The neurotrophins and their receptors

m Cell. Biol. 4, 766--771 34 PLOW,E. F. and GINSBERG,M. H. (1989) Prog. Hemost. Thromb. 9,117-156 35 FRELINGER,A. L., DU, X., PLOW, E. F. and GINSBERG,M. H. (1991) 1. Biol. Chem.266,17106-17111 36 HUANG,M. M., LIPFERT,L., CUNNINGHAM, M., BRUGGE, J. S., GINSBERG, M. H. and SHAI"I'IL,S. J. I. Cell Biol. (in press) 37 DAVIS,S. et al. (1991) Science252, 712-715 38 KANNER,S. B., REYNOLDS,A. 8., VINES,R. R. and PARSONS,J. T. (1990) Proc. Natl Acad. Sci. USA 87, 3328-3332

The neurotrophins and their receptors

The neurotrophins, which include nerve growth factor (NGF) and its relatives, were discovered and characterized for their distinctive ability to promote survival and differentiation of postmitotic neurons. Perhaps surprisingly, the neurotrophins have recently been found to utilize a family of receptor tyrosine kinases (the Trks) similar to those used by normally mitogenic growth factors. In fact, ectopic expression of the Trks in non.neuronal cells allows them to mediate conventional mitogenic responses to the neurotrophins. Despite similarities with other receptor tyrosine kinases, the Trks are rather unique in that they are almost exclusively expressed in the nervous system, and they also display a number of novel structural features. In addition to the Trks, the neurotrophins all bind to another cell surface receptor (known as p75 or the low.affinity NGF receptor), whose role remains quite controversial.

Over the last few decades, several families of peptide growth factors have been characterized for their ability to cause cellular proliferation 1. Nerve The authors are at growth factor (NGF), in contrast, was discovered Regeneron and characterized for its actions on postmitotic Pharmaceuticals, neurons; rather than promoting proliferation, NGF Inc., 777 Old Saw serves as a 'survival' and 'differentiation' factor for Mill River Road, these cellsz, Recently, several other factors have Tarrytown, NY been discovered that share -50% amino acid 10591, USA. identity with NGF: brain-derived neurotrophic 262

© 1993 ElsevierSciencePublishersLtd (UK) 0962-8924/93/$06.00

39 PASQUALE,E. B., MAHER,P. A. and SINGER,S. I. (1986) Proc. Notl Acad Sci. USA 83, 5507-5511 40 DECLUE,J. E. and MARTIN,G. S. (1987) Mol. Cell. BioL 7, 371-378 41 SEFTON,8. M. and HUNTER,T. (1981) Ce1124, 165-174 42 TAPLEY,P., HORWlTZ,A., BUCK,C., DUGGAN, K. and ROHRSCHNEIDER,L. (1989) Oncogene4, 325-333 43 ZACHARY,I., SINNEIT-SMITH,J. and ROZENGURT,E. (1992) J. BioL Chem. 267, 19031-19034 44 TURNER,C. E., GLENNEY,J. R. and BURRIDGE,K. (1990) I. Cell BioL 111,1059-1068

