- Email: [email protected]
Neurotrophic factors and their receptors
Neurotrophic
factors and their receptors Mariano Barbacid
Bristol-Myers Several
Squibb Pharmaceutical
new members
which comprises
Research Institute, Princeton,
of the nerve growth
factor family
nerve growth factor itself, brain-derived
and neurotrophins-3,
-4 (also known
isolated in recent years. Their
signaling
Trk family of tyrosine
kinases, thus facilitating
signaling
pathways
protein
responsible
of neurotrophins, neurotrophic
as neurotrophin5)
factor
and -6, have been
receptors have been identified
for mediating
USA
the dissection
their trophic
properties.
as the of the More
recently, the advent of gene targeting has made it possible to generate strains of mice lacking neurotrophins
and their receptors. Analysis of the phenotypes
of these mutant animals
has provided
neurotrophins
receptors
and their
detailed information
play in the ontogeny
on the role that
of the mammalian
nervous system. Current
Opinion
in Cell Biology
Introduction Growth factors play a critical role in the development and maintenance of the mammalian nervous system. Some of these factors (e.g. platelet-derived growth factor and fibroblast growth factor) have pleiotropic activities and are often studied in systems other than the nervous system [l-3]. Other factors have a more restricted activity on neuronal cells and are classified as ‘neurotrophic factors’. Among them, the most intensively studied include the ciliary neurotrophic factor (CNTF) [4], the recently discovered glial-derived neurotrophic factor (GDNF) [5*] and the members of the nerve growth factor (NGF) family, now known as ‘neurotrophins’ [6,7]. Each of these neurotrophic factors exerts its biological activities through a different class of receptors. Neurotrophins signal through the Trk family of tyrosine protein kinases [8-lo]. In addition, neurotrophins interact with a second receptor, ~75, a member of the tumor necrosis factor receptor superfamily [ll]. CNTF signals though a multimeric receptor complex which includes binding and signaling elements [12,13]. The binding subunit is specific for CNTF and does not have transmembrane or cytoplasmic domains, but instead anchors to the plama membrane by a glycosyl phosphatidylinositol linkage [14]. The signaling subunits include gp130, a transmembrane glycoprotein previously found in various cytokine receptors [15], and the p-subunit of the receptor for leukemia inhibitory factor [16]. The cytoplasmic domains of both of these molecules interact with members of the Jak family of cytoplasmic tyrosine kinases, which are likely to be the primary
1995,
7:148-l
55
mediators of CNTF activity [12,13]. CNTF shares these signaling subunits with a variety of cytokine receptors, including the receptors for leukemia inhibitory factor, interleukin-6, oncostatin M and, possibly, interleukin-1 1 [12,13]. How these growth factors can elicit such different biological responses by activating the same signaling receptor subunits is a subject of intense study at this time [12,13]. Finally, the receptor for GDNF has not as yet been identified; however, the limited structural similarities between GDNF and the transforming growth factor-p superfamily [5*] raise the possibility that GDNF may signal through multimeric serine/threonine kinase receptors. During the past year, several important developments have occured in the field of neurotrophins and their receptors. The generation of strains of mice carrying targeted mutations in the genes encoding several neurotrophins, as well as in those encoding the ~75 and the Trk kinase receptors, has provided a wealth of information regarding the role that these molecules play in the ontogeny of the mammalian nervous system [17]. In consideration of the space limitations, this review will focus on neurotrophins and their receptors. Other exciting developments regarding CNTF and its signal transduction pathways have been recently summarized in several reviews [4,12,13] and will not be discussed here. GDNF, initially thought to be specific for dopaminergic neurons [5-l, appears to have a broader specificity, as it can also support the survival of motorneurons [18]. Further studies are needed to define the full range of neurotrophic activities of this novel growth factor.
Abbreviations BDNF-brain-derived neurotrophic factor; CNTF-ciliary neurotrophic factor; EGFGpidermal growth factor; GDNF-glial-derived neurotrophic factor; MAP-mitogen-activated protein; NCF-nerve growth factor; NT-neurotrophin; PLC-phospholipase C. 148
0 Current
Biology
Ltd ISSN
0955-0674
Neurotrouhic factors and their receutors Barbacid
The NCF family of neurotrophins This family of growth factors includes NGF, brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3 and NT-4 (also known as NT-4/5 and NT-5) [2]. A molecule recently isolated from the teleost fish Xiphophorus, NT-6, probably represents a novel member of this f&nily [19*]. These neurotrophins bind specifically to the three known Trk tyrosine kinase receptors, T&A, TrkB and TrkC [8-lo]. Whereas NGF binds to T&A, BDNF and NT-4 interact with TrkB (Fig. 1). NT-3 appears to be more promiscuous and binds to each of the Trk receptors, but its primary biological responses are mediated by TrkC (see below). In addition, each of these neurotrophins binds with low affinity to ~75, a receptor structurally unrelated to the Trks and whose precise role in mediating neurotrophin activity remains to be elucidated (see below) [ll] (Fig. 1). Whether NT-6 binds to any of these receptors is not known at the present time. NGF recruitment ?
Qo NGF
TrKA
BDNF
NT-4
TrkB
NT-3
TrKC
Signal transduction 0 1995 Current Opinion in Cell Biology
Fig. 1. Functional interactions between neurotrophins and their cognate receptors. Each of the neurotrophins binds with similarly low affinity (nanomolar range) to ~75. The neuronal defects observed in p75 null mice, however, suggest a functional interaction of this receptor with NGF (solid arrow) but not with BDNF or NT-3 (dashed arrows). The interactions of NCF with TrkA and of BNDF and NT-4 with TrkB are highly specific. NT-3 primarily activates TrkC receptors; however, this neurotrophin can also interact with the TrkA and TrkB receptors (see text).
The three-dimensional structure of NGF has been resolved by X-ray crystallography (at 2.3A resolution; [20]) and shown to consist of two identical subunits. Based on previous sequence analysis, each subunit has 118 amino acids residues and is generated by cleavage of a precursor polypeptide of 252 residues [1,7]. The NGF protomers are elongated and contain three twisted anti-parallel p-sheet segments linked by turns. These protomers are linked to each other by a ‘cysteine knot’, a novel structure also found in transforming growth factor-b and platelet-derived growth factor B-subunit homodimers, and display a twofold symmetry with parallel long axes [20]. The structure of the other neurotrophins remains to be elucidated; however, their close similarities to each other (47 residues are identical in all four mammalian neurotrophins) suggest that they are likely to have similar tertiary structures. Site-directed mutagenesis studies have provided important information regarding those residues critical for the
interaction of neurotrophins with the p75 and TrkA receptors. The interaction of NGF with p75 is mediated by a positively charged region that consists of at least three lysine residues located in two spatially adjacent loops (I and V) [21]. These positively charged residues are conserved in NT-3 and NT-4, but not in BDNF, which contains three consecutive positively charged residues in loop V that may compensate for the absence of the two lysines present in loop I in the other neurotrophins [22]. These differences may explain the somewhat distinct dissociation kinetics shown by BDNE The interaction of NGF with TrkA is more complex and requires cooperativity of multiple contact sites derived horn at least five different domains within the NGF molecule [21,22]. These sites are concentrated on one side of the NGF dimer with both protomers contributing some of the critical residues. Overall, the TrkA binding site appears as a flat, continuous surface approximately parallel to the twofold axis of the molecule. Because of its twofold symmetry, the active NGF dimers have two potential T&A binding sites which may facilitate the formation of T&A receptor homodimers, a step required for receptor activation [23]. Historically, the biological activity of the neurotrophins has been studied using explants of primary neurons or neuronal progenitors in culture, as well as certain immortal cell lines such as PC12 and MAH cells [6,24]. In the case of NGF, pioneering in viva studies using neutralizing antibodies (either passively administered into the placenta or generated by immunization of pregnant rats with NGF) provided the first conclusive evidence for a role of neurotrophins during the development of the mammalian nervous system [6]. These studies have been described in detailed elsewhere [6,24] and will not be discussed here. In this review, I will focus on recent results obtained with ‘knockout’ mice devoid of either NGF, BDNF or NT-3 (see below).
Trk receptors The structural features of the Trk family of receptors have been reviewed recently [25]. The unique combination of structural motifs, particularly in their extracellular, ligand-binding domains, indicate that they constitute a novel receptor subfamily (Fig. 2). Their kinase catalytic domain is most closely related to a series of novel orphan receptors that include the mammalian RORl, ROR2, DDR (also known as Ptk-3, Nep or T&E), Tyro-10 and TKT as well as the Torpedo RTK tyrosine kinases [l 11. Several Trk receptor isoforms have been identified 111,251. Two TrkA kinases differ by the presence of six amino acid residues (VSFSPV, in the single letter code for amino acids) located in the extracellular domain near the transmembrane region [26,27]. Both isoforms appear to have similar biological properties, but they have different patterns of expression. Whereas the TrkA isoform carrying the VSFSPV sequence is primarily expressed in
149
150
Cell reaulation
Signal peptide Cysteine cluster 1
Signal peptide
Leucine-rich motif Cysteine cluster 2 Cysteine repeats
Ig-like domains
Transmembrane
Transmembrane
Juxtamembrane
Cytoplasmic domain
Kinase domain
COOH Tai I
TrkA
TrkB
TrkC
P’5 0 1995
Current Opinion in Cell Biology
Fig. 2. Neurotrophin receptors. Representation of the main structural motifs of the Trk and p75 receptors [lo,11,251. Only one TrkB and one TrkC non-catalytic receptor isoform is depicted. Likewise, only one of the three TrkC tyrosine kinase isoforms containing additional sequences in the kinase domain is shown (see text). The black box in one of the TrkA isoforms represents the VSFSPV sequence.
neuronal cells, the other isoform has been found in cells of non-neuronal origin [26,27]. Four TrkC kinase isoforms may exist on the basis of analysis of t&C cDNA clones. These isoforms differ from the canonical TrkC tyrosine kinase by the presence of 14 (TrkC K14), 25 (TrkC K25) or 39 (TrkC K39) additional amino acid residues in the middle of their kinase catalytic domains [28-301. The additional residues present in the TrkC K14 and TrkC K25 isoforms are unique and do not contain informative structural motifs. TrkC K39 contains the combined 25 and 14 amino acid long sequences (in this order) of TrkC K25 and TrkC K14. A second class of Trk receptor isoforms lacking a tyrosine kinase catalytic domain has also been described [l l] (Fig. 2). These receptors have the same extracellular and transmembrane domains as the Trk kinases described above, but they have distinct and unique cytoplasmic domains that are devoid of distinctive structural motifs. So far, two T&B [31,32] and four TrkC [29,30] non-catalytic receptors have been identified (Fig. 2). Among these putative receptors, TrkB.Tl (also known as gp95trkB) has been identified at the protein level [31]. It remains to be determined whether other Trk non-catalytic receptors are expressed in t&o. The role of these receptors remains to be determined. It has been proposed, however, that gp95trkB may play a role in BDNF transport (or clearing) from the blood and the spinal fluid to (or from) the brain, on the basis of the high levels of expression of this receptor in the choroid plexus and the lining of the ventricles [31]. More recently, it has been speculated that non-catalytic
receptors may play a role in recruiting circulatory neurotrophins in a manner similar to that proposed for the p75 receptor (see below).
