project, sequence and chromosomal localization of cDNAs are to be collected, whereas in the shotgun analysis of brain cDNA, sequence and expression data are determined. Clearly, the goals of the projects overlap, and, ideally, these three types of data should be collected simultaneously. Analysis of cDNA will reveal many new genes that function in the nervous system. In particular, shotgun analysis will identify a large number of such genes. Eventually we should have an extensive catalogue of cDNA sequences and protein structures, plus gene expression data, and we should be able to generate and test hypotheses using this catalogue. If the final goal of the reductionist approach is to understand a tissue by understanding its molecules, having a catalogue of DNA sequences, protein structures and expression information should move us much closer to this goal. Selected references

1 Goodman, C. S. (1985) Trends Neurosci. 8, 229-230 2 Hawkes, R., Niday, E. and Matus, A. (1982) Proc. IVatlAcad. Sci. USA 79, 2410-2414 3 Sternberger, L. A., Harwell, L. W. and Stemberger, N. H. (1982) Proc. Natl Acad. Sci. USA 79, 1326-1330 4 McKay, R. D. G. and Hockfield, S. (1982) Proc. NatlAcad. 5ci. USA 79, 6747-6751 5 Barnstable, C. J., Akagawa, K., Holstein, R. and Horn, J. P. (1983) COld Spring Harbor Syrnp. Quant. Biol. 48~ 863-876 6 Deneris, E. S. et'al. (1988) Neuron 1, 45-54 7 Grenningloh, G. et al. (1990) EMBO J. 9, 771-776 8 Butler, A., Wei, A., Baker, K. and Salkoff, L. (1989) Science 243,943-947 9 Gould, S. J., Subramani, S. and Scheffler, I. E. (1989) Proc. Nat/Acad. Sci. USA 86, 1934-1938 10 Strathmann, M., Wilkie, T. M. and Simon, M. I. (1989) Proc. Nat/Acad. Sci. USA 86, 7407-7409

11 Wilks, A. F., Kurban, R. R., Hovens, C. M. and Ralph, S. J. AcCents (1989) Gene 85, 67-74 Theau~orthanks 12 Suzuki, S. and Naitoh, Y. (1990) EMBOJ. 9, 757-763 DrSydneyBrenner 13 Libert, F. etat. (1969) Science 244, 569-572 for hisadvice,support 14 Keininen, K. et al. (1990) Science 2.49, 556-560 and encouragement 15 Buck, L. and Axet, R. (1991) Cell65, 175--187 and Prof Kenichi 16 Anderson, D. J. and Axel, R. (1985) Ce# 42, 649-662 Mat~ubarafor critical 17 Rogers,J. H. (1987) J. Ce//. Biol. 105, 1343-1353 18 Oberdick, J., Levinthal, F. and Levinthal, C. (198~) Neuron 1, readingof this 367-376 manuscriptand 19 Miller, F. D., Naus, C. C. G., Higgins, G. A., Bloom, F. E. and supportduringthe Milner, R. J. (1987) J. Neurosci. 7, 2433-2444 earlystageof the 20 Clayton, D. F., Huecas, M. E., Sinclair-Thompson, E. Y., author's work. The Nastiuk, K. L. and Nottebohm, F. (1988) Neuron 1,249--261 authoralsothanks 21 Richter, K., Grunz, H. and Dawid, I. B. (1988) Proc Natl Dr Mike Brownstein Acad. ScL USA 85, 8086-8090 22 Zopf, D., Hermans-Borgmeyer, I., Gundelfinger, E. D. and for adviceon the manuscript.Themain Betz, H. (1987) Genes Dev. 1,699-708 23 Sambrook, J., Fritch, E. F. and Maniatis, T. (1989) Molecular partof theauthor's Cloning: A Laboratory Manual, Book 2, pp. 10.38-10.43, work wasdonein Cold Spring Harbor Laboratory Press the MRCMolecular 24 Travis, G. H. and Sutcliffe, J. G. (1988) Proc. NatlAcad. Sci. GeneticsUnit, USA 85, 1696-1700 Cambridge,UK, and 25 Nordquist, D. T., Kozak, C. A. and Orr, H. T. (1988) wassupportedby a J. Neurosci. 8, 4780-4789 26 Milner, R. J. and Sutcliffe, J. G. (1983) Nucleic Adds Res. 11, grant from E.I. Du Pontde Nemours 5497-5517 27 Sutctiffe, J. G., Milner, R. J., Shinnick, T. M. and Bloom, F. E. andCo. (1983) Ceil 33, 671-682 28 Kato, K. (1990) Eur. J. Neurosci. 2, 704-711 29 Kaplan, B. B., Shachter, B. S., Osterburg, H. H., de Vellis, J. S. and Finch, C. E. (1978) Biochemistry 17, 55165524 30 Kato, K. (1990) J. MOI. Biol. 214, 619-624 31 Kato, K. (1990) FEB$ Lett. 271, 137-140 32 Umekage, T. and Kato, K. (1991) FEB5 Lett. 286, 147-151 33 Palazzoto, M. J. et a/. (1989) Neuron 3,527-539 34 Hyde, D. R. et a/. (1990) Proc. Nat/Acad. Sci. USA 87, 1008-1012 35 Adams, M. D. eta/. (1991)Science 252, 1651-1656 36 Adams, M. D. eta/. (1992) Nature 355, 632-634 37 Smith, L. M. eta/. (1986) Nature 321,674-679

