels of the hormone, as measured by a two-site radioimmunoassay, and a subsequent return to normal levels nine weeks after the operation, without any regeneration of the excised tissue. In a similar experiment, Murphy et al (24) observed that the circulating levels

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of the hormone were unaffected by sialoadenectomy and concluded that the levels of systemic NGF are not controlled by the submandibular gland. Their results also differed from those of Hendry & Iverson (77) in that differences in the serum levels of male and female mice were not observed. As a result of these experiments, Murphy et al (24) suggest an exocrine role for the mouse submandibular gland with regard to NGF, which is sup­ ported by their observations that the saliva of both male and female mice contain extraordinarily high concentrations of the hormone. Wallace & Partlow (78) have reported a similar finding, but only after preferential stimulation of the gland by a-adrenergic agents. Whether salivary NGF simply represents disposed material or whether it has a direct physiologic role in the digestive process is not presently appreciated. The relationships of submaxillary and blood NGF in the mouse is further confused by the lack of agreement on the normal levels of serum NGF. Of particular interest in this regard is the observation of Ishii & Shooter (79) that injected 1251-NGF is cleared from mouse serum in vivo with a half-time of 10-30 min. Although it cannot be ruled out that this value is affected by the prior iodination of the hormone, it seems reasonable to conclude, based on the fact that this modification does not affect any other biologic or immunologic behavior, that NGF displays a rather short lifetime in serum. This raises the question whether there is any appreciable quantity of cir­ culating NGF in the mouse, or any other organism. Attempts to isolate the hormone from the blood have been singularly unproductive and recent, improved analyses (H. Thoenen, personal communication) have found that normal circulating levels of the hormone are immeasurably low. The ab­ sence of serum NGF would be consistent with the proposal that NGF is a "diffusion" hormone that is not transported by the blood system. As such, submaxillary NGF, at the prepubescent or female level, would affect only sympathetic neurons innervating that organ, and its excision in the adult animal would not materially affect other elements of the peripheral nervous system, a conclusion which has already been substantiated (6). The much higher levels in the male would not alter this primary role. By virtue of the amounts produced, the submaxillary gland is the primary producer of NGF in adult male mouse. Since it seems reasonable to assume that this increased activity is basically an amplification of the normal pro­ cess, studies on the biosynthesis of NGF in that tissue should have general applications. Levi-Montalcini and Angeletti (75), using slices of adult male mouse submaxillary gland, demonstrated the incorporation of labeled amino acids into material that was immunologically precipitable with anti­ NGF. They also showed a differential incorporation of labeled amino acids into the immunoprecipitate in one of the two lobes of the gland in vivo.

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Berger & Shooter (80), using [35S]-L-cystine, have demonstrated the incor­ poration of this amino acid by submaxillary glands in vitro into material that is specifically precipitated by anti-NGF. This material was shown to have a tryptic peptide profile, with respect to the migration of half-cystine containing peptides, indistinguishable from that of native NGF. Similar results were obtained in vivo. When the labeled products were examined as a function of time (10-240 min) by SDS-gel electrophoresis, Berger & Shooter (81) observed that a 22,000 mol wt precursor appeared first, followed by the production of the 13,000 mol wt polypeptide chain of {3-NGF. The amount of 22,000 mol wt material reached a maximum at I hr and remained constant, while the amount of ,B-NGF (as 13,000 mol wt units) rose rapidly. After four hours, it was present in a tenfold greater amount and after six hours the larger material had essentially disappeared. The 22,000 mol wt precursor was shown to contain all of the half-cystine peptides of {3-NGF and it could be converted to the 13,000 mol wt unit by the action of either NGF y-subunit or the EGF binding protein. These results clearly demonstrate the biosynthesis of a proNGF species containing some 80 additional amino acids (as judged by molecular weight differences). Although it remains to be established whether these residues are removed from the amino or carboxyl terminal or both, it is interesting that the comparison of NGF and proinsulin (see above) resulted in the prediction of an additional carboxyl-terminal segment that would be sus­ ceptible to the catalytic action of the y-subunit (61). It has not been estab­ lished whether proNGF is biologically competent or if limited proteolysis is a prerequisite for the production of active hormone. It is also unknown whether the structural gene for NGF contains a region coding for a prepro­ hormone segment. In related systems, the removal of this peptide from the amino terminal of the nascent chain ensures the proper "packaging" of the hormone for ultimate export from the cell of origin (82). BIOLOGIC PROPERTIES