Signal transduction As indicated above, the Trk family of tyrosine protein kinases is responsible for mediating neurotrophin signaling. PC12 cells lacking TrkA receptors do not respond to NGF in spite of containing abundant p75 receptors; however, transfection of TrkA restores full NGFresponsiveness to these cells [33]. Likewise, transfection of either Trkl3 or TrkC kinases (but not the TrkC K14 or K25 isoforms) renders PC12 cells responsive to either BDNF (and NT-4) or NT-3, respectively. Moreover, activation of Trk kinase receptors by their cognate ligands in proliferating cells such as rodent frbroblasts elicits a mitogenic response that leads to their malignant transformation [25]. Trk kinase receptors become activated by a two-step process that involves their ligand-mediated dimerization followed by autophosphorylation of their tyrosine residues [22], a mechanism common to other tyrosine kinase receptors [34]. To date, several Trk substrates have been identified [35]. They include phospholipase C (PLC-y, phosphatidylinositol-3 kinase and the adaptor protein She. Other potential substrates for the Trk receptor are the Ras GTPase activating protein and the mitogen-activated protein (MAP) kinase ERKl (but not the highly related ERK2 kinase). It is not known, however, whether these two proteins are directly phosphorylated by the Trk receptors [35].
Neurotrophic factors and their receptors Barbacid
PLC-‘y binds to a conserved tyrosine residue located in the short carboxy-terminal tail characteristic of the Trk receptor family. Phosphatidylinositol-3 kinase interacts with a neighboring tyrosine residue located at the carboxyl terminus of the kinase domain. The physiological significance of these interactions remains obscure, however, as Trk receptors carrying Tyr+Phe mutations in these residues retain their ability to transform NIH3T3 cells and to cause PC12 cells to differentiate [36*,37*]. The She binding site has been mapped to a conserved tyrosine residue located in the juxtamembrane domain [37*]. Mutation of this residue decreases significantly the mitogenic (NIH3T3 cells) and differentiative (PC12 cells) activity of the Trk receptors. Complete ablation of Trk signaling, however, requires the additional mutation of the tyrosine residue responsible for PLC-y binding [36*,37*]. The TrkC kinase isoforms, TrkC K14 and TrkC K25, have normal autocatalytic kinase activity but they do not phosphorylate PLC-), or phosphatidylinositol-3 kinase [28]. These receptors mediate NT-3 dependent signal transduction, as determined by the induction of c-Fos expression and DNA synthesis, but they cannot elicit full mitogenic or PC12 differentiation responses. These results, taken together, indicate that neurotrophin activity may require activation of multiple signaling pathways. One of these pathways includes the well characterized signaling elements of the Ras/Raf/MAP kinase pathway [38]. Addition of NGF to wild-type, but not to Trk-deficient, PC12 cells results in the rapid activation of Ras as well as of the downstream Raf and MAP kinases [35]. Moreover, expression of oncogenic Ras and Raf proteins in PC12 cells results in their neuronal differentiation in the absence of NGE A second pathway utilized by Trk receptors may involve neuron-specific signaling elements such as the recently identified SNT protein [39]. This 90 kDa protein binds to ~13, a subunit of the cell cycle regulatory complex that includes the cdc2 kinase and cyclin. SNT is rapidly phosphorylated on tyrosine residues upon treatment of PC12 with NGF, but not upon treatment with mitogenic factors such as epidermal growth factor (EGF).
Neurotrophins
and their Trk receptors:
in vivo
function The generation of mice carrying germ line mutations in the genes encoding NGF [40”], BDNF [410*,42**] and NT-3 [43**,44**] by homologous recombination in embryonic stem (ES) cells has opened up a unique opportunity to study the role of these neurotrophins in the development and maintenance of the mammalian nervous system. Similar studies involving knockout mice carrying mutations in the catalytic domains of each of the Trk kinase receptors have also provide critical information regarding the role of these receptors in viva [45**-47**]. Moreover, the striking similarities between the phenotypes of mice defective for each Trk receptor and its cognate neurotrophin (Table 1) represent the
most compelling evidence to date supporting the concept that the Trk receptors mediate most, if not all, of the biological activities of the neurotrophins [17]. Mice defective for either NGF [40**] or TrkA receptors [46**] display severe sensory defects characterized by a complete loss of nociceptive activity (Table 1). These mice fail to react to deep pinpricks in their whisker pads and rear paws [40”,46**]. In addition, they exhibit deficiencies in thermoception as they can stay on top of a 60°C hot plate for at least 10s. Neuroanatomical examination of the TrkA- and NGF-null mice revealed extensive neuronal cell loss in trigeminal, dorsal root and sympathetic ganglia [40**,46**]. In the dorsal root ganglia, the vast majority of the missing neurons correspond to those of small size, a population known to be NGF-dependent. The sympathetic ganglia are severely shrunken and contain only a few neurons [40”,46”]. A similar correlation can be found between the phenotypes of mice targeted in the genes encoding NT-3 [43**,44**] and TrkC [47**] (Table 1). These mice also display severe sensory defects, but of a nature distinct h-om those observed in NGF- and T&A-defective mice. Mice lacking NT-3 or its cognate TrkC kinase receptors have normal nociception, but they are defective in proprioception, the sensory activity responsible for localizing the limbs in space [43**,44**,47**]. As a consequence, these targeted mice display abnormal movements of an athetotic nature that result in highly abnormal limb postures. This sensory defect is due to the complete absence of Ia muscle afferents, the projections derived from large proprioceptive dorsal root ganglion neurons that connect primary endings of muscle spindles in the periphery to motor pools in the ventral region of the spinal cord [43**,44**,47**]. TrkC and NT-3 mutant mice have limited life spans most probably because of additional neuronal defects; however, a few TrkC (but not NT-3) mutant mice have survived for over six months. The apparently more severe phenotype of the NT-3 targeted mice might simply be attributed to differences in strain or in the animal colony However, the TrkC defective mice may express non-catalytic TrkC receptor isoforms, as the gene targeting took place within their t&C tyrosine kinase sequences. Whether mutant mice carrying a completely inactivated t&C gene display the shorter life span of the NT-3 knockouts remains to be investigated. Comparison of the phenotypes of mutant mice deficient in BDNF [41**,42**] and in its TrkB kinase receptor [45**] has been less straightforward (Table 1). TrkB mutants display a severe phenotype that results in the death of most animals in the first postnatal week due to their inability to feed [45**]. BDNF-defective mice also have short life spans; however, many animals survive at least two weeks. These mice display defective movement coordination and balance, with head bobbing and spinning followed by long periods of inactivity. This defect is likely to be due to atrophy and loss of vestibular ganglion neurons, which results in defective innervation of the inner ear [41**,42**]. No such defects were observed
151
152
Cell regulation Table 1. Summary of the defects observed in mice targeted in genes encoding various neurotrophins and their receptors.*
Knockout strain Phenotype
p75
NGF
TrkA
BDNF
Sensory activity Nociception Balance Proprioception
Partial Normal Normal
Very low Very low Normal Normal
Necmal . Normal
PNS defects~::[: Superior cervical ganglion Trigeminal ganglion Nodose-petrosal ganglion Vestibular ganglion Dorsal root ganglia la Afferents
Normal Normal ND ND Smaller ND
5% 30% Normal ND 30% Normal
5% 30% Normal ND 30% Normal
CNS defects Facial motor neurons Spinal cord motor neurons Cholinergic projections
ND ND ND
ND ND ND ND Reduced(b) Reduced
TrkB
NT-3
TrkC
Normal Normal Impaired** ND-~:]: Normal Normal
Normal Normal Impaired
Normal Normal Impaired
Normal 60% 40% 15% 70% Normal
Normal 40% 10% (a) ND:]: 70% Normal
50% 40% 60% 80% 35% Lost
75% ND ND ND 80% Lost
Normal Normal ND
30% 70% ND
Normal Normal ND
ND ND ND
*Data obtained from [40°'~7°',57]. **Characters in bold indicate significant differences found in mice defective for a neurotrophin and its cognate Trk receptor. LND, not determined. :[:Animals die too early. :[::[:Approximatepercentage of remaining neurons. (a)JT Erickson, DT Katz, personal communication. (b)H Phillips, personal communication. PNS, peripheral nervous system; CNS, central nervous system.
in the TrkB mutant mice as they do not survive long enough to undergo maturation of their vestibular neurons (Table 1). The TrkB-defective mice display more extensive neuronal cell loss in the motor facial nucleus (50% cen loss compared with none in the BDNF mutant) and/or in the nodose-petrosal complex (>90% cell loss compared with ~40°A in BDNF mutant mice) ([45°°]; JT Erickson, RJ Smeyne, M Barbacid, DM Katz, unpublished data). Neurons in the nodose-petrosal complex control sensory information from cardiovascular, respiratory and gastrointestinal systems and therefore their absence may account for the early death of the TrkB mutant mice. These results are not entirely surprising, as the TrkB tyrosine kinase is also a receptor for NT-4. Whether double knockout BDNF and NT-4 mice display the same phenotype as the TrkB-defective animals remains to be determined. Likewise, it is not known whether mice that also lack expression of the TrkB noncatalytic isoforms display additional neuronal defects.