viewpoint

Thenervegrowthfader familyof receptors Susan O. M e a k i n and Eric M . S h o o t e r

The neurotrophins,of which nervegrowth factor(NGF)is the best known example,support the survivaland differentiationof chick embryosensoryneuronsat extremelylow concentrations,I0-12mor tess. Thesesame neurons display two different classesof neurotrophin receptors with dissodation constants of 10- 11M and 10- 9M, respective~,, implying that only low occupancy of the higher afh'nity receptor is required to medate the blb/ogical actions of the neurotrophins. Two structurally unrelated receptors have now been characterizedfor NG/:, and one of ther~ p75 N~R, servesas a receptor for a# the known neurotm~in~ This is the receptor with a dissociation constant of I0- M. The second NGF receptor is a member of the trk family of tyr~i=nekinasereceptors,p140 trk.4. Other members, p145 t~Band p145 trkC, are receptors for brain,derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) and neurotrophin.3 (NT.3), respe~'vely, when assayedin h'brob/asts. The spedhcity of neurotrophin binding to these receptors appears to be much higher in neurons than in the non.neuronal cells. The receptor p140~*Ahas many of the prope~es of the higher affinity cla~sof NGF receptors, and is able to mediate survival and differentiation of the PCI 2 cell line, and cell growth and transformation in h'broblastcells. On the other hand, expression of p75 NGFRin several types of cells displaying p140 ~ induces a component of higher a~nily NGF binding not seen in its absence. Since it is unlikely that p75 ~FR and pt40 ~ interact at the level of the receptors, the crosstalk between TINS, Vol. 15, No. 9, 1992

~

receptors probably occurs through their signal transduction mechan. isms. Characterizing the signal transduction mechanism of p75 NGFR and determining how this receptor influences the binding of NGF, especially the specificity of t~nding, to primary sensory neurons remains a considerable challenge in the held. Work on the first neurotrophin, nerve growth factor (NGF), has spanned 40 years since its initial discovery by Levi-Montatcini and Hamburger ~. Within the past few years the field has been greatly stimulated by several exciting developments.

SusanO. Meakinand EricM. Shooterareat the Deptof Neurobioto~, StanfordUnive~ty Schoolof Medidne, Stanford, CA943055401,USA.

(1) The discovery of a family of neurotrophins related to NGF, namely brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4) and neurotrophin-5 (NT-5) 2-~. (2) The characterization of two receptors for NGF, namely p75 NGFRand the proto-oncogene, p140 tr~ (Ref. 7), and of several members of the b/rosine kinase trk family, namely p145 trkB and p145 trkc. (3) The solution of the three-dimensional structure of NGF s. (4) The discovery that NGF can rescue appropriate adult neurons from injury or age-related

1992, Elsevier Science Publishers Ltd. (UK)