factor (BDNF), neurotrophin-3 (NT-3), and a factor referred to as either neurotrophin-4 or neurotrophin-5 (see Box; here called NT-4/$) 3-11. Together with NGF these proteins constitute the 'neurotrophin family'. Like NGF, the other neurotrophins induce neurite outgrowth and rescue distinct subpopulations of neurons from programmed cell death. Conventional mitogenic factors, such as the fibroblast growth factors (FGFs), the epidermal growth factors (EGFs), and the platelet-derived growth factors (PDGFs), initiate signal transduction by activating the intrinsic tyrosine kinase activity of their cognate transmembrane receptors 1. Activation of these growth-factor receptors, known collectively as receptor tyroslne klnases (RTKs), depends on llgand-medlated receptor dl. merlzatlon and usually results In cell growth and proliferation1. By contrast, it seemed likely that the distinctive effects of the neurotrophlns on neuronal survival and differentiation would be mediated by a different class of cellular receptors. The initial receptor discovered for NGF, now known as the low-affinity NGF receptor (LNGFR), lacked any recognizable catalytic motifs within its cytoplasmic domain, and did little to discourage the view that there might be something unique about the cellular signalling mechanism of neurotrophins 12-14, However, recent studies have revealed that the neurotrophins are in fact quite similar to other classes of peptide growth factors in that they mediate their actions by utilizing a family of RTKs known as the TrkslS, 16. NGF is the preferred ligand for the first member of this family to be identified, known initially as Trk (here called TrkA). BDNF and NT-4/5 are the preferred ligands for TrkB, while NT-3 is a secondary ligand for TrkB as well as the only known ligand that can effectively activate TrkC (Fig. 1). The role of the LNGFR, and the manner by which it may collaborate with the Trks to mediate neurotrophin actions, remain controversial. The "Irks resemble the RTKs used by conventional growth factors both structurally and in the way they activate intracellular signalling pathways. However, the Trks differ from other RTKs in that they are prominently expressed only within the nervous system is. It is the restricted distribution of TRENDS IN CELL BIOLOGY VOL. 3 AUGUST 1993

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NEUROTROPHIN-4 OR NEUROTROPHIN-5? the Trks to postmitotic neurons, rather than any unique signalling mechanism of these receptors, that underlies the discovery of the neurotrophins as neuronal survival and differentiative factors. In fact, ectopic expression of the Trks in nonneuronal cells allows them to act very much like conventional mitogens, while the neuronal expression of RTKs for conventional growth factors allows them to have neurotrophic actions. However, the Trks do display a number of novel features that distinguish them from other families of RTKs.

High- and low-affinity NGF receptors Early studies with NGF revealed that chick neurons display two distinct classes of NGF receptors: one of low affinity (the LNGFR, with a Kd of 10-8 -10 9 M) and the other of high affinity (the highaffinity NGF receptor, called the HNGFR, with a Kd of 10-10-10 -11 M)17. Although in other cells, such as the NGF-responsive PC12 pheochromocytoma cell line, these two receptor classes are much more similar in their affinities for NGF 17, they can easily be distinguished by other characteristics 18. For example, the HNGFR is trypsin-stable and displays a slow dissociation rate for NGF (with a half-life of approximately 10 min), while the LNGFR is trypsinlabile and displays a very rapid dissociation rate for NGF (with a half-life of a few seconds). Furthermore, crosslinking analysis revealed that the HNGFR and the LNGFR have distinct molecular masses of about 140 kDa and 75 kDa, respectively. A variety of early data suggested that the bio. logical actions of NGF were mediated by the HNGFR. Distribution of the HNGFR, and not the LNGFR, correlated with NGF responsiveness; a variety of cells were found that expressed the LNGFR but did not respond to NGF, whereas at least some NGF-responslve cells expressed only the HNGFR19,20. Furthermore, only the HNGFR could be crossllnked to NGF under conditions that restricted NGF binding to the biologically active receptor classlS, and only the HNGFR could internalize bound NGFZLzz. Although these findings clearly pointed to the HNGFR as the receptor that mediated functional NGF responses, they left open the intriguing possibility that, while the LNGFR might not be sufficient to mediate responses to NGF, it might under certain circumstances be contained within, or converted into, a functional HNGFR. However, intensive efforts aimed at demonstrating physical association between the LNGFR and the HNGFR failed, and antibodies directed against the LNGFR were not immunoreactive towards the HNGFR 17,23. Is the LNGFR required to form a functional neurotrophin receptor? Antibodies specific for the LNGFR allowed the molecular cloning of a gene (from both rat and human) encoding a transmembrane protein exhibiting all the characteristics of the LNGFR12,13: it had a molecular mass of -75 kDa, it bound to NGF with low affinity, and it exhibited the expected rapid dissociation rate for NGF and sensitivity to TRENDS IN CELL BIOLOGY VOL. 3 AUGUST 1993