The p75 neurotrophin receptor p75 is a cell surface glycoprotein that belongs to the tumor necrosis factor receptor superfam~y [11] (Fig. 1). The p75 receptor binds each of the neurotrophins with similarly low nanomolar affinity [11]; however, its precise biological function remains to be elucidated. A significant body of evidence indicates that p75 does not mediate neurotrophin signaling, at least as it relates to neuronal survival and/or differentiation [11,48]. For instance, PC12 cells expressing high levels of p75 receptors
in the absence of TrkA do not differentiate in response to NGE Moreover, p75 is expressed in many cell types that do not respond to neurotrophins. There are several reports, however, that suggest that p75 may mediate some aspects of signal transduction [11]. For instance, ectopic expression of wild-type, but not mutant, p75 receptors in PC12 cells lacking NGF receptors led to the induction oftyrosine phosphorylation (of as yet unidentified substrates) and c-fos expression [11]. In addition, wild-type PC12 cells expressing a chimeric EGF-p75 receptor whose cytoplasmic sequences are derived from p75 can undergo neuronal-like differentiation in the presence of EGF [11]. More recently, it has been reported that p75 mediates the activation of the sphingomyelin cycle [49] and the glycosyl-phosphatidylinositol/inositol phosphoglycan pathway [50] as well as Schwann cell migration [51] and matrix invasion by melanoma cells [52]. Finally, two recent reports indicate that unliganded p75 may mediate signal transduction in apoptotic pathways [53,54]. NGF binding appears to block this apoptotic activity, perhaps contributing to the well known effect of NGF in neuronal survival. A more generally accepted view is that p75 may facilitate NGF signaling through TrkA receptors [11,48]. For instance, it has been proposed [11] that p75 interacts and/or cooperates with the signaling Trk kinases to generate 'high-affinity' binding sites thought to be responsible for mediating neurotrophin function. More recently, it has been shown that transfection of p75 into TrkA-expressing MAH sympathoadrenal cells
Neurotrophic factors and their receutors
enhances their response to NGF [55]. Likewise, blocking p75 binding sites in PC12 cells with either a p75 monoclonal antibody or with BDNF decreases their response to NGF [56].
Role of p75
receptors
in vivo
The most relevant information regarding the function of the p75 receptor in neurotrophin signaling has been provided by analysis of mutant mice carrying a targeted p75 gene [57]. These mice display sensory and sympathetic defects, thus demonstrating that the p75 receptor is indeed required for proper neuronal development (Table 1). Interestingly, these mice do not display the defects characteristic of mice lacking BDNF (or TrkB receptors) and NT-3 (or TrkC receptors). These observations indicate that ~75, despite serving as a receptor for BDNF and NT-3 in vitro, may not be involved in mediating their biological activities in viva (Fig. 1). Alternatively, the absence of p75 might be compensated for by other molecules such as the TrkI3 and TrkC noncatalytic receptors. The observed defects in the p75-null mice appear to be limited to NGF-dependent neurons, mainly sensory (nociceptive) and sympathetic neurons [58*]. Moreover, these defects are much more limited than those observed in either NGF or TrkA mutant mice (Table 1). For instance, NGF- and T&A-null mice have lost most (>95%) of their sympathetic neurons, whereas, in contrast, the p75 null mice have normal sympathetic ganglia as well as normal innervation patterns to all their target tissues with the exception of the pineal and the sweat glands [57,58’]. These observations raise the possibility that TrkA signaling may require p75 receptors in only a subset of NGF-dependent neurons. A more plausible interpretation of these results, however, is that p75 plays a role in recruiting circulating NGF molecules rather than in NGF signaling [48]. In support of this hypothesis, p75-defective sensory and sympathetic neurons survive well in culture in the presence of NGF [59*,60], but they need four times more NGF to achieve the same response as wild-type neurons [59*,60]. Considering the rapid dissociation kinetics of NGF binding to p75 [6], it is possible that expression of p75 receptors either in neurons or in adjacent glial cells may increase the local concentration of diffusible NGE If so, the lack of p75 receptors will only have phenotypic consequences in those neurons for which availability of NGF is limiting. Crossing p75-null mice with transgenic strains overexpressing NGF should provide the experimental tools to test this hypothesis.
means to study their function in viva. Characterization of strains of transgenic mice lacking neurotrophic factors and their receptors is making it possible to define precisely the physiological role of each of these molecules, at least during development. Some of the results obtained (i.e. the observation of sympathetic and sensory defects in NGF- and T&A-null mice) were partly expected on the basis of earlier studies using immunological approaches to neutralize NGF activity. Others, such as the exquisite specificity of the NT-3-TrkC pathway in proprioception or the limited defects displayed by the p75null mice, were unexpected. Another important lesson learned from these gene-targeted mice is the differential role that neurotrophins play in the peripheral versus the central nervous systems. In the peripheral nervous system, ablation of neurotrophin or Trk receptor genes results in massive neuronal cell death, whereas the central nervous system neurons of these mutant mice appear to be, for the most part, unaffected in spite of widespread neurotrophin and receptor expression. These observations illustrate the complex mechanisms involved in the development and survival of the nervous system and predict the existence of additional neurotrophic factors that await discovery.
References and recommended
reading
Papers review, . ..
of particular interest, published have been highlighted as: of special interest of outstanding interest
1.
Westermark B, Heldin CH: Platelet-derived growth factor. Structure, function and implications in normal and malignant cell growth. Acta Oncol 1993, 32:101-l 05.
2.
Eckenstein FP: Fibroblast growth factors in the nervous system. Neurobiology 1994, 25:1467-l 480.
3.
Patterson PH: The emerging neuropoietic cytokine family: first CDF/LIF, CNTF and L-6; next ONC, MCF, CCSF? Curr Opin Neurobiol 1992, 2:94-97.
4.
Sendtner M, Carroll P, Holtmann 6, Hughes RA, Thoenen H: Ciliary neurotrophic factor. / Neurobiol 1994, 25:1436-l 453.
within the annual
period of
5. .
Lin LF, Doherty DH, Lile JD, Bektesh S, Collins F: CDNF: a glial cell line-derived neurotroohic factor for midbrain dooaminer’ gic neurons. Science 1993, 260:1130-l 132. This paper describes the isolation and cloning of a new neurotrophic factor, glial-derived neurotrophic factor. Although initially thought to be specific for dopaminergic neurons, recent studies [l81 indicate that it may have a broad spectrum of action. 6.
Levi-Montalcini R: The nerve growth Science 1987, 237:1154-1162.
7.
Barde Y-A: Neurotrophic factors: an evolutionary / Neurobiol 1994, 25:1329-l 333.
8.
Meakin SO, Shooter EM: The nerve growth factor family of receptors. Trends Neurosci 1992, 15:323-331.
9.
Chao MV: Neurotrophin receptors: a window differentiation. Neuron 1992, 9:583-593.
10.
Barbacid M: The Trk family of neurotrophin robiol 1994, 25:1386-l 403.
11.
Chao MV: The p75 neurotrophin 25:1373-l 385.
12.
Stahl N, Yancopoulos CD: The alphas, betas, and kinases of cytokine receptor complexes. Cell 1993, 74:587-590.
Conclusions Most of the information we had to date regarding the role of neurotrophic factors has been derived corn in vitro studies. The advent of gene targeting has provided new
Barbacid
factor
35 years later.
perspective.
into neuronal
receptors. ) Neu-
receptor. / Neurobiol
1994,
153
154
Cell reeulation 13.
Davis S, Yancopoulos CD: The mofecufat biifogy of the CNTF receptor. Curr Opin Ccjl &of 1993, 5:281-285.
forms of rat TrfK with different functiunaf capabilities. Neuron 1993, 10:963-974.
14.
Davis S, Aldrich TH, Valenzuela DM, Wong VV, Furth ME, Squint0 SP, Yancopoulos CD: The recqtor for ciliiry neurotrof&ic factor. hence 1991, 253:59-63.
31.
Klein R, Conway D, Parada LF, Barbacid M: The trkB tyrosine protein fdnase gene codes for a second neurogenic receptor Ff& facks the catalytic fcinasedon&n. Ce# 1990, 61:647-656.
15.
Kishimoto T, Akira S, Taga T: lnlerleukin-6 and its receptor: a paradigm for cytokiies, Scieoce 1992, 258~593-597.
32.
16.
Gearing DP, Thut CJ, VandeBos T, Gimpef SD, Delaney PB, King J, Price V, Cosman D, Beckmann MP: Leukemia inhibitory factor receptor is structurally refated to the IL-6 signal &ansducer, gp130. EMBO f 1991, ?0:2839-2848.
Middlemas DS, Lindberg RA, Hunter T: trkB, a neural recep tar protein-tyrosine fdise: evidence for a full-fengtf~and two trunca&d receptors. Mof Cell &of 1991, It:7 43-l 53.
33.
Loeb DM, Maragos J, Martin-Zanca D, Chao MV, Parada LF, Greene LA: The trk proto-oncogene rescues NGF responsiveness in mutant NGF-nonresponsive PC12 cuff fhtes. Ceff 1991, 66:961-966.
34.
Schlessinger J, Utirich A: Growth factor signaling by receptor tyrosine kinases. Neuron 1992, 9~383-391.
17.
Snider
WD:
Functions
system ~vel~~~
of the nwrotropbins
what the k&outs
during nervous are teaching us. Cefi
1994, 77627638. 18.
19. .
Henderson CE, Phillips HS, Pollock RA, Davies AM, Lemeulie C, Armanini M, Simpson LC, Moffet B, Vandien RA, Koiiatsos VE, Rosenthal A: GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle. Science 1994, 266: 10&Z-1 064. Gotz R, Koster R, Winkler C, Raulf F, Lottspeich F, Schartl M, Thoenen H: Neurotrof&in-6 is a new member of the nerve
grow& factor family. &&We $994, 372:26L%9. Describes the isolation of a novel neurotroohin. neurotroohin (NT)-6. Extensive efforts using approaches based on the polymeras;! chain reaction to isolate new neurotrophins, after the rapid discovery of NT-3 and NT-4, had failed, thus sugg&ing that there might not be additional members of the neurotrophin family. it remains to be determined, however, whether NT-6 homologues exist in mammals. 20.
McDonald NQ, Lapatto R, Murray-Uust f, Gunning J, ylodawer A, Blundell TL: New protein fold revealed by a 2.3A resofu-
tion crystal structure of nerve growth factor. Nature 1991, 354:411-414. 21.
22.
ibanez CF, Ebendal T, Barbany G, Murray-Rust 1, Blundell Tl, Persson Ii: Disruption of * low affinity ~eptor~~i~ site in NGF allows neuronal survival and differentiation by binding to the trk gene product. Cell “1992, 69:329-342. lbanez CF, flag LL, Murray-Rust J, Persson i-l: An extended surface of binding to Trk tyrosine kinase receptors in NCF and BDNF allows the engineering of a multifunctional pan-
neurotiophin. EMBO I 1993, 12:2281-2293. 23.
Jing S, Tapley P, Barbacid M: Nerve growth factor mediates signal transduction through trk homodimer receptors. Neuron 1992, 9:1067-1079.
24.
Chao MV: Growth factor signaling: where is the specificity? Celi 1992, 68:995-Y%‘.
25.