323

degeneration, raising the question of potential therapies for the neurodegeneration diseases9'1°. NGF is the prototypic neurotrophin that defines the properties and functions of this class of growth factors. NGF is synthesized and released from the target tissues of sympathetic neurons, a proportion of sensory neurons, and the cholinergic basal forebrain neurons in the CNS. In the periphery, the target tissues are typically non-neuronal cells, while in the CNS the targets are neurons 5. During development, a retrograde flow of NGF is established, transporting NGF from the target into the nerve terminal and up the axon to the cell body 11. Those neurons which establish this flow survive the period of neuronal cell death, while those that do not, rapidly degenerate. If the supply of NGF to the target is augmented some of the neurons which would normally die are rescued12. Once the retrograde flow of NGF is established it must continue for the lifetime of the neuron in order to develop and maintain the functional differentiated state of the neuron 13. In order to initiate the retrograde flow, NGF binds to its receptors on the nerve terminal, the receptors then cluster and are internalized in membrane-bound vesicles. The vesicles are transported along microtubules to deliver intact, biologically active NGF to the neuronal cell body 11. The interaction of NGF with its receptors also initiates signal transduction events that are the key to the mechanism of action of NGF. Whether signal transduction occurs only at the level of the nerve terminal membrane or also during transport of the NGF - NGF-receptor complexes remains an interesting question. While the paradigm that a neurotrophin is synthesized in the target and neurotrophin receptors are synthesized in the neuron might be accurate for NGF, it will need to be modified for other members of the family. BDNF mRNA has been detected in sensory neurons and NT-3 mRNA in sensory and motor neurons (Bothwell, M. A., unpublished observations), while both types of receptors appear in the targets 14. One current challenge in the NGF field is to understand how two structurally unrelated NGF receptors mediate the biological actions of this neurotrophin and this is the major topic of the review.

The pharmacologyof the neurotrophin receptors Two classes of NGF receptors were originally distinguished on chick embryonic sensory neurons by binding and kinetic measurements ~,~6. The major population of receptors, the class II receptors, have a Kd of 10-9M, while the minor population, the class I receptors, have a Kd of 10 -~1 M. These receptors are also known as the Iow-(LNGFR) and high-(HNGFR) affinity NGF receptors, respectively (Table I). While the rates of association of NGF to the two receptors are similar and fast, the rates of dissociation are markedly different, being relatively slow from the HNGFR (t~ of approximately lOmin) and fast from the LNGFR (t~ of approximately 3 s). This difference has given rise to another operational definition of the two receptors; slow (SNGFR) and 324

fast (FNGFR) rates of dissociation for the HNGFR and LNGFR, respectively 17. The dissociation constants measured from the ratio of the rates of dissociation and association agree well with those determined by steady-state binding. Both classes of receptors can be found in binding experiments at 37°C and O°C, indicating that the HNGFR binding component is not, in these neurons, a reflection of NGF internalization. Direct measurement of NGF internalization at 37°C confirms that it accounts for only a small percentage of the total binding 1~. The NGF receptors on chick embryo sympathetic neurons have the same properties as those on chick sensory neurons 18. Two classes of receptors are also present on chick sensory neurons for BDNF19 and for NT-3 (Ref. 20) and they display dissociation constants similar to those of the NGF receptors (Table I). While the absolute numbers of receptors vary for each neurotrophin, the number of HNGFR is always less by a factor of 4-10 than the number of LNGFR. Dissociation of BDNF or NT-3 from their HNGFR is also slower than from the LNGFR, although the differences are not as large as for the NGF receptors. For all three neurotrophins the concentrations required for half-maximal survival of, or neurite outgrowth from, the chick sensory neurons are significantly below the Kd of the high affinity receptors (Table I), suggesting that the biological responses of these neurotrophins are mediated by the HNGFR. Most importantly, the data show that occupancy of only a few hundred HNGFR (possibly reaching as high as a thousand receptors for NT-3) is required for a half-maximal biological response. This is especially striking in the case of NT-3 where positive cooperativity reduces NT-3 binding to its LNGFR to negligible levels at concentrations which support neuronal survival 2°. In contrast, the dissociation constants of the two classes of NGF receptors on rat pheochromocytoma (PC12) cells21 are nearly the same, with a value of approximately 10-9M (Refs 22, 23). For this reason PC12 cells show only one dose-dependent NGF displacement curve centered at approximately 10-9M, in contrast to chick sensory neurons which show two curves centered at approximately 10 -11 M and 10-9M, depending on the concentration of [~2Sl]NGF initially used to label the cells1~'24. As a result, induction of half-maximal neurite outgrowth from PC12 cells requires an NGF concentration (4 x 10-11M) higher than that required for neurite outgrowth from chick sensory neurons. These cells have approximately 50000 NGF receptors per cell, of which 5-10% are HNGFR. PC12 cells also internalize NGF efficiently at 37°C (Ref. 25). The slow rate of [12Sl]NGF release from these cells therefore comprises two components, one rep~2s resenting the actual release of [ I]NGFR from the HNGFR and the other the lysosomal degradation of the [1251]NGF and release of [1251]tyrosine. The finding that part of the NGF bound to PC12 cells at 0°C dissociates slowly at either 0°C or 37°C confirms the presence of HNGFR. The two classes of receptors differ in other ways. NGF bound to the LNGFR is rapidly released by mild trypsin treatment, presumably because of the rapid TINS, Vol. 15, No. 9, 1992