Is a recently identified mammalian neurotrophin the counterpart of XenopusNT-4, or is it a novel neurotrophin? It is significantly more diverged from its putative Xenopus counterpart than the other neurotrophins BUT: It is the closest mammalian homologue of XenopusNT-4 AND: !t parallels XenopusNT-4 in its specific interactions with TrkB and not TrkA or TrkC CONCLUSION: It is the mammalian counterpart of XenopusNT-4

trypsin. The LNGFR binds NGF through four cysteine-rich loops in its extracellular domain, which bears significant homology to the extracellular regions of the two receptors for tumour necrosis factor, the CD40 antigen, the Fas antigen, the Tcell antigens OX40 and mu4-1BB, and the B-cell antigen CD30 (Ref. 17). The intracytoplasmic region of the LNGFR fails to imply a signalling mechanism; there is no tyrosine kinase domain, nor is there any evidence that the receptor is phosphorylated upon binding neurotrophin. While there is a single mastoparan-like domain, which is a consensus sequence for the binding of G proteins, there is no evidence to date for G-protein-linked signal transduction via the LNGFR. Cloning of the LNGFR allowed the notion that this protein contributes to the formation of the functional HNGFR to be tested. Early reconstitution experiments, which showed that binding of NGF and NGF-induced phosphorylation in mutant PC12 cells required introduction of the LNGFR14,24, seemed to support this possibility, and suggested a receptor model in which the LNGFR is absolutely required for the formation of the HNGFR, as well as for its ability to mediate functional responses 14. Further reconstitution studies, involving a chimeric receptor in which the ectodomain of the EGF receptor was fused to the intracytoplasmic domain of the LNGFR, claimed that activation of the LNGFR intracytoplasmic domain (by EGF stimulation of this chimeric receptor) was sufficient to induce neuronal differentiation of PC12 cells 2s. A variety of more recent findings seriously challenge the interpretations and implications of these reconstitution experiments. Most importantly, it is now clear that the neurotrophins can directly bind to and activate the Trk family of RTKs, by inducing Trk dimerization 26, even in the absence of the LNGFRlSA6,36. Furthermore, the finding that all the neurotrophins can bind to the LNGFR9,27-29, but that only NGF can mediate neuronal differentiation of PC12 cells 16,28 (which express the LNGFR and TrkA) reveals that ligand-mediated activation of the LNGFR intracytoplasmic domain is not sufficient to induce neuronal outgrowth in PC 12 cells. 263

NGF

BDNF

NT-4/5

NT-3

Binds all the neurotrophins

either qualitatively or quantitatively 37. Similarly, a mutant NGF that cannot bind the LNGFR but which retains its ability to bind to TrkA can also elicit neuronal differentiation responses from PC12 cells at normal doses 38. Other potential roles for the LNGFR

,

I

LNGFR

TRKA

1/1 TRKB

TRKC FIGURE 1

Neurotrophin homodimers activate their cognate Trks by inducing receptor dimerization; while TrkA is specifically activated by NGF, and TrkC is specifically activated by NT-3, TrkB can be activated by both BDNF and NT4/5, and even NT-3 in some cases. All of the neurotrophins also bind the LNGFR;the functional significance of this binding, and whether the LNGFRcan somehow interact with the Trks, remains very controversial.