Barbacid M: The Trk family of ~eurotrop~in receptors: mofecufar characterization and oncogenic activation in human tumors. in Mo~~u~ar genetics of nervous system tumors. Edited by Levine AJ, Schmidek HH. 1993:123-l 35.
26.
27.
New York: Wiley and Sons, Inc;
Barker PA, Lomen-i-ioerth C, Gensch EM, Meakin SO, Glass DJ, Shooter EM: Tissue-specific alternative splicing generates two isoforms of the trkA receptor. 1 Biol Chem 1993, 268:1515&15157. Horigome K, Pryor JC, Bullock ED, Johnson EM Jr: Mediator release from mast cells by nerve growth factor. Neurotrophm specifiiify and receptor mediation. f Biol Chem 1993, 26~1488~-14887.
28.
Lamb&e F, Tapley P, Barbacid M: t&C encodes multiple neurotrophin-3 receptors with distinct biological properties and substrate speeifidties. EMBO / 1993, 12:3083-3094.
29.
Tsoulfas Ramirez
P, Soppet 0, Escandon E, Tessarollo L, MendozaIL. Rosenthal A. Nikolics K. Parada LF: The rat trkC
locus en&es multiple burogenic receptors that exhibit dif&e&al response to n~~rophi~~ in PC12 cells. Neuron 1993, l&975-990. 30.
Valenruela DM, Maisonpierre PC, Glass DJ, Rojas E, Nunez L, Kong Y, Gies DR, St&t TN, lp NY, Yancopoulos GD: AltemaGe
35.
Kaplan DR, Stephens RM: Neurotrophin signal transduction by 1994, 25:1404-1417.
the Trk receptor. j ~~r~i~i 36.
Stephens RM, Loeb DM, Copeland TD, Pawson T, Greene LA, Kaplan DR: Trk recep&rs use redundant signaf trans. duction pathways involving WC and PLC-yl to mediate NCF responses. Neuron 1994, 12:691-705. See I37.1 annotation.
l
37. .
Obermeier A, Bradshaw RA, Seedotf K, Choidas A, Schlessinger J, Ullrich A: Neuronaf differentiation signals are cnntrolfed by
nerve growth factor receptor/Tilt binding sites for WC and P&y* EMBO f 1994, f 3:t 58%1590. In this paper and 136.1, site-directed mutagenesis was used to define the role of individual tyrosine residues in the cytoplasmic domain of the TrkA receptor. These tyrosine residues serve as specific anchors for various TrkA substrates. Therefore, the biological activity of these mutant recep tors provides useful information regarding the relative contributions of these substrates {and their respective signaling pathways) to signailing by nerve growth factor. 38.
Egan SE, Weinberg RAz The pathway to signal a&iivement. Nature 1993, 365~781-783.
39.
Rabin SJ, Cleghon V, Kaplan DR: SNT, a differentiation-specific taq+ of neurotrophic factor-fnduced tyrosine fdnase activity in neurons and PC12 cells. Mel Ceff EGoi1993, 13~2203-22 13.
40.
Crowley C, Spencer SD, Nishimura MC, Chen KS, Pitts-Meek S, Armanini MP, Ling LH, McMahon SB, Shelton DL, Levinson AD, Phillips HS: Mice lacking nerve growth factor dispfay perinataf
l
*
loss of sensory and sympathetic neurons yet develop basal forebrain chofinergic neurons, Cell 1994, 76:1001-l 011. Disruption of the nerve growth factor JNGFJ gene by homologous recombination in embryonic stem cells results in the generation of mice that do not respond to noxious mechanical stimuli. These mice display severe loss of sensory and sympathetic neurons, confirming the critical dependence of these neurons for NCF previously shown in in vitro studies. 41.
..
Jones KR, Farinas I, Backus C, Reichardt LF: Targeted disrup
tion of the BDNF gene perturbs brain and sensory neuron development but not motor neuron development. Cetl 1994, 76:989-999. See [42**1 annotation.
42. l*
Ernfors P, Lee KF, Jaenisch R: Mice lacking brain-derived neuro&opftic factor develop with sensory de&&s. Naiure 1994,
368: 147-l 50. This study and [41**] independently report the generation of mice lacking a functional brain-derived neurotrophic factor gene. Most of these mice die after birth, although some survive for 2-4 weeks, These mice display severe deficiencies in coordination and balance, a phenotype that may result from their significantly reduced levels of vestibular sensory neurons. These mice also display neuronal deficiencies in other cranial and spinal sensory ganglia, but not in motor neurons. 43. .
l
Farinas
I,
Jones
KR,
Backus
C,
Wang
XV, Reichardt
LF:
Severe sensory and sympathetic deficits in mice facking neurotrophin-3. Nature 1994, 369:658-661.
See [44**1 annotation. 44.
..
Ernfors P, Lee KF, Kucera J, Jaenisch R: lack of weurotrophiw-3 leads to deficiencies in the peripheral nervous system and loss of lib mioceptive afi+ren&. C&i 1994, 7R503-512.
N e u r o t r o p h i c factors and their receptors Barbacid Mice lacking neurotrophin-3 die within a few weeks of birth and display abnormal limb movements. This defect is likely to be due to the lack of spinal proprioceptive afferents and muscle spindles. These mice display additional defects in other sensory (trigeminal and cochlear) and sympathetic ganglia, but not in enteric or motor neurons. In addition, the central nervous system of these mice appears to be normal, at least anatomically. Klein R, Smeyne RJ, Wurst W, Long LK, Auerbach BA, Ioyner AL, Barbacid M: Targeted disruption of the trkB neurotrophin receptor gene results in nervous system lesions and neonatal death. Cell 1993, 75:113-122. Partial disruption of the trkB gene led to the generation of mice lacking TrkB tyrosine kinase receptors but not their non-catalytic isoform gp95trkB. Most of these mice die during the first postnatal week due to lack of feeding activity. They display neuronal defects in the peripheral (trigeminal and dorsal root ganglia) and central (facial motor nucleus and spinal cord) nervous system. However, most structures of the central nervous system known to express high levels of trkB transcripts (cortex, hippocampus, etc.) appear to be normal, at least anatomically.
affinity nerve growth factor receptor. Proc Nat/Acad Sci USA 1991, 88:8016-8019. 51.
Anion ES, Weskamp G, Reichardt LF, Matthew WD: Nerve growth factor and ils low-affinity receptor promote Schwann cell migration. Proc Nat/Acad Sci USA ]994, 91:2795-2799.
52.
Herrmann JL, Menter DG, Hamada J, Marchetti D, Nakajima M, Nicolson GL: Mediation of NGF-stimulated extracelluiar matrix invasion by the human melanoma Iow-affinlty p75 neurotrophln receptor: melanoma p75 functions independently of trkA. Mol Biol Cell 1993, 4:]205-]216.
53.
Rabizadeh S, Oh l, Zhong LT, Yang J, Bitler CM, Butcher LL, Bredesen DE: Induction of apoptosis by the low-affinity NGF receptor. Science 1993, 261:345-348. Barrett GL, Bartlett PF: The p75 nerve growth factor receptor mediates survival or death depending on the stage of sensory neuron development. Proc Nat/ Acad Sci USA 1994, 91:6501-6505.
45. *•
Smeyne RJ, Klein R, Schnapp A, Long LK, Bryant S, Lewin A, Lira SA, Barbacid M: Severe sensory and sympathetic neoropathies in mice carrying a disrupted Trk/NGF receptor gene. Nature 1994, 368:246-249. Mice lacking TrkA receptors display the same sensory and sympathetic defects as those carrying a disrupted nerve growth factor (NGF) gene, thus demonstrating that TrkA mediates NGF activity in vivo. TrkA-defective mice also display deficiencies in the cholinergic projections that connect basal forebrain neurons (known to be NGF-dependent) with the hippocampus and cortex.
54.
55.
Verdi JM, Birren SJ, Ibanez CF, Persson H, Kaplan DR, Benedetti M, Chao MV, Anderson DJ: p75LNGFR regulates Irk signal transduction and NGF-induced neuronal differentiation in MAH cells. Neuron 1994, 12:733-745.
56.
Barker PA, Shooter EM: Disruption of NGF binding to the low affinity neurotrophln receptor p75LNTR reduces NGF binding to TrkA on PC12 cells. Neuron ]994, 13:203-215.
57.
Lee KF, Li E, Huber LI, Landis SC, Sharpe AH, Chao MV, Jaenisch R: Targeted mutation of the gene encoding the low affinity NGF receptor p75 leads to deficits in the peripheral sensory nervous system. Cell 1992, 69:737-749.
46. "•
47. ••
Klein R, Silos-Santiago I, Smeyne RJ, Lira SA, Brambilla R, Bryant S, Zhang L, Snider WD, Barbacid M: Disruption of the neurotruphln-3 receptor gene trkC eliminates ia muscle afferents and results in abnormal movements. Nature 1994, 368:249-251. Specific disruption of those sequences encoding the catalytic domain of the TrkC tyrosine kinase receptor results in mice with severe proprioceplive sensory defects, most likely caused by the total absence of la muscle afferent projections to spinal motor neurons. These mice also have fewer large myelinated axons in the dorsal root and posterior columns of the spinal cord. Unlike mice lacking neurotrophin-3, some of the TrkC-defective mice survive for long periods of time (>6months). 48.
Barbacid M: Nerve growth factor: a tale of two receptors. Oncogene 1993, 8:2033-2042.
49.
Dobrowsky RT, Werner MH, Castellino AM, Chao MV, Hannun YA: Activation of the sphingomyelln cycle through the lowaffinity neurotrophin receptor. Science 1994, 265:1596-1599.
50.
Represa J, Avila MA, Miner C, Giraldez F, Romero G, Clemente R, Mato JM, Varela-Nieto I: Glycosyl-phosphatidylinositol/inositol phosphoglycan: a signaling system for the low-
58. •
Lee KF, Bachman K, Landis S, Jaenisch R: Dependence on p75 for innervation of some sympathetic targets. Science 1994, 263:1447-] 449. See [59 •] annotation. 59. .
Davies AM, Lee KF, laenisch R: p75-deficlent trigemlnal sensory neurons have an altered response to NGF but not to other neurotrophins. Neuron 1993, 11:565-574. This study and [58 •] extend the initial characterization of the p75-null mice [57] and provide support for the concept that p75 receptors might only be indispensible for those neurons (and innervations) that have access to limiting amounts of nerve growth factor. 60.
Lee KF, Davies AM, laenisch R: p75-deflcient embryonic dorsal root sensory and neonatal sympathetic neurons display a decreased sensitivity to NGF. Development 1994, 120:1027-1033.