degradation of the NGF binding site. The binding site of the occupied HNGFR, on the other hand, is stable to trypsin treatment, although the receptor decreases in molecular weight by approximately 10000 (Ref. 26). The HNGFR is insoluble in Triton X-lO0 under specific conditions, while the LNGFR is soluble. However, both receptors can be solubitized by appropriate detergent treatment 27-29. Also, both receptors contain sialic acid residues which can be released by neuraminidase treatment without affecting NGF binding 3°.

The molecular properties of the two NGF receptors Crosslinking of [1251]NGF to rabbit sympathetic neurons with hydroxysuccinimidyl-4-azidobenzoate reveals two different sizes of NGF receptors 3~. Similar results were obtained with chick embryo sensory neurons and PC12 cells 26 and, indeed, a wide range of crosslinking agents identify the same two species27'29. Although it was originally thought that some crosslinking agents are specific for one or other NGF receptor, e.g., ethyldimethylaminopropyl carbodiimide (EDAC) for LNGFR, essentially any crosslinker will react with either receptor under appropriate conditions. In the most widely studied system, the PC12 cell, the two NGF-receptor complexes have molecular weights of 160000 and 100000, leading to estimates for the molecular weights of the receptors themselves of approximately 140000 and 80000, respectively. The high molecular weight complex contains the slow dissociating, trypsin-resistant receptor, and the low molecular weight complex contains the fast dissociating, trypsin-labile receptor. On this basis it was concluded that the larger complex contains the HNGFR, and the smaller complex the LNGFR26. The HNGFR on PC12 cells internalize NGF while the LNGFR do not 22'32. Several lines of evidence originally suggested that the HNGFR contains the LNGFR. First, the two receptors were found together on NGF-responsive cells, sensory and sympathetic neurons, and PC12 cells, and in approximately the same ratio. Given the doseresponse curves of NGF biological activity, it was thought that the only way the LNGFR could be involved was by being present in the HNGFR. Secondly, agents which cluster receptors, like the lectins (in particular, wheat germ agglutinin) and antibodies against the ligand, convert most of the receptors on PC12 cells to a form displaying the slow-dissociating, Triton X-lOO-insoluble state characteristics of HNGFR 33. In addition, the LNGFREDAC crosslinked complex was observed under conditions where NGF binding was only to the HNGFR 34. However, there is now a significant body of evidence to suggest that the HNGFR does not contain the LNGFR, but is a separate protein, p140 trkA. This evidence will be discussed in more detail below. The low affinity NGF receptor The availability of monoclonal antibodies against the human and rat LNGFR permitted cloning of these receptors by gene transfer and rescue by differential library screening 35'36. In the isolation of TINS, Vol. 15, No. 9, 1992

TABLE I. The characterization of NGF, BDNF and NT-3 receptors on chick embryonic sensory neurons by binding assays Property

NGF 2.3 X 10-11 1000-3000 1.7 x 10-9

BDNF 1.7 X 10-11 230 1.3 X 10-9

Kd of high affinity binding (M) Number of sites per neuron Kd of lOW affinity binding (M) Number of sites per neuron 23 0 0 0 - 4 5 000 3200 2 x 10-12 6.6 x 10-12 Concentration for halfmaximal survival or neurite outgrowth (M)