The physiologic effects of NGF have been extensively reviewed (2, 6, 7, 9) and are summarized here only as a prerequisite for mechanistic consider­ ations. The initial appreciation of the striking morphologic effects of NGF was actually the basis for the discovery and subsequent isolation of the hormone. In experiments involving the transplantation of sarcomas 37 and 180 into mice, it was observed that the sympathetic nervous system of the host was stimulated to innervate the tumor (83-86). The transfer of this growth-

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stimulating ability to an in vitro culture using check embryonic ganglia provided the basis for the bioassay still used today (86). This assay also demonstrated that two types of peripheral neurons will respond, i.e. extend neurites, in culture: the adrenergic neurons of sympathetic ganglia and the mediodorsal sensory neurons of dorsal root ganglia (6). In fact, NGF is absolutely required to maintain both cell types in vitro. In vivo, it is unsure whether or not these sensory neurons have a direct dependence on the factor. However, the requirement for NGF of sympathetic neurons is clearly maintained throughout the lifetime of the animal. Perhaps the most compelling demonstration of this fact was the experiment by Levi-Montal­ cini & Booker (87) which showed that injection of anti- NGF into neonatal animals causes the destruction of the sympathetic nervous system. This procedure, commonly used as a pharmacologic model (88), is termed im­ munosympathectomy. Positive effects can also be demonstrated by the injection of NGF into similar animals, most notably in increased levels of tyrosine hydroxylase and dopamine-,B -hydroxylase, enzymes involved in the biosynthesis of catecholamines (89). Morphologic changes, such as the increase in cell volume and innervation of sympathetic end organs, also occur (6). The permanancy of these effects is, however, questionable. The stimulation of embryonic neurons to produce neurites and the re­ quirement of adult neurons for maintenance of viability can be translated into defined effects on the metabolic processes of these cells. In simplest terms, NGF may be viewed as a trophic agent that acts as a pleiotypic activator of various anabolic and catabolic pathways (90). Among other effects, NGF causes: increases in uridine uptake and polysome formation; protein, R NA, and lipid synthesis; and glucose utilization (6, 91, 92). It also causes microtubule (neurotubule) polymerization and in a limited way, can act as a mitogenic factor (6). As already noted, some of these effects can be translated into very specific events such as the stimulation of the two enzymes of catecholamine biosynthesis. NGF can also act as a tropic agent to attract growing axons to their target tissues. Although it is often difficult to separate such effects from trophic actions, evidence from experiments involving transplantation of iris into the brain (93) and the unnatural position of sympathetic nerve fibers after intracerebral injection of NGF (9, 94) clearly support this facet of NGF action. A variety of effects of NGF on cells not normally classed as responsive to NGF have been reported. In addition to effects on noradrenergic fibers in brain (95-97), it also inhibits the biosynthesis of mucopolysaccharide in chondrocytes (98) and stimulates the temporal conversion of cell surface adhesive specificity in embryonic optic tectal cells (99). It has also been

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reported that NGF directly affects an adenyl cyclase in the chromaffin granules of adrenal medulla (100). Three types of neoplastic tissues of neural crest origin, neuroblastoma (10 1), melanoma (102), and pheo­ chromocytoma (103) have,in specific lines,been reported to bind 125I- NGF. In the last case, neurite proliferation, induced by the hormone, was also observed. Finally, specific receptors for NGF have been observed in brain and a number of sympathetic end organs (104, 105). In brain, the receptors are apparently largely associated with synaptosomal elements (106, 107). The significance of any of these observed activities to the presence of specific plasma membrane receptors in unresponsive tissues cannot be read­ ily explained. These situations may result from artifacts induced by in vitro conditions, may represent analog activities of other closely related but as yet unidentified growth factors, or may reflect true physiologic activities of the hormone not yet appreciated. MECHANISM OF ACTION

As with other polypeptide hormones,much more is known of the molecular and biologic properties of NGF than is known of its mechanism of action. Nonetheless, several aspects have been clarified recently that offer distinct clues as to the nature of the overall process. In general, they suggest an initial interaction with a plasma membrane receptor followed by internali­ zation and complexation with a second type of receptor, which is located in the nucleus. Since direct effects on cellular metabolism occur as the result of hormone binding to both recognitive entities, the mechanism of NGF can be viewed as biphasic with information transfer occurring at two levels. The salient evidence in support of the three main steps and a plausible model to accommodate them are summarized below.