Altogether, current evidence indicates that the LNGFR is neither sufficient to mediate responses to the neurotrophlns, nor required to allow the Trks to interact functionally with their ligands. However, it remains possible that the LNGFR may In some manner collaborate with Trks, converting them into higher-affinity receptors for the neurotrophins; such a role would be of crucial importance, since it might enable lower concentrations of neurotrophins to activate signalling by Trks. However, binding studies addressing this question remain very controversial, with a number of studies both supporting30, 31 and refuting26,32-34 such a possibility. Apart from the controversial binding data, all other biochemical evidence indicates that Trks, in the absence of the LNGFR, display the characteristic features of the HNGFP, (for example, slow dissociation, trypsin-insensitivity and ligand internalization) 26,3s. Furthermore, ectopic expression of the Trks in fibroblasts (which do not normally express the LNGFR) allows biochemical and biological responses to their primary neurotrophin ligands with dose-dependencies similar to those of the same Trks expressed in PC12 cells (although dose-dependencies for secondary ligands may differ - see below)16,26,36; co-expression of the LNGFR in these fibroblasts does not alter the nature of the biological response, nor does it affect the dose dependencies of the Trks for their primary neurotrophin ligands26. In addition, an antibody that blocks binding of NGF to the LNGFR does not affect the ability of PC12 cells to respond to NGF 264

Thus the available data do not convincingly

support early models that suggested the LNGFR was absolutely required for the formation of a functional neurotrophin receptor 14. What is not disputed is the functional importance of the LNGFR, as evidenced by the conservation among vertebrates of its structure and ability to bind the neurotrophins 17. Although it may yet be demonstrated that the LNGFR plays a role in modulating the dose dependencies of the Trks for their ligands, new potential roles for this neurotrophin receptor should also be explored. Such roles may come to light from the study of LNGFR-deficient mice generated from embryonic stem cells in which the LNGFR gene has been disrupted 39. These animals exhibit a marked sensory deficit that can be correlated with decreased sensory innervation of the skin. The animals do not. however, suffer from extensive death of either sensory neurons or even sympathetic neurons, whose survival in vivo depends on NGF as demonstrated by using an NGF-blocking antibody. Thus, studies with the LNGFR-deficient mice are consistent with the notion that the LNGFR is not absolutely required to allow neurons to respond to the neurotrophins, but may rather play a more subtle role in the normal development and function of neurotrophin-dependent neurons. Potential roles for the LNGFR need not involve direct interaction with the Trks. Since the LNGFR can bind to each of the neurotrophlns, it seems logical to search for roles that may generally apply to all members of the neurotrophin family. One possible function for the LNGFR, based on the finding that it is often expressed not only by neurons but also by specific target cells of neurons as well as by gila, is that the LNGFR serves a role as a 'neurotrophin presentation' molecule40; such a role is also consistent with the finding that the LNGFR binds a portion of the NGF molecule distinct from that bound by TrkA38. Thus the LNGFR may localize the neurotrophins to the surface of non-neuronal cells, and present these factors to axonai processes bearing Trks. Although the fast dissociation rate of the LNGFR may make such a presentation role seem unlikely, this fast dissociation occurs only in the presence of large amounts of excess competing ligand; when challenged with physiological levels of the neurotrophins, cells bearing only LNGFR binding sites release the neurotrophin quite slowly. Alternatively, the LNGFR may play a role in modulating the specificity of the Trks for the neurotrophins (see below). The Trks as functional neurotrophin receptors The finding that NGF induces rapid tyrosine

phosphorylations in responding cells41,4z, coupled TRENDS IN CELL BIOLOGYVOL. 3 AUGUST 1993