NT-3 1.8 x 10-11 11 000 0.8 x 10-9 39 000

~2 x 10-12

The data are taken from Refs 15, 19 and 20 for NGF, BDNF and NT-3, respectively. Binding assayswere performed at both 0°C and 37°C for NGF,and at 4°C for BDNFand NT-3. the rat LNGFR a stable L cell line, expressing approximately 2 x 106 LNGFR per cell, was isolated by fluorescence-activated cell sorting using a monoclonal antibody against the rat LNGFR (MC192) that actually increases NGF binding to this receptor 37. The LNGFR is a single peptide chain of approximately 400 amino acid residues, with a single membrane-spanning domain separating a slightly longer extracellular domain from a shorter cytoplasmic domain (Fig. 1). A comparison of the amino acid sequences of human, rat and chick LNGFR shows significant sequence identities in the four cysteine-rich regions of the extracellular domain, in the membrane-spanning domain and in the C-terminus in the cytoplasmic domain 38. The four cysteine-rich regions (loops) determine the NGFbinding domain 39, and each one of these loops is required and arranged in the correct order to create the NGF-binding site4°'41 . It is reasonable to assume that the six cysteine residues in each of these loops form three disulfide bridges within the loop. The conserved C-terminal region contains a single mastoparan-like domain, a consensus sequence for the binding of G proteins 42. Whether or not the LNGFRs have a G-protein-linked signal transduction mechanism remains to be seen. A soluble, truncated p75 LNGFR

p140 t~A

NOFf D O D

Tyrosine kinase 75 kDa 140 kDa G Protein ? Fig. 1. The NGF receptors. 325

FAS

TNFR-I

TNFR-II

p75 "GFR

CD40

OX40

F===,

i

i Fig. 2. The p75 NGI;R family of receptors. The double fine represents the membrane with the extracel/ular domain above and the intraceltular domain below. The boxes with rounded comers are the cysteine-dch re&ions with the cysteine residues indicated within the boxes by horizontal lines. The shaded boxes below the fines indicate regions of homology. (Reproduced, with permission, from Ref. 44.) form of LNGFR, which still binds NGF, is observed in conditioned media of cells that synthesize LNGFR and in various biological fluids 43. The cloned LNGFR has all the properties of the low affinity receptor as defined by binding and kinetic measurements. When expressed in L cells, it has a Kd of I0-9M, NGF dissociates from it rapidly and it is trypsinqabile. The molecular weight of the NGF-LNGFR complex is approximately 100000 (Ref. 36). Similar observations have been made of the human LNGFR3s. It has been renamed p75 NGFR based on the approximate molecular weight of the human receptor of 75 000. Although the rat receptor is slightly larger at approximately 80000 molecular weight, the same terminology will suffice to describe the receptors from both species. The p75 NGFRis a member of a family of related proteins, which includes the two receptors for tumor necrosis factor, TNFRI and TNFRII, and the Fas antigen (receptor) (Fig. 2). A characteristic feature of each of these receptors is the presence of three or four cysteine-rich extracellular regions, with cysteine residue placement similar to that seen in p75 N G F R . Interestingly, some of these receptors mediate cell death; the Fas antigen mediates apoptotic cell death in human cells44, while TNFRI and TNFRII play the same role in many cells. This similarity suggests that p75 NCFRdoes play a role in determining neuronal cell survival. Another extraordinary aspect of p75 NGFR has recently emerged. Using sensory neurons and the cell line PCNA, which expresses large numbers of p75 NGFR(Ref. 36), it was observed that the receptor binds not only NGF but also BDNF with the same Kd of approximately 10-9M (Ref. 45). However, significant differences in the observed association and 326

dissociation rates of NGF and BDNF to and from p75 NCFRindicate that either the three-dimensional structures of NGF and BDNF differ in detail or there are differences in the small conformational changes that might occur on the binding of the two ligands to p75 NcFR, or even that both are possible. These observations have been extended to NT.3 (Refs 20, 46, 47) and NT-4 (Ref. 4) with the same results. All these neurotrophins bind to p75 NGFRwith a Kd of approximately 10-9M, but all show differences in association and dissociation rates, and for two of them, BDNF and NT-3, the binding is positively cooperative 2°'4s. It is perhaps not surprising, given this information, that p75 NG~R has a broad distribution not only within the CNS but also in other tissues48. The high affinity NGF receptor