Plasma Membrane Receptors The first direct identification of cell surface receptors for NGF on respon­ sive neurons was made by Frazier et al (38). In these experiments,NGF was attached to Sepharose beads that had been activated with BrC N. The coupling reaction was carried out in 6M guanidine-HC l which effectively prevented subsequent release of soluble NGF from the resin, presumably because of the formation of multiple attachment sites between the dena­ tured protein and the carbohydrate matrix. The NGF-Sepharose conjugates were found to be active as judged by their ability to elicit neurite prolifera­ tion. Thus,it was concluded that it was not necessary for NGF to enter cells to mediate this phase of its biologic activity, although these experiments did

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not rule out that NGF could be internalized. It was also noted that the possibility of controlled cleavage of the protein (and/or matrix) after com­ plexation of the conjugate with receptor could not be eliminated. If such material were directly taken into the cell it would not be detected in the diffusion controls. This limitation is germane in view of the subsequent demonstration that NGF can be internalized ( lOS, 109), vide infra, and a recent report claiming that a fragment ofNGF, generated by limited tryptic digestion, possesses full biologic activity (110). The characteristics of the plasma membrane receptor for NGF have been investigated using 125I-Iabeled hormone (45, 111, 112). In all studies, high affinity specific binding, defined as labeled NGF that can be displaced by an excess of unlabeled hormone, was observed. In one study (112), low affinity binding ("'1061/mol as opposed to "'10lO/mol) was observed at higher concentrations of added !25I- NGF. The curvilinear Scatchard analy­ sis of this binding data was similar to that observed for the binding of 125I-insulin to cultured lymphocytes (113). The high affinity asymptote corresponded well to the concentration range required to maximally pro­ duce neurites. Kinetic analyses of the association and dissocation of the hormone and receptor gave constants of 7.5 X 106 M-1 sec-I and 3.S X 104 sec-J, at 24° C , respectively (112). Most interesting was the observation that an excess of native NGF greatly accelerated the dissociation process. This deviation from the law of mass action had been previously observed by De Meyts et at (113) for the dissociation of labeled insulin from lymphocytes and was attributed by these workers to negatively cooperative interactions between receptor molecules. Thus, the similarity between insulin and NGF appears to extend to molecular features of their receptors as well. Similar observa­ tions have now been made for a number of hormone receptor systems (114), which suggests negative cooperativity as a common, although not universal, feature of cell-hormone interactions. The potential advantage of this property in regulating hormonal response have been discussed else­ where (10, 115). The plasma membrane receptor for NGF has been solubilized by non­ ionic detergents from both sensory (116) and sympathetic (117) ganglia. The solubilized preparations can be assayed for specific binding using gel filtration or precipitation by polyethylene glycol to separate unbound from complexed hormone. The affinity constant of the solubilized receptor from rabbit superior cervical ganglia was found to be 5 X 109liter/mol, in excel­ lent agreement with the value for the high affinity binding in the membrane bound state, which indicates that the solubilization process does not appear to materially affect the receptor molecule (117).

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Internalization (Retrograde Axonal Transport)