M Barbacid, Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543-4000, USA.
155
factors and their receptors Mariano Barbacid
Bristol-Myers Several
Squibb Pharmaceutical
new members
which comprises
Research Institute, Princeton,
of the nerve growth
factor family
nerve growth factor itself, brain-derived
and neurotrophins-3,
-4 (also known
isolated in recent years. Their
signaling
Trk family of tyrosine
kinases, thus facilitating
signaling
pathways
protein
responsible
of neurotrophins, neurotrophic
as neurotrophin5)
factor
and -6, have been
receptors have been identified
for mediating
USA
the dissection
their trophic
properties.
as the of the More
recently, the advent of gene targeting has made it possible to generate strains of mice lacking neurotrophins
and their receptors. Analysis of the phenotypes
of these mutant animals
has provided
neurotrophins
receptors
and their
detailed information
play in the ontogeny
on the role that
of the mammalian
nervous system. Current
Opinion
in Cell Biology
Introduction Growth factors play a critical role in the development and maintenance of the mammalian nervous system. Some of these factors (e.g. platelet-derived growth factor and fibroblast growth factor) have pleiotropic activities and are often studied in systems other than the nervous system [l-3]. Other factors have a more restricted activity on neuronal cells and are classified as ‘neurotrophic factors’. Among them, the most intensively studied include the ciliary neurotrophic factor (CNTF) [4], the recently discovered glial-derived neurotrophic factor (GDNF) [5*] and the members of the nerve growth factor (NGF) family, now known as ‘neurotrophins’ [6,7]. Each of these neurotrophic factors exerts its biological activities through a different class of receptors. Neurotrophins signal through the Trk family of tyrosine protein kinases [8-lo]. In addition, neurotrophins interact with a second receptor, ~75, a member of the tumor necrosis factor receptor superfamily [ll]. CNTF signals though a multimeric receptor complex which includes binding and signaling elements [12,13]. The binding subunit is specific for CNTF and does not have transmembrane or cytoplasmic domains, but instead anchors to the plama membrane by a glycosyl phosphatidylinositol linkage [14]. The signaling subunits include gp130, a transmembrane glycoprotein previously found in various cytokine receptors [15], and the p-subunit of the receptor for leukemia inhibitory factor [16]. The cytoplasmic domains of both of these molecules interact with members of the Jak family of cytoplasmic tyrosine kinases, which are likely to be the primary
1995,
7:148-l
55
mediators of CNTF activity [12,13]. CNTF shares these signaling subunits with a variety of cytokine receptors, including the receptors for leukemia inhibitory factor, interleukin-6, oncostatin M and, possibly, interleukin-1 1 [12,13]. How these growth factors can elicit such different biological responses by activating the same signaling receptor subunits is a subject of intense study at this time [12,13]. Finally, the receptor for GDNF has not as yet been identified; however, the limited structural similarities between GDNF and the transforming growth factor-p superfamily [5*] raise the possibility that GDNF may signal through multimeric serine/threonine kinase receptors. During the past year, several important developments have occured in the field of neurotrophins and their receptors. The generation of strains of mice carrying targeted mutations in the genes encoding several neurotrophins, as well as in those encoding the ~75 and the Trk kinase receptors, has provided a wealth of information regarding the role that these molecules play in the ontogeny of the mammalian nervous system [17]. In consideration of the space limitations, this review will focus on neurotrophins and their receptors. Other exciting developments regarding CNTF and its signal transduction pathways have been recently summarized in several reviews [4,12,13] and will not be discussed here. GDNF, initially thought to be specific for dopaminergic neurons [5-l, appears to have a broader specificity, as it can also support the survival of motorneurons [18]. Further studies are needed to define the full range of neurotrophic activities of this novel growth factor.
Abbreviations BDNF-brain-derived neurotrophic factor; CNTF-ciliary neurotrophic factor; EGFGpidermal growth factor; GDNF-glial-derived neurotrophic factor; MAP-mitogen-activated protein; NCF-nerve growth factor; NT-neurotrophin; PLC-phospholipase C. 148
0 Current
Biology
Ltd ISSN
0955-0674
Neurotrouhic factors and their receutors Barbacid
The NCF family of neurotrophins This family of growth factors includes NGF, brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3 and NT-4 (also known as NT-4/5 and NT-5) [2]. A molecule recently isolated from the teleost fish Xiphophorus, NT-6, probably represents a novel member of this f&nily [19*]. These neurotrophins bind specifically to the three known Trk tyrosine kinase receptors, T&A, TrkB and TrkC [8-lo]. Whereas NGF binds to T&A, BDNF and NT-4 interact with TrkB (Fig. 1). NT-3 appears to be more promiscuous and binds to each of the Trk receptors, but its primary biological responses are mediated by TrkC (see below). In addition, each of these neurotrophins binds with low affinity to ~75, a receptor structurally unrelated to the Trks and whose precise role in mediating neurotrophin activity remains to be elucidated (see below) [ll] (Fig. 1). Whether NT-6 binds to any of these receptors is not known at the present time. NGF recruitment ?
Qo NGF
TrKA
BDNF
NT-4
TrkB
NT-3
TrKC
Signal transduction 0 1995 Current Opinion in Cell Biology
Fig. 1. Functional interactions between neurotrophins and their cognate receptors. Each of the neurotrophins binds with similarly low affinity (nanomolar range) to ~75. The neuronal defects observed in p75 null mice, however, suggest a functional interaction of this receptor with NGF (solid arrow) but not with BDNF or NT-3 (dashed arrows). The interactions of NCF with TrkA and of BNDF and NT-4 with TrkB are highly specific. NT-3 primarily activates TrkC receptors; however, this neurotrophin can also interact with the TrkA and TrkB receptors (see text).
The three-dimensional structure of NGF has been resolved by X-ray crystallography (at 2.3A resolution; [20]) and shown to consist of two identical subunits. Based on previous sequence analysis, each subunit has 118 amino acids residues and is generated by cleavage of a precursor polypeptide of 252 residues [1,7]. The NGF protomers are elongated and contain three twisted anti-parallel p-sheet segments linked by turns. These protomers are linked to each other by a ‘cysteine knot’, a novel structure also found in transforming growth factor-b and platelet-derived growth factor B-subunit homodimers, and display a twofold symmetry with parallel long axes [20]. The structure of the other neurotrophins remains to be elucidated; however, their close similarities to each other (47 residues are identical in all four mammalian neurotrophins) suggest that they are likely to have similar tertiary structures. Site-directed mutagenesis studies have provided important information regarding those residues critical for the
interaction of neurotrophins with the p75 and TrkA receptors. The interaction of NGF with p75 is mediated by a positively charged region that consists of at least three lysine residues located in two spatially adjacent loops (I and V) [21]. These positively charged residues are conserved in NT-3 and NT-4, but not in BDNF, which contains three consecutive positively charged residues in loop V that may compensate for the absence of the two lysines present in loop I in the other neurotrophins [22]. These differences may explain the somewhat distinct dissociation kinetics shown by BDNE The interaction of NGF with TrkA is more complex and requires cooperativity of multiple contact sites derived horn at least five different domains within the NGF molecule [21,22]. These sites are concentrated on one side of the NGF dimer with both protomers contributing some of the critical residues. Overall, the TrkA binding site appears as a flat, continuous surface approximately parallel to the twofold axis of the molecule. Because of its twofold symmetry, the active NGF dimers have two potential T&A binding sites which may facilitate the formation of T&A receptor homodimers, a step required for receptor activation [23]. Historically, the biological activity of the neurotrophins has been studied using explants of primary neurons or neuronal progenitors in culture, as well as certain immortal cell lines such as PC12 and MAH cells [6,24]. In the case of NGF, pioneering in viva studies using neutralizing antibodies (either passively administered into the placenta or generated by immunization of pregnant rats with NGF) provided the first conclusive evidence for a role of neurotrophins during the development of the mammalian nervous system [6]. These studies have been described in detailed elsewhere [6,24] and will not be discussed here. In this review, I will focus on recent results obtained with ‘knockout’ mice devoid of either NGF, BDNF or NT-3 (see below).
Trk receptors The structural features of the Trk family of receptors have been reviewed recently [25]. The unique combination of structural motifs, particularly in their extracellular, ligand-binding domains, indicate that they constitute a novel receptor subfamily (Fig. 2). Their kinase catalytic domain is most closely related to a series of novel orphan receptors that include the mammalian RORl, ROR2, DDR (also known as Ptk-3, Nep or T&E), Tyro-10 and TKT as well as the Torpedo RTK tyrosine kinases [l 11. Several Trk receptor isoforms have been identified 111,251. Two TrkA kinases differ by the presence of six amino acid residues (VSFSPV, in the single letter code for amino acids) located in the extracellular domain near the transmembrane region [26,27]. Both isoforms appear to have similar biological properties, but they have different patterns of expression. Whereas the TrkA isoform carrying the VSFSPV sequence is primarily expressed in
149
150
Cell reaulation
Signal peptide Cysteine cluster 1
Signal peptide
Leucine-rich motif Cysteine cluster 2 Cysteine repeats
Ig-like domains
Transmembrane
Transmembrane
Juxtamembrane
Cytoplasmic domain
Kinase domain
COOH Tai I
TrkA
TrkB
TrkC
P’5 0 1995
Current Opinion in Cell Biology
Fig. 2. Neurotrophin receptors. Representation of the main structural motifs of the Trk and p75 receptors [lo,11,251. Only one TrkB and one TrkC non-catalytic receptor isoform is depicted. Likewise, only one of the three TrkC tyrosine kinase isoforms containing additional sequences in the kinase domain is shown (see text). The black box in one of the TrkA isoforms represents the VSFSPV sequence.
neuronal cells, the other isoform has been found in cells of non-neuronal origin [26,27]. Four TrkC kinase isoforms may exist on the basis of analysis of t&C cDNA clones. These isoforms differ from the canonical TrkC tyrosine kinase by the presence of 14 (TrkC K14), 25 (TrkC K25) or 39 (TrkC K39) additional amino acid residues in the middle of their kinase catalytic domains [28-301. The additional residues present in the TrkC K14 and TrkC K25 isoforms are unique and do not contain informative structural motifs. TrkC K39 contains the combined 25 and 14 amino acid long sequences (in this order) of TrkC K25 and TrkC K14. A second class of Trk receptor isoforms lacking a tyrosine kinase catalytic domain has also been described [l l] (Fig. 2). These receptors have the same extracellular and transmembrane domains as the Trk kinases described above, but they have distinct and unique cytoplasmic domains that are devoid of distinctive structural motifs. So far, two T&B [31,32] and four TrkC [29,30] non-catalytic receptors have been identified (Fig. 2). Among these putative receptors, TrkB.Tl (also known as gp95trkB) has been identified at the protein level [31]. It remains to be determined whether other Trk non-catalytic receptors are expressed in t&o. The role of these receptors remains to be determined. It has been proposed, however, that gp95trkB may play a role in BDNF transport (or clearing) from the blood and the spinal fluid to (or from) the brain, on the basis of the high levels of expression of this receptor in the choroid plexus and the lining of the ventricles [31]. More recently, it has been speculated that non-catalytic
receptors may play a role in recruiting circulatory neurotrophins in a manner similar to that proposed for the p75 receptor (see below).