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with the finding that NGF could be complexed to a molecule with tyrosine kinase activityz3, provided the first clues that the NGF receptor might be an RTK. This possibility was verified by the demonstration that a receptor-like tyrosine kinase initially known as Trk (here called TrkA), whose expression characterizes NGF-responsive neuronal sensory ganglia 43, could directly bind to and be activated by NGF 30,32,44. TrkA is a member of a family of closely related RTKs that includes TrkB4S,46 and TrkC 33,63, both of which are also neurotrophin receptors28,33,34,36,47,48,63, 64. The three Trks have -50% amino acid identity in their extracellular portions, and -80% identity in their cytoplasmic domains is. A variety of systems, including PC12 and fibroblast cell lines, have been used to explore the specificity of the three Trks for the neurotrophins. As discussed above, PC12 cells normally express TrkA and the LNGFR; among the neurotrophins, only NGF can induce neuronal differentiation and survival responses in parental PC12 cells16,28. Transfection of TrkB into PC12 cells allows them to differentiate in response to BDNF and NT-4/S, while transfection of TrkC confers responsiveness to NT-316,28. These TrkB- and TrkC-mediated neuronal differentiation responses appear quite similar to those normally mediated by TrkA, but they have yet to be exhaustively compared. Perhaps surprisingly, the ectopic expression of Trks in NIH3T3 fibroblasts (in the absence of the LNGFR) has also provided a useful system for the study of neurotrophin-Trk interactions a6,49. By contrast to their effects on PC12 cells (and normal cultured neurons), the neurotrophins act as mitogenlc factors on flbroblasts expressing Trks, although they can also be shown to affect survival of these cells. The dose responses of Trk.expressing fibroblasts for their primary neurotrophln ligands are quite similar to those of Trk.expresslng PC12 cells, both for biochemical activation of the Trk receptor (as assayed by receptor autophosphorylation) and for ultimate biological responses 16,26,36. These results suggest a similar mechanism of Trk activation presumably ligand-mediated receptor dimerization - in both PC12 cells and fibroblasts for the preferred ligands of each Trk. -

Trks in flbroblasts are more promiscuous than Trks in neurons Interestingly, the Trks expressed in fibroblasts are less selective than the same receptors expressed in PC12 cells 16. For example, TrkB-expressing fibroblasts respond to NT-3 almost as well as they respond to BDNF and NT-4/5, while TrkB-expressing PC12 cells display only negligible responses to NT-3. Thus, the PC12 cell environment appears to be more restrictive than that of fibroblasts with respect to secondary ligands for each of the Trks, even though the dose-responses displayed by Trkexpressing fibroblasts for their primary neurotrophin ligands are quite similar to those of Trkexpressing PC12 cells. These data raise the possibility that accessory molecules (such as the TRENDSIN CELLBIOLOGYVOL. 3 AUGUST1993

LNGFR) expressed on PC12 cells might restrict the ability of the Trks to interact with their nonpreferred ligands. The LNGFR could achieve this restriction by acting in a manner analogous to truncated RTK forms, which can undergo ligandmediated heterodimerization with normal RTK forms and thus prevent their ligand-induced activation in a 'dominant negative' fashion 1. According to such a model, heterodimerization with the LNGFR might prevent activation of a Trk (such as TrkB) by a nonpreferred ligand (such as NT-3) for which the LNGFR has a higher affinity than does the Trk receptor. Alternatively, either other accessory molecules (candidates could include cytoplasmic proteins as well as cell surface proteins) or PC12-specific receptor modifications may serve to restrict the ability of the Trks to respond to their nonpreferred neurotrophin ligands when expressed in PC12 cells. At least some neuronal populations (for example, sensory neurons isolated from embryonic chick dorsal root ganglia) are similar to PC12 cells in limiting the ability of their Trk receptors to respond to nonpreferred ligands 16. Pleiotropy of Trk responses is determined by the cell context in which they are expressed The finding that Trks mediate very different biological responses when ectopically expressed in fibroblasts does not by any means suggest that these responses are in some way inappropriate or lacking in physiological significance. In fact, the ability of a given factor to have different effects (i.e. 'pleiotropic' effects) on different cells is becoming more of a rule than an exception - and the neurotrophins are just one of many examples. It seems that the cellular context in which a receptor is activated is most important in determining the ultimate effect that activation will eventually produce - the same receptor can seemingly plug into different signalling pathways in different cells, leading to diverse biological readouts. Although neurotrophin receptors are not normally expressed on fibroblasts, there is growing evidence for naturally occurring targets of neurotrophin action in which the Trk receptors do indeed mediate proliferative responses s°-s2. Conversely, factors initially studied for their mitogenic effects on fibroblasts (for example, FGFs, EGFs and PDGFs) have now been shown to have neurotrophic effects on a variety of neuronal populations, with FGF and PDGF able to mimic the effects of NGF on PC12 cellsS3-SS; in fact, the prediction that the neurotrophins could, like FGF, have growth effects on fibroblasts if their functional receptors were introduced into these cells suggested a strategy that led to the cloning of the Trks via a function-based assay in fibroblasts s6. TRK-medlated signal transduction The binding of neurotrophins to Trks results in