The most extensive documentation ofthe internalization of NGF in respon­ sive neurons is found in the experiments of Hendry, Thoenen, and their co- workers ( 108, 1 1 8- 1 23). By injection of microgram quantities into the anterior chamber of the eye, they demonstrated that small amounts of hormone were specifically taken up by the sympathetic terminals innervat­ ing this organ and transported intraaxonally to the perikaryia located in the ipsilateral superior cervical ganglia. The contralateral ganglia, which serves as an internal control, contained significantly less radioactivity. In more recent experiments utilizing labeled NGF of much higher specific activity ( 1 1 2), this phenomenon has been confirmed using only nanogram quantities with virtually no radioactivity reaching the contralateral ganglia (E. M. Johnson, R. Y. Andres, and R. A. Bradshaw, unpublished experiments). In these latter experiments, maximal accumulation was seen at 1 2 hr in ham­ sters and 1 6 hr in rats with the uptake system half-saturable at 1 5 ng of injected hormone. The uptake process was abolished by transection of the postganglionic fibers and by colchicine injections, 12 hr prior to the administration of the labeled hormone. The process was also shown to be specific for NGF. C ytochrome c, insulin, horseradish peroxidase, ovalbumin, bovine serum albumin, and ferritin were taken up to an extremely small extent ( 1 1 8, 1 1 9). Furthermore, inactivation of NGF by oxidation of two or more tryptophan residues, which destroys biologic activity (43), also prevented the uptake process which suggests that only the active species can be properly recog­ nized by the receptors responsible for the specific uptake. This was con­ firmed by the demonstration that excess amounts of unlabeled NGF effectively block the uptake of iodinated hormone. Although the nature of the uptake process at the presynaptic membrane is unclear, it may be presumed that a pinocytoti c mechanism, which results i n the vesicularization of the internalized hormone, probably occurs. This is supported by electron microscopic autoradiography of superior cervical ganglia after retrograde transport of 125I-NGF. The transported material is localized in vesicles attached to smooth endoplasmic reticulum and in secondary lysosomes (1 23). In addition, as judged by radioactive analysis of subcellular fractions of similarly treated superior cervical ganglia, 1 5-30 percent of the NGF is found in nuclear fractions (E. M. Johnson, R . Y. Andres, and R . A: Bradshaw, unpublished experiments). Of primary importance in the studies on retrogradily transported NGF was the demonstration that hormone specifically transported in superior cervical neurons causes specific increase in the levels of tyrosine hydroxy-

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lase ( 120). The induction of this enzyme, which occurs after administration of a few micrograms of NGF to intact sympathetic neurons (89), can be demonstrated to occur with internalization of only picograms of the hor­ mone. Stoeckel et al (124) have also demonstrated that dorsal root neurons will transport NGF injected into the forepaw of adult rats, in 10 days. There were, however, significant differences between this process and that in the sympathetic neurons, which may be explained by the fact that these neurons appear to lose their responsiveness to NGF postembryonically ( 125). Norr & Varon ( 126) have also reported a time-linear uptake (pinocytosis) ofNGF by chick embryonic dorsal root neurons. The clear demonstration of the uptake and retrograde transport of NGF by mature neurons still leaves the question of whether these processes occur during embryonic development, before axonal extension and synapse for­ mation is complete. The possibility remains that this internalization occurs only as a feature of mature neurons which have a special need to communi­ cate between remote termini and their cell bodies. Nuclear Receptors

Although Triton X-loo will readily solubilize the p lasma membrane recep­ tors of embryonic dorsal root neurons, a significant amount of specific NGF binding is still found in the insoluble pellet ( 1 1 6). Following subcellular fractionation, this binding was localized in the nuclear fraction. Microsomal contamination was eliminated by reconstitution experiments, and purified chromatin, prepared from these nuclei, showed identical binding properties ( 1 16). In contrast to the plasma membrane receptors, the nuclear receptors showed only saturable high affinity binding with an association constant of approximately 5 X 109 liter/mol (1 1 6). Binding to these receptors is not inhibited by lOoo -fold molar excesses of insulin, cytochrome c, and lyso­ zyme. The highest levels are found in nuclei derived from sympathetic and sensory ganglia, with only relatively low amounts in avian erythrocytes or optic tectum cells. Interestingly, nuclear receptors for insulin have been reported in lymphocytes and liver cells with properties essentially identical to those found for NGF ( 1 27, 1 28). A Mechanistic Model

Any model for the action ofNGF must take into account its known biologic properties. Restricting these considerations to cells generally classified as responsive, that is sympathetic and embryonic sensory neurons, these in­ clude the uptake of metabolites, stimulation of anabolic metabolism, the polymerization of microtubules, the specific induction of enzyme synthesis,