Signal transduction As indicated above, the Trk family of tyrosine protein kinases is responsible for mediating neurotrophin signaling. PC12 cells lacking TrkA receptors do not respond to NGF in spite of containing abundant p75 receptors; however, transfection of TrkA restores full NGFresponsiveness to these cells [33]. Likewise, transfection of either Trkl3 or TrkC kinases (but not the TrkC K14 or K25 isoforms) renders PC12 cells responsive to either BDNF (and NT-4) or NT-3, respectively. Moreover, activation of Trk kinase receptors by their cognate ligands in proliferating cells such as rodent frbroblasts elicits a mitogenic response that leads to their malignant transformation [25]. Trk kinase receptors become activated by a two-step process that involves their ligand-mediated dimerization followed by autophosphorylation of their tyrosine residues [22], a mechanism common to other tyrosine kinase receptors [34]. To date, several Trk substrates have been identified [35]. They include phospholipase C (PLC-y, phosphatidylinositol-3 kinase and the adaptor protein She. Other potential substrates for the Trk receptor are the Ras GTPase activating protein and the mitogen-activated protein (MAP) kinase ERKl (but not the highly related ERK2 kinase). It is not known, however, whether these two proteins are directly phosphorylated by the Trk receptors [35].
Neurotrophic factors and their receptors Barbacid
PLC-‘y binds to a conserved tyrosine residue located in the short carboxy-terminal tail characteristic of the Trk receptor family. Phosphatidylinositol-3 kinase interacts with a neighboring tyrosine residue located at the carboxyl terminus of the kinase domain. The physiological significance of these interactions remains obscure, however, as Trk receptors carrying Tyr+Phe mutations in these residues retain their ability to transform NIH3T3 cells and to cause PC12 cells to differentiate [36*,37*]. The She binding site has been mapped to a conserved tyrosine residue located in the juxtamembrane domain [37*]. Mutation of this residue decreases significantly the mitogenic (NIH3T3 cells) and differentiative (PC12 cells) activity of the Trk receptors. Complete ablation of Trk signaling, however, requires the additional mutation of the tyrosine residue responsible for PLC-y binding [36*,37*]. The TrkC kinase isoforms, TrkC K14 and TrkC K25, have normal autocatalytic kinase activity but they do not phosphorylate PLC-), or phosphatidylinositol-3 kinase [28]. These receptors mediate NT-3 dependent signal transduction, as determined by the induction of c-Fos expression and DNA synthesis, but they cannot elicit full mitogenic or PC12 differentiation responses. These results, taken together, indicate that neurotrophin activity may require activation of multiple signaling pathways. One of these pathways includes the well characterized signaling elements of the Ras/Raf/MAP kinase pathway [38]. Addition of NGF to wild-type, but not to Trk-deficient, PC12 cells results in the rapid activation of Ras as well as of the downstream Raf and MAP kinases [35]. Moreover, expression of oncogenic Ras and Raf proteins in PC12 cells results in their neuronal differentiation in the absence of NGE A second pathway utilized by Trk receptors may involve neuron-specific signaling elements such as the recently identified SNT protein [39]. This 90 kDa protein binds to ~13, a subunit of the cell cycle regulatory complex that includes the cdc2 kinase and cyclin. SNT is rapidly phosphorylated on tyrosine residues upon treatment of PC12 with NGF, but not upon treatment with mitogenic factors such as epidermal growth factor (EGF).
Neurotrophins
and their Trk receptors:
in vivo
function The generation of mice carrying germ line mutations in the genes encoding NGF [40”], BDNF [410*,42**] and NT-3 [43**,44**] by homologous recombination in embryonic stem (ES) cells has opened up a unique opportunity to study the role of these neurotrophins in the development and maintenance of the mammalian nervous system. Similar studies involving knockout mice carrying mutations in the catalytic domains of each of the Trk kinase receptors have also provide critical information regarding the role of these receptors in viva [45**-47**]. Moreover, the striking similarities between the phenotypes of mice defective for each Trk receptor and its cognate neurotrophin (Table 1) represent the
most compelling evidence to date supporting the concept that the Trk receptors mediate most, if not all, of the biological activities of the neurotrophins [17]. Mice defective for either NGF [40**] or TrkA receptors [46**] display severe sensory defects characterized by a complete loss of nociceptive activity (Table 1). These mice fail to react to deep pinpricks in their whisker pads and rear paws [40”,46**]. In addition, they exhibit deficiencies in thermoception as they can stay on top of a 60°C hot plate for at least 10s. Neuroanatomical examination of the TrkA- and NGF-null mice revealed extensive neuronal cell loss in trigeminal, dorsal root and sympathetic ganglia [40**,46**]. In the dorsal root ganglia, the vast majority of the missing neurons correspond to those of small size, a population known to be NGF-dependent. The sympathetic ganglia are severely shrunken and contain only a few neurons [40”,46”]. A similar correlation can be found between the phenotypes of mice targeted in the genes encoding NT-3 [43**,44**] and TrkC [47**] (Table 1). These mice also display severe sensory defects, but of a nature distinct h-om those observed in NGF- and T&A-defective mice. Mice lacking NT-3 or its cognate TrkC kinase receptors have normal nociception, but they are defective in proprioception, the sensory activity responsible for localizing the limbs in space [43**,44**,47**]. As a consequence, these targeted mice display abnormal movements of an athetotic nature that result in highly abnormal limb postures. This sensory defect is due to the complete absence of Ia muscle afferents, the projections derived from large proprioceptive dorsal root ganglion neurons that connect primary endings of muscle spindles in the periphery to motor pools in the ventral region of the spinal cord [43**,44**,47**]. TrkC and NT-3 mutant mice have limited life spans most probably because of additional neuronal defects; however, a few TrkC (but not NT-3) mutant mice have survived for over six months. The apparently more severe phenotype of the NT-3 targeted mice might simply be attributed to differences in strain or in the animal colony However, the TrkC defective mice may express non-catalytic TrkC receptor isoforms, as the gene targeting took place within their t&C tyrosine kinase sequences. Whether mutant mice carrying a completely inactivated t&C gene display the shorter life span of the NT-3 knockouts remains to be investigated. Comparison of the phenotypes of mutant mice deficient in BDNF [41**,42**] and in its TrkB kinase receptor [45**] has been less straightforward (Table 1). TrkB mutants display a severe phenotype that results in the death of most animals in the first postnatal week due to their inability to feed [45**]. BDNF-defective mice also have short life spans; however, many animals survive at least two weeks. These mice display defective movement coordination and balance, with head bobbing and spinning followed by long periods of inactivity. This defect is likely to be due to atrophy and loss of vestibular ganglion neurons, which results in defective innervation of the inner ear [41**,42**]. No such defects were observed
151
152
Cell regulation Table 1. Summary of the defects observed in mice targeted in genes encoding various neurotrophins and their receptors.*
Knockout strain Phenotype
p75
NGF
TrkA
BDNF
Sensory activity Nociception Balance Proprioception
Partial Normal Normal
Very low Very low Normal Normal
Necmal . Normal
PNS defects~::[: Superior cervical ganglion Trigeminal ganglion Nodose-petrosal ganglion Vestibular ganglion Dorsal root ganglia la Afferents
Normal Normal ND ND Smaller ND
5% 30% Normal ND 30% Normal
5% 30% Normal ND 30% Normal
CNS defects Facial motor neurons Spinal cord motor neurons Cholinergic projections
ND ND ND
ND ND ND ND Reduced(b) Reduced
TrkB
NT-3
TrkC
Normal Normal Impaired** ND-~:]: Normal Normal
Normal Normal Impaired
Normal Normal Impaired
Normal 60% 40% 15% 70% Normal
Normal 40% 10% (a) ND:]: 70% Normal
50% 40% 60% 80% 35% Lost
75% ND ND ND 80% Lost
Normal Normal ND
30% 70% ND
Normal Normal ND
ND ND ND
*Data obtained from [40°'~7°',57]. **Characters in bold indicate significant differences found in mice defective for a neurotrophin and its cognate Trk receptor. LND, not determined. :[:Animals die too early. :[::[:Approximatepercentage of remaining neurons. (a)JT Erickson, DT Katz, personal communication. (b)H Phillips, personal communication. PNS, peripheral nervous system; CNS, central nervous system.
in the TrkB mutant mice as they do not survive long enough to undergo maturation of their vestibular neurons (Table 1). The TrkB-defective mice display more extensive neuronal cell loss in the motor facial nucleus (50% cen loss compared with none in the BDNF mutant) and/or in the nodose-petrosal complex (>90% cell loss compared with ~40°A in BDNF mutant mice) ([45°°]; JT Erickson, RJ Smeyne, M Barbacid, DM Katz, unpublished data). Neurons in the nodose-petrosal complex control sensory information from cardiovascular, respiratory and gastrointestinal systems and therefore their absence may account for the early death of the TrkB mutant mice. These results are not entirely surprising, as the TrkB tyrosine kinase is also a receptor for NT-4. Whether double knockout BDNF and NT-4 mice display the same phenotype as the TrkB-defective animals remains to be determined. Likewise, it is not known whether mice that also lack expression of the TrkB noncatalytic isoforms display additional neuronal defects.