Trk dimerization 26, which then apparently activates the intrinsic tyrosine kinase activity of the Trk receptor - an activation process common to RTKs. The autophosphorylation of specific tyrosine 265

residues within the cytoplasmic domains of RTKs While differences in receptor substrate choices provides anchoring sites for a variety of intracellumay prove critical, an alternative explanation has been offered for why EGF induces a different ullar signalling proteins that can in turn be activated by association with, or phosphorylation by, the timate response from the other RTKs - this explaactivated Trk 1. The topic of RTK-induced signal nation suggests that the EGF receptor need not transduction is beyond the scope of this review; choose a different set of substrates, but that it here, we briefly note that Trks have much in commerely activates them to differing extents or for mon with other RTKs1 in terms of the signalling differing time periodsSS, sg. Just to provide more molecules they activate, either directly or indirectly. confusion, but perhaps a useful research tool, various Many of these activations have been shown to groups have now reported the existence of variant occur both in PC12 cells and in fibroblasts ectopiPC12 subclones that respond to EGF with neuronal cally expressing the Trks. Similarly, other RTKs differentiation60, 61. Clearly, elucidating the mechhave also been shown to activate many of these anisms by which cellular context and individual signalling molecules both in cells in which they RTKs influence the type of responses elicited is mediate a proliferative signal and in cells in which critical to understanding how a multicellular orthey mediate a differentiative signal 1. ganism undergoes development, maintains homeoHow then does ligand stimulation of the same stasis, and responds to environmental cues. RTK in different cellular contexts result in such distinct biological effects? Many RTKs mediate mitoAlternative forms of the Trk receptors genic responses in fibroblasts but mediate neurAfter the initial characterization of TrkA, TrkB and onal differentiation in PC12 cells16,s3,ss. It seems TrkC, variant forms of TrkB and TrkC that lack most likely that different cell types present a given the tyrosine kinase domains were characterRTK with different menus of signalling substrates, ized34,46,62-64. These truncated forms of TrkB and allowing the same RTK to elicit fundamentally dis- TrkC are widely distributed throughout the nertinct biological responses dependent upon the cell vous system; while the fuU-length forms of the type in which they are expressed; the choices pre- Trks are prominently expressed only in neurons, sented to the RTK seem to be the most important the truncated forms of TrkB and TrkC are highly determinants of the type of response elicited, expressed not only in neurons but also in glia. In explaining why different RTKs often produce the addition to its widespread expression throughout same effect when activated in the same cellular the nervous system, the truncated form of TrkB context. exhibits strikingly high expression in the ependyThe different signalling molecules that might ma! lining of the cerebral ventricles and in the distinguish a ftbroblast and a neuronal cell have choroid plexus 62. The structure and distribution of not yet been elucidated, and it is not clear whether the truncated forms of the Trks have suggested a these would consist of molecules that directly variety of functional roles, many of which are interact with RTKs or those much further along in similar to those proposed for the LNGFR. These the signalling cascade (e.g. in the nucleus). How- truncated Trks may act to establish gradients of the ever, it is certainly true that the same RTK will neurotrophins and/or to present neurotrophins to induce tyroslne phosphorylation of a distinct functional receptors on other cells. Alternatively, (albeit overlapping) set of proteins in fibroblasts as as with the LNGFR, such truncated Trks might be compared with neuronal cellsST; identification of particularly effective as dominant negatives in prethese proteins might go a long way towards deter- venting activation of a full-length Trk (such as mining why a flbroblast and a neuron can respond TrkB) by a nonpreferred ligand (such as NT-3) for so differently to the activation of the same RTK. which the truncated Trk (i.e. TrkC) has a higher Despite the fact that different RTKs often induce affinity than does the full-length Trk Finally, the the phosphorylation of similar sets of proteins in striking expression of truncated TrkB within the the same cellular context, differences in the in. lining of the ventricles and in the choroid plexus duced set of proteins can often be noted (see, for has led to the suggestion that it may act to transexample, Ref. 57). Thus, not only could the output port its ligand across the blood-brain barrier, or be of RTK stimulation be determined by the menu a scavenger of excess ligand 62. presented to the RTK, but it could also be deterIn addition to its truncated forms, isoforms of mined by the choices made by that particular RTK. TrkC have been described that have variable-sized This possibility can conveniently explain why dif- inserts within their kinase domain34,63,64; such inferent RTKs can occasionally elicit very distinct sertions have not been found in the corresponding responses from the same cell. PC12 cells offer one regions of other RTK domains, nor have any variof the most dramatic examples of this situation. able-sized inserts ever been described in other While most RTKs (including those for the neuroRTKs. Like the truncated forms of TrkC, inserttrophins, the FGFs and PDGF) elicit neuronal dif- containing forms are widely expressed throughout ferentiation in PCl2 cells, EGF-receptor activation the nervous system. Remarkably, insert-containing results in a mild mitogenic effect. Interestingly, forms of TrkC retain the ability to autophosphorylwhile all these RTKs induce the tyrosine phosate in response to NT-3, but cannot mediate prophorylation of a very similar set of proteins in liferation in fibroblasts or neuronal differentiation PC12 cells sT, a target that cannot be phosphorylated in PC12 cells; thus they present a perfect example by the EGF receptor has recently been identified sS. of different RTKs (in this case isoforms of the same 266