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the proliferation of neurites, and the long-term maintenance of cell viability both in vitro and in vivo. These effects do not all occur on the same time scale. Metabolite uptake is relatively rapid and occurs in the first few hours of incubation, whereas neurite proliferation cannot be observed until 1 2 hr and does not reach maturity until 36 to 48 hr. Long term maintenance, by definition, must be judged over several days. It is interesting to note that this somewhat extended period of response is also typical of other growth factors, including insulin, and is markedly distinct from the response that is approximately proportional to receptor occupancy. It is in fact possible to subdivide hormonal agents into two general classes based on these char­ acteristics (129). One class elicits essentially instantaneous responses, often through the generation of cyclic nucleotide second messengers, is rapidly degraded at the plasma membrane, and the duration of the effect is approxi­ mately proportional to the actual occupancy of the receptor. The second class, which have been termed maintenance or permissive hormones, show extended periods of response that last far longer than actual receptor occu­ pancy, are degraded slowly, usually by lysosomal action, and ultimately effect protein biosynthesis. It seems clear that the primary hormones act externally exclusively through complexation with plasma membrane recep­ tors and their associated effectors. On the other hand, growing evidence suggests that the second class, of which NGF is representative, act through a combination of events, initiated by plasma membrane receptor complexa­ tion and terminated inside the cell by combination with nuclear receptors ( 10). This mechanism (130) can be described by the following steps: 1 . Complexation of the hormone with plasma membrane receptors, which results in metabolite uptake and the stimulation of anabolic and cata­ bolic pathways. Subsequent events include the polymerization of tubulin to microtubules which initiates the second step. 2. Vesicularization of the hormone-receptor complex, perhaps engineered by microtubules, with the resultant internalization of the hormone. 3. Transport of the vesicle to and fusion with internal membrane struc­ tures. As a secondary phase of this process, vesicles can also be directed to lysosomes, in which case degradation of the hormone ensues. 4. Transfer of the hormone to the nucleus, followed by complexation with nuclear receptors to directly effect ongoing transcriptional events. This mechanism finds support in many experimental observations on NGF. The presence of plasma membrane and nuclear receptors, and the capacity of neurons to internalize NGF have already been described. This mechanism also accounts for the established fact that neurite proliferation can occur without RNA synthesis but cell maintenance cannot (6). The polymerization of microtubules is probably sufficient to initiate fiber out-

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growth, a process that is extensively initiated before occupation of the nuclear receptors occurs. In this regard, the observation that NGF can directly affect microtubule polymerization may indicate another role for the internalized hormone (131, 1 32). This model also predicts that a loss of plasma membrane receptors will occur during the transfer process. Al­ though "down regulation," the decrease of receptors due to chronic expo­ sure of cells to hormone has not been demonstrated for NGF, it has been clearly shown for insulin (133) and epidermal growth factor (134). It is also noteworthy that this mechanism is closely similar to that of steroid hor­ mones (1 35). Although these substances do not have plasma membrane receptors they do have cytoplasmic binding proteins that serve a similar function. The subsequent binding of the steroid-receptor complex to a chromatin-bound receptor would be entirely analogous to the binding of "maintenance" hormones to their nuclear receptors. It should be noted that although this model accounts for the many facets of NGF activity, detailed information of many of the proposed steps is lacking. As with other similar hormones, such as insulin, it is unclear how the complexation with the plasma membrane receptor causes the uptake of metabolites and the resul­ tant microtubule polymerization. It is equally unclear how the vesicle trans­ port and transfer of the hormone to the nucleus is accomplished. Finally, the manner in which the formation of the nuclear receptor complex allows changes in RNA transcription is unknown. In additional to these problems, many other questions remain to be answered before this mechanism can be integrated into a description of NGF action in the organism as a whole. Definitive localization of the sites of NGF synthesis must be made and the factors that regulate that synthesis determined. It will also be necessary to determine the mode of transport between the sites and the target neurons. However, the model also provides some insight into these questions. Specifically, it suggests that tissues receiv­ ing sympathetic innervation synthesize and export the hormone, which reaches the embryonic neuron by a diffusion process. Uptake occurs at the plasma membrane of the growing neuron and as neurites are extended toward this source, the uptake continues but with an additional internal transport phase. The terminal stage occurs when the growing axon has reached the end organ where the diffusion process is limited to a trans­ synaptic passage. In this way, only a single mechanism of action is required that accommodates the dramatic morphologic changes that occur in the target tissue during development. This mechanism also provides these neu­ rons with a direct means for communicating between synapse and cell body, i.e. NGF becomes the "chromalytic messenger" for cells responsive to it. Although much experimental verification is required, this model provides an excellent working hypothesis for further research into the nature ofNGF and other growth factors.

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ACKNOWLEDGMENTS

The portions of this review dealing with studies i n the author's laboratory

were supported by US Public Health Service research grant NS 10229. During the preparation of this article the aut hor was a Josiah Macy Jr. Foundation Faculty Scholar at the Howard Florey Institute of Physiology

and Experimental Medicine, University of Melbourne.

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