The p75 neurotrophin receptor p75 is a cell surface glycoprotein that belongs to the tumor necrosis factor receptor superfam~y [11] (Fig. 1). The p75 receptor binds each of the neurotrophins with similarly low nanomolar affinity [11]; however, its precise biological function remains to be elucidated. A significant body of evidence indicates that p75 does not mediate neurotrophin signaling, at least as it relates to neuronal survival and/or differentiation [11,48]. For instance, PC12 cells expressing high levels of p75 receptors
in the absence of TrkA do not differentiate in response to NGE Moreover, p75 is expressed in many cell types that do not respond to neurotrophins. There are several reports, however, that suggest that p75 may mediate some aspects of signal transduction [11]. For instance, ectopic expression of wild-type, but not mutant, p75 receptors in PC12 cells lacking NGF receptors led to the induction oftyrosine phosphorylation (of as yet unidentified substrates) and c-fos expression [11]. In addition, wild-type PC12 cells expressing a chimeric EGF-p75 receptor whose cytoplasmic sequences are derived from p75 can undergo neuronal-like differentiation in the presence of EGF [11]. More recently, it has been reported that p75 mediates the activation of the sphingomyelin cycle [49] and the glycosyl-phosphatidylinositol/inositol phosphoglycan pathway [50] as well as Schwann cell migration [51] and matrix invasion by melanoma cells [52]. Finally, two recent reports indicate that unliganded p75 may mediate signal transduction in apoptotic pathways [53,54]. NGF binding appears to block this apoptotic activity, perhaps contributing to the well known effect of NGF in neuronal survival. A more generally accepted view is that p75 may facilitate NGF signaling through TrkA receptors [11,48]. For instance, it has been proposed [11] that p75 interacts and/or cooperates with the signaling Trk kinases to generate 'high-affinity' binding sites thought to be responsible for mediating neurotrophin function. More recently, it has been shown that transfection of p75 into TrkA-expressing MAH sympathoadrenal cells
Neurotrophic factors and their receutors
enhances their response to NGF [55]. Likewise, blocking p75 binding sites in PC12 cells with either a p75 monoclonal antibody or with BDNF decreases their response to NGF [56].
Role of p75
receptors
in vivo
The most relevant information regarding the function of the p75 receptor in neurotrophin signaling has been provided by analysis of mutant mice carrying a targeted p75 gene [57]. These mice display sensory and sympathetic defects, thus demonstrating that the p75 receptor is indeed required for proper neuronal development (Table 1). Interestingly, these mice do not display the defects characteristic of mice lacking BDNF (or TrkB receptors) and NT-3 (or TrkC receptors). These observations indicate that ~75, despite serving as a receptor for BDNF and NT-3 in vitro, may not be involved in mediating their biological activities in viva (Fig. 1). Alternatively, the absence of p75 might be compensated for by other molecules such as the TrkI3 and TrkC noncatalytic receptors. The observed defects in the p75-null mice appear to be limited to NGF-dependent neurons, mainly sensory (nociceptive) and sympathetic neurons [58*]. Moreover, these defects are much more limited than those observed in either NGF or TrkA mutant mice (Table 1). For instance, NGF- and T&A-null mice have lost most (>95%) of their sympathetic neurons, whereas, in contrast, the p75 null mice have normal sympathetic ganglia as well as normal innervation patterns to all their target tissues with the exception of the pineal and the sweat glands [57,58’]. These observations raise the possibility that TrkA signaling may require p75 receptors in only a subset of NGF-dependent neurons. A more plausible interpretation of these results, however, is that p75 plays a role in recruiting circulating NGF molecules rather than in NGF signaling [48]. In support of this hypothesis, p75-defective sensory and sympathetic neurons survive well in culture in the presence of NGF [59*,60], but they need four times more NGF to achieve the same response as wild-type neurons [59*,60]. Considering the rapid dissociation kinetics of NGF binding to p75 [6], it is possible that expression of p75 receptors either in neurons or in adjacent glial cells may increase the local concentration of diffusible NGE If so, the lack of p75 receptors will only have phenotypic consequences in those neurons for which availability of NGF is limiting. Crossing p75-null mice with transgenic strains overexpressing NGF should provide the experimental tools to test this hypothesis.
means to study their function in viva. Characterization of strains of transgenic mice lacking neurotrophic factors and their receptors is making it possible to define precisely the physiological role of each of these molecules, at least during development. Some of the results obtained (i.e. the observation of sympathetic and sensory defects in NGF- and T&A-null mice) were partly expected on the basis of earlier studies using immunological approaches to neutralize NGF activity. Others, such as the exquisite specificity of the NT-3-TrkC pathway in proprioception or the limited defects displayed by the p75null mice, were unexpected. Another important lesson learned from these gene-targeted mice is the differential role that neurotrophins play in the peripheral versus the central nervous systems. In the peripheral nervous system, ablation of neurotrophin or Trk receptor genes results in massive neuronal cell death, whereas the central nervous system neurons of these mutant mice appear to be, for the most part, unaffected in spite of widespread neurotrophin and receptor expression. These observations illustrate the complex mechanisms involved in the development and survival of the nervous system and predict the existence of additional neurotrophic factors that await discovery.
References and recommended
reading
Papers review, . ..
of particular interest, published have been highlighted as: of special interest of outstanding interest
1.
Westermark B, Heldin CH: Platelet-derived growth factor. Structure, function and implications in normal and malignant cell growth. Acta Oncol 1993, 32:101-l 05.
2.
Eckenstein FP: Fibroblast growth factors in the nervous system. Neurobiology 1994, 25:1467-l 480.
3.
Patterson PH: The emerging neuropoietic cytokine family: first CDF/LIF, CNTF and L-6; next ONC, MCF, CCSF? Curr Opin Neurobiol 1992, 2:94-97.
4.
Sendtner M, Carroll P, Holtmann 6, Hughes RA, Thoenen H: Ciliary neurotrophic factor. / Neurobiol 1994, 25:1436-l 453.
within the annual
period of
5. .
Lin LF, Doherty DH, Lile JD, Bektesh S, Collins F: CDNF: a glial cell line-derived neurotroohic factor for midbrain dooaminer’ gic neurons. Science 1993, 260:1130-l 132. This paper describes the isolation and cloning of a new neurotrophic factor, glial-derived neurotrophic factor. Although initially thought to be specific for dopaminergic neurons, recent studies [l81 indicate that it may have a broad spectrum of action. 6.
Levi-Montalcini R: The nerve growth Science 1987, 237:1154-1162.
7.
Barde Y-A: Neurotrophic factors: an evolutionary / Neurobiol 1994, 25:1329-l 333.
8.
Meakin SO, Shooter EM: The nerve growth factor family of receptors. Trends Neurosci 1992, 15:323-331.
9.
Chao MV: Neurotrophin receptors: a window differentiation. Neuron 1992, 9:583-593.
10.
Barbacid M: The Trk family of neurotrophin robiol 1994, 25:1386-l 403.
11.
Chao MV: The p75 neurotrophin 25:1373-l 385.
12.
Stahl N, Yancopoulos CD: The alphas, betas, and kinases of cytokine receptor complexes. Cell 1993, 74:587-590.
Conclusions Most of the information we had to date regarding the role of neurotrophic factors has been derived corn in vitro studies. The advent of gene targeting has provided new
Barbacid
factor
35 years later.
perspective.
into neuronal
receptors. ) Neu-
receptor. / Neurobiol
1994,
153
154
Cell reeulation 13.
Davis S, Yancopoulos CD: The mofecufat biifogy of the CNTF receptor. Curr Opin Ccjl &of 1993, 5:281-285.
forms of rat TrfK with different functiunaf capabilities. Neuron 1993, 10:963-974.
14.
Davis S, Aldrich TH, Valenzuela DM, Wong VV, Furth ME, Squint0 SP, Yancopoulos CD: The recqtor for ciliiry neurotrof&ic factor. hence 1991, 253:59-63.
31.
Klein R, Conway D, Parada LF, Barbacid M: The trkB tyrosine protein fdnase gene codes for a second neurogenic receptor Ff& facks the catalytic fcinasedon&n. Ce# 1990, 61:647-656.
15.
Kishimoto T, Akira S, Taga T: lnlerleukin-6 and its receptor: a paradigm for cytokiies, Scieoce 1992, 258~593-597.
32.
16.
Gearing DP, Thut CJ, VandeBos T, Gimpef SD, Delaney PB, King J, Price V, Cosman D, Beckmann MP: Leukemia inhibitory factor receptor is structurally refated to the IL-6 signal &ansducer, gp130. EMBO f 1991, ?0:2839-2848.
Middlemas DS, Lindberg RA, Hunter T: trkB, a neural recep tar protein-tyrosine fdise: evidence for a full-fengtf~and two trunca&d receptors. Mof Cell &of 1991, It:7 43-l 53.
33.
Loeb DM, Maragos J, Martin-Zanca D, Chao MV, Parada LF, Greene LA: The trk proto-oncogene rescues NGF responsiveness in mutant NGF-nonresponsive PC12 cuff fhtes. Ceff 1991, 66:961-966.
34.
Schlessinger J, Utirich A: Growth factor signaling by receptor tyrosine kinases. Neuron 1992, 9~383-391.
17.
Snider
WD:
Functions
system ~vel~~~
of the nwrotropbins
what the k&outs
during nervous are teaching us. Cefi
1994, 77627638. 18.
19. .
Henderson CE, Phillips HS, Pollock RA, Davies AM, Lemeulie C, Armanini M, Simpson LC, Moffet B, Vandien RA, Koiiatsos VE, Rosenthal A: GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle. Science 1994, 266: 10&Z-1 064. Gotz R, Koster R, Winkler C, Raulf F, Lottspeich F, Schartl M, Thoenen H: Neurotrof&in-6 is a new member of the nerve
grow& factor family. &&We $994, 372:26L%9. Describes the isolation of a novel neurotroohin. neurotroohin (NT)-6. Extensive efforts using approaches based on the polymeras;! chain reaction to isolate new neurotrophins, after the rapid discovery of NT-3 and NT-4, had failed, thus sugg&ing that there might not be additional members of the neurotrophin family. it remains to be determined, however, whether NT-6 homologues exist in mammals. 20.
McDonald NQ, Lapatto R, Murray-Uust f, Gunning J, ylodawer A, Blundell TL: New protein fold revealed by a 2.3A resofu-
tion crystal structure of nerve growth factor. Nature 1991, 354:411-414. 21.
22.
ibanez CF, Ebendal T, Barbany G, Murray-Rust 1, Blundell Tl, Persson Ii: Disruption of * low affinity ~eptor~~i~ site in NGF allows neuronal survival and differentiation by binding to the trk gene product. Cell “1992, 69:329-342. lbanez CF, flag LL, Murray-Rust J, Persson i-l: An extended surface of binding to Trk tyrosine kinase receptors in NCF and BDNF allows the engineering of a multifunctional pan-
neurotiophin. EMBO I 1993, 12:2281-2293. 23.
Jing S, Tapley P, Barbacid M: Nerve growth factor mediates signal transduction through trk homodimer receptors. Neuron 1992, 9:1067-1079.
24.
Chao MV: Growth factor signaling: where is the specificity? Celi 1992, 68:995-Y%‘.
25.