TRENDSIN CELLBIOLOGYVOL. 3 AUGUST1993

RTK) being able to mediate distinct effects w i t h i n the same cell, presumably by distinguishing between the various substrates available in that cell. The physiological roles of the various forms of the Trks will provide fertile areas for future investigation, and should provide important insights into the mechanism of action of the neurotrophins in vivo. Summary Although they were discovered and characterized based on their neuronal survival and differentiation activities, the neurotrophins utilize a family of RTKs similar to those used by no:really mitogenic growth factors. When expressed in non-neuronal cells, the Trks mediate mitogenic responses indistinguishable from those of traditional growth factors. Thus it seems likely that the cellular context in w h i c h an RTK is expressed dictates, to a large degree, the type of response that RTK will mediate. Despite the similarities w i t h other families of RTKs, the Trks are rather unique in that they are almost exclusively expressed in the nervous system; this restricted expression is presumably responsible for the discovery and characterization of the neurotrophins as neurotrophic factors rather than as mitogenic agents. The Trks are also unique in that they have naturally occurring forms (both truncated forms and those w i t h variable inserts w i t h i n the kinase domain) that have not been described for other RTKs and that presumably have novel physiological roles. In addition to the "Irks, the neurotrophins utilize another cell surface receptor, k n o w n as the LNGFR, which binds to all known members of the neurotrophin family with low affinity. The role of the LNGFR remains quite controversial. ?lthough not essential for the formation of a functional neurotrophin receptor, it may modulate the binding, specificity or signalling capacities of the Trks. Alternatively, it may serve a variety of roles (such as the establishment of fixed gradients of the neurotrophins) that do not require direct interaction with the Trks. Further analysis of the interactions between the neurotrophins and their receptors, and how these operate w i t h i n the context of the animal, will continue to provide insights into the mechanisms by which the nervous system develops, is maintained, and becomes susceptible to degenerative disease. References 1 SCHLESSINGER,l, and ULLRICH,A. (1992) Neuron 9, 383-391 2 THOENEN,H. (1991) Trends Neurosci. 14, 165-170 3 LEIBROCK,]. et al. (1989) Nature 341,149-152 4 HAHN,A., LEIBROCK,J., BAILEY,K. and BARDE,Y-A.(1990) Nature 344, 339-341 S ERNFORS,P., IBAI~IEZ,C. F., EBENDAL,T., OLSON,L. and PERSSON,H. (1990) Proc. Natl Acad. Sci. USA 87, 5454-5488 6 MAISONPIERRE,P. C. et al. (1990) Science 247, 1446-1451 7 IONES,K. R. and REICHARDT,L. F. (1990) Proc.Natl Acad. Sci. USA 87, 8060-8064 8 ROSENTHAL,A. et al. (1990) Neuron 4, 763-773 9 HALLBOOK,F., IBA~EZ,C. F. and PERSSON,H. (1991) Neuron 6, 845-858