Barbacid M: The Trk family of ~eurotrop~in receptors: mofecufar characterization and oncogenic activation in human tumors. in Mo~~u~ar genetics of nervous system tumors. Edited by Levine AJ, Schmidek HH. 1993:123-l 35.
26.
27.
New York: Wiley and Sons, Inc;
Barker PA, Lomen-i-ioerth C, Gensch EM, Meakin SO, Glass DJ, Shooter EM: Tissue-specific alternative splicing generates two isoforms of the trkA receptor. 1 Biol Chem 1993, 268:1515&15157. Horigome K, Pryor JC, Bullock ED, Johnson EM Jr: Mediator release from mast cells by nerve growth factor. Neurotrophm specifiiify and receptor mediation. f Biol Chem 1993, 26~1488~-14887.
28.
Lamb&e F, Tapley P, Barbacid M: t&C encodes multiple neurotrophin-3 receptors with distinct biological properties and substrate speeifidties. EMBO / 1993, 12:3083-3094.
29.
Tsoulfas Ramirez
P, Soppet 0, Escandon E, Tessarollo L, MendozaIL. Rosenthal A. Nikolics K. Parada LF: The rat trkC
locus en&es multiple burogenic receptors that exhibit dif&e&al response to n~~rophi~~ in PC12 cells. Neuron 1993, l&975-990. 30.
Valenruela DM, Maisonpierre PC, Glass DJ, Rojas E, Nunez L, Kong Y, Gies DR, St&t TN, lp NY, Yancopoulos GD: AltemaGe
35.
Kaplan DR, Stephens RM: Neurotrophin signal transduction by 1994, 25:1404-1417.
the Trk receptor. j ~~r~i~i 36.
Stephens RM, Loeb DM, Copeland TD, Pawson T, Greene LA, Kaplan DR: Trk recep&rs use redundant signaf trans. duction pathways involving WC and PLC-yl to mediate NCF responses. Neuron 1994, 12:691-705. See I37.1 annotation.
l
37. .
Obermeier A, Bradshaw RA, Seedotf K, Choidas A, Schlessinger J, Ullrich A: Neuronaf differentiation signals are cnntrolfed by
nerve growth factor receptor/Tilt binding sites for WC and P&y* EMBO f 1994, f 3:t 58%1590. In this paper and 136.1, site-directed mutagenesis was used to define the role of individual tyrosine residues in the cytoplasmic domain of the TrkA receptor. These tyrosine residues serve as specific anchors for various TrkA substrates. Therefore, the biological activity of these mutant recep tors provides useful information regarding the relative contributions of these substrates {and their respective signaling pathways) to signailing by nerve growth factor. 38.
Egan SE, Weinberg RAz The pathway to signal a&iivement. Nature 1993, 365~781-783.
39.
Rabin SJ, Cleghon V, Kaplan DR: SNT, a differentiation-specific taq+ of neurotrophic factor-fnduced tyrosine fdnase activity in neurons and PC12 cells. Mel Ceff EGoi1993, 13~2203-22 13.
40.
Crowley C, Spencer SD, Nishimura MC, Chen KS, Pitts-Meek S, Armanini MP, Ling LH, McMahon SB, Shelton DL, Levinson AD, Phillips HS: Mice lacking nerve growth factor dispfay perinataf
l
*
loss of sensory and sympathetic neurons yet develop basal forebrain chofinergic neurons, Cell 1994, 76:1001-l 011. Disruption of the nerve growth factor JNGFJ gene by homologous recombination in embryonic stem cells results in the generation of mice that do not respond to noxious mechanical stimuli. These mice display severe loss of sensory and sympathetic neurons, confirming the critical dependence of these neurons for NCF previously shown in in vitro studies. 41.
..
Jones KR, Farinas I, Backus C, Reichardt LF: Targeted disrup
tion of the BDNF gene perturbs brain and sensory neuron development but not motor neuron development. Cetl 1994, 76:989-999. See [42**1 annotation.
42. l*
Ernfors P, Lee KF, Jaenisch R: Mice lacking brain-derived neuro&opftic factor develop with sensory de&&s. Naiure 1994,
368: 147-l 50. This study and [41**] independently report the generation of mice lacking a functional brain-derived neurotrophic factor gene. Most of these mice die after birth, although some survive for 2-4 weeks, These mice display severe deficiencies in coordination and balance, a phenotype that may result from their significantly reduced levels of vestibular sensory neurons. These mice also display neuronal deficiencies in other cranial and spinal sensory ganglia, but not in motor neurons. 43. .
l
Farinas
I,
Jones
KR,
Backus
C,
Wang
XV, Reichardt
LF:
Severe sensory and sympathetic deficits in mice facking neurotrophin-3. Nature 1994, 369:658-661.
See [44**1 annotation. 44.
..
Ernfors P, Lee KF, Kucera J, Jaenisch R: lack of weurotrophiw-3 leads to deficiencies in the peripheral nervous system and loss of lib mioceptive afi+ren&. C&i 1994, 7R503-512.
N e u r o t r o p h i c factors and their receptors Barbacid Mice lacking neurotrophin-3 die within a few weeks of birth and display abnormal limb movements. This defect is likely to be due to the lack of spinal proprioceptive afferents and muscle spindles. These mice display additional defects in other sensory (trigeminal and cochlear) and sympathetic ganglia, but not in enteric or motor neurons. In addition, the central nervous system of these mice appears to be normal, at least anatomically. Klein R, Smeyne RJ, Wurst W, Long LK, Auerbach BA, Ioyner AL, Barbacid M: Targeted disruption of the trkB neurotrophin receptor gene results in nervous system lesions and neonatal death. Cell 1993, 75:113-122. Partial disruption of the trkB gene led to the generation of mice lacking TrkB tyrosine kinase receptors but not their non-catalytic isoform gp95trkB. Most of these mice die during the first postnatal week due to lack of feeding activity. They display neuronal defects in the peripheral (trigeminal and dorsal root ganglia) and central (facial motor nucleus and spinal cord) nervous system. However, most structures of the central nervous system known to express high levels of trkB transcripts (cortex, hippocampus, etc.) appear to be normal, at least anatomically.
affinity nerve growth factor receptor. Proc Nat/Acad Sci USA 1991, 88:8016-8019. 51.
Anion ES, Weskamp G, Reichardt LF, Matthew WD: Nerve growth factor and ils low-affinity receptor promote Schwann cell migration. Proc Nat/Acad Sci USA ]994, 91:2795-2799.
52.
Herrmann JL, Menter DG, Hamada J, Marchetti D, Nakajima M, Nicolson GL: Mediation of NGF-stimulated extracelluiar matrix invasion by the human melanoma Iow-affinlty p75 neurotrophln receptor: melanoma p75 functions independently of trkA. Mol Biol Cell 1993, 4:]205-]216.
53.
Rabizadeh S, Oh l, Zhong LT, Yang J, Bitler CM, Butcher LL, Bredesen DE: Induction of apoptosis by the low-affinity NGF receptor. Science 1993, 261:345-348. Barrett GL, Bartlett PF: The p75 nerve growth factor receptor mediates survival or death depending on the stage of sensory neuron development. Proc Nat/ Acad Sci USA 1994, 91:6501-6505.
45. *•
Smeyne RJ, Klein R, Schnapp A, Long LK, Bryant S, Lewin A, Lira SA, Barbacid M: Severe sensory and sympathetic neoropathies in mice carrying a disrupted Trk/NGF receptor gene. Nature 1994, 368:246-249. Mice lacking TrkA receptors display the same sensory and sympathetic defects as those carrying a disrupted nerve growth factor (NGF) gene, thus demonstrating that TrkA mediates NGF activity in vivo. TrkA-defective mice also display deficiencies in the cholinergic projections that connect basal forebrain neurons (known to be NGF-dependent) with the hippocampus and cortex.
54.
55.
Verdi JM, Birren SJ, Ibanez CF, Persson H, Kaplan DR, Benedetti M, Chao MV, Anderson DJ: p75LNGFR regulates Irk signal transduction and NGF-induced neuronal differentiation in MAH cells. Neuron 1994, 12:733-745.
56.
Barker PA, Shooter EM: Disruption of NGF binding to the low affinity neurotrophln receptor p75LNTR reduces NGF binding to TrkA on PC12 cells. Neuron ]994, 13:203-215.
57.
Lee KF, Li E, Huber LI, Landis SC, Sharpe AH, Chao MV, Jaenisch R: Targeted mutation of the gene encoding the low affinity NGF receptor p75 leads to deficits in the peripheral sensory nervous system. Cell 1992, 69:737-749.
46. "•
47. ••
Klein R, Silos-Santiago I, Smeyne RJ, Lira SA, Brambilla R, Bryant S, Zhang L, Snider WD, Barbacid M: Disruption of the neurotruphln-3 receptor gene trkC eliminates ia muscle afferents and results in abnormal movements. Nature 1994, 368:249-251. Specific disruption of those sequences encoding the catalytic domain of the TrkC tyrosine kinase receptor results in mice with severe proprioceplive sensory defects, most likely caused by the total absence of la muscle afferent projections to spinal motor neurons. These mice also have fewer large myelinated axons in the dorsal root and posterior columns of the spinal cord. Unlike mice lacking neurotrophin-3, some of the TrkC-defective mice survive for long periods of time (>6months). 48.
Barbacid M: Nerve growth factor: a tale of two receptors. Oncogene 1993, 8:2033-2042.
49.
Dobrowsky RT, Werner MH, Castellino AM, Chao MV, Hannun YA: Activation of the sphingomyelln cycle through the lowaffinity neurotrophin receptor. Science 1994, 265:1596-1599.
50.
Represa J, Avila MA, Miner C, Giraldez F, Romero G, Clemente R, Mato JM, Varela-Nieto I: Glycosyl-phosphatidylinositol/inositol phosphoglycan: a signaling system for the low-
58. •
Lee KF, Bachman K, Landis S, Jaenisch R: Dependence on p75 for innervation of some sympathetic targets. Science 1994, 263:1447-] 449. See [59 •] annotation. 59. .
Davies AM, Lee KF, laenisch R: p75-deficlent trigemlnal sensory neurons have an altered response to NGF but not to other neurotrophins. Neuron 1993, 11:565-574. This study and [58 •] extend the initial characterization of the p75-null mice [57] and provide support for the concept that p75 receptors might only be indispensible for those neurons (and innervations) that have access to limiting amounts of nerve growth factor. 60.
Lee KF, Davies AM, laenisch R: p75-deflcient embryonic dorsal root sensory and neonatal sympathetic neurons display a decreased sensitivity to NGF. Development 1994, 120:1027-1033.
M Barbacid, Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543-4000, USA.
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