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Mx proteins: GTPases with antiviral activity Pete r s ta e h eli, Fe r n an d o Pito ssi and:Jgvan Pavovic Mx proteins are synthesized in interferon.treated vertebrate cells. They have attracted much attention because some of them can block the multiplication of influenza A virus and certain other negative.stranded RNA viruses. Recently, Mx proteins have been shown to be GTPases with significant homology to dynamins and yeast VPS1, enzymes involved in intraceilular protein trafficking. Several biochemical properties of dynamin and VPS1 are similar to those of Mx, prompting new speculation about how Mx proteins might interfere with virus multiplication.

When cells are infected by virus, they rapidly synthesize interferons. Secretion of these cytokines signals to other cells of the organism that a virus is about to invade, and that they should prepare for viral attack. Cells respond to the interferon signal by synthesizing at least 50 new proteins, some of which are able to inhibit specific viral multiplication steps. The mouse Mxl protein was identified as an interferon-induced factor that determines resistance of mice to influenza A viruses 1-3. It also confers a high degree of resistance to influenza A viruses in mammalian and chick cells in tissue cul268

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ture2, 4. Mxl is a nuclear protein s that acts by blocking primary transcription of the viral RNA genome6;, a process that takes place in the nudeus. Interferon-treated human cells contain two Mxrelated proteins, now designated MxA and MxBS,9, which, in contrast to mouse Mxl, accumulate in the cytoplasm. When constitutively expressed in mouse 3T3 cells, MxA effectively blocks the multiplication of influenza A virus and vesicular stomatitis virus (VSV), whereas MxB has no detectable activity against these two viruses lo. Unlike mouse Mxl, human MxA does not inhibit primary transcription of influenza A virus7; rather, it blocks a poorly defined multiplication step after primary transcription but before genome replication. This step may be transport of viral mRNAs to ribosomes, translation, or transport of newly synthesized viral proteins to the site of genome replication. Interestingly, when MxA is localized to the nucleus by fusion with a heterologous nuclear translocation signal, MxA does block primary transcription of influenza A virus 1l, thereby mimicking the action of mouse Mxl. Thus, the viral target of MxA must be present in both cell compartments at early times of infection. By contrast to its normal action on influenza virus, MxA inhibits primary genome transcription of VSV12. Since transcription and replication of VSV take place entirely in the cytoplasm, this action is consistent with the intracellular distribution of MxA. The mechanistic details of MxA-mediated inhibition of VSV are unknown. Mx proteins are found in all interferon-treated vertebrate cells, but only some of these proteins have been shown to possess antiviral activity. For example, rat Mxl inhibits multiplication of influenza virus but not of VSV; rat Mx2 inhibits multiplication of VSV but not of influenza virus, and rat Mx3 inhibits neither 13. Ducks have a single Mx protein that fails to inhibit multiplication of either influenza virus o r VSV 14. Since only a limited number of viruses have been tested for Mx sensitivity, it is unclear whether Mx proteins inhibit viruses TRENDS IN CELL BIOLOGY VOL. 3 AUGUST 1993