One of the most important advances of the past year was the demonstration by two groups 49-51 that the product of the proto-oncogene trk, p140trkA, is an NGF receptor (the designation trkA is used to distinguish it from other related receptors). In another study, antibodies to NGF were used to immunoprecipitate the HNGFR, and a search was made for other antibodies that would precipitate the HNGFR and its associated proteins 29. Interestingly, one of the antibodies which precipitated the HNGFR was an anti-phosphotyrosine antibody, suggesting that the HNGFR contains tyrosine kinase activity 2~. This was later confirmed by using the immunoprecipitates of the NGF-HNGFR complex to phosphorylate the artificial tyrosine kinase substrate, poly[(Glu)4(Tyr)l)] and to phosphorylate a protein of approximately 135000 molecular weight contained within the complex s2. The kinase activity is blocked by 5~-5-methyladenosine, an inhibitor that also blocks NGF-induced differentiation of PC12 cells but does not affect NGF binding to its receptorss2. The identification of p140trkA as an NG F receptor was made after it was noted that the distribution of p140trkA, first identified as an oncogene fusion protein with tropomyosin s3, was restricted to targets of NGF action in neural crestderived sensory neurons54. Indeed, p140trkA proved to be an NGF receptor whose tyrosine kinase activity and autophosphorytation is stimulated on binding NGF49'~°. The property of autophosphorylation was used to show expression of p140trkA on NGFresponsive neurons, such as dorsal root ganglion sensory neurons, the cell line PC12 and the neuroblastomas, SY5Y and LA-N-5, and on a 3T3 cell line transfected with a mouse-rat p140trkA cDNA hybrid 49, [1251]NGF,crosslinked to PC12 cells or the transfected 3T3 cell line, was also precipitated by anti-p140trkA antibodies 49. The second study was also prompted by the observed preferential distribution of p140trkA and other members of this family in neural tissuesss, and by the observation that HNGFR is immunoprecipitated with anti-phosphotyrosine antibodies 29. Specific NGF binding to human or mouse p140tr~ expressed in 3T3 cells was observed, as was the immunoprecipitation of the complex with anti-p140 t~kA antibodies and the autophosphorylation of p140trkA in PC12 cells5~. ~N5, VoL 1~No.~1992

Interaction of NGF with endogenous p140trl~ in PC12 cellss° or with exogenously expressed p140trkA in the Sf9 insect cells~1 significantly increases its kinase activity, demonstrating that p140trkA is almost certain to be important in the signal transduction events induced by NGF. In keeping with these observations, Radeke and Feinstein27 also demonstrated that a semi-purified fraction of the HNGFR contains a single peptide chain of approximately 135 000 Da. The crosslinking experiments and immunoprecipitation with anti-p140 trkA antibodies clearly identify the receptor in the 160000 molecular weight HNGFR complex as p140trkA (Refs 28, 49, 51). Does this mean that p140trkA is the biologically active NGF receptor? The p140trkA receptor has at least two of the other properties which define the HNGFR. NGF bound to the rat p140trkA, as expressed in COS cells, is trypsin-stable, although the molecular weight of the complex is slightly reduced28. NGF also dissociates slowly from the receptor expressed in COS cells, under conditions (0°C) where no internalization of NGF is observed. What about the dissociation constant of p140trkA? Interestingly, when expressed in 3T3 cells, the mouse-rat hybrid p140trkA has a Kd of approximately 10- 9 M (Ref. 49), while the rat p140trick expressedin COS cells also has a Kd of 10-9M (Meakin, S. et al., unpublished observations), both measured by steady-state binding. Although these receptors have been referred to as low-affinity NGF receptors49, it should be recalled that the HNGFR receptor on rat PC12 cells also has a Kd of 10-9M when measured by displacement binding. Therefore, it can equally well be argued by all the above criteria, that the rat p140trkA is the HNGFR. The mouse p140trkA expressed in 3T3 cells has a similar Kd to that of the rat p140trkA, but the data on the human p140trkA expressed in 3T3 cells are more complicated. Scatchard analysis of steadystate binding showed two populations of receptors, a major population with Kd in the range of 2.5-7.3 x 10-~M and a minor population (but still numbering 14000-31 000 receptors per cell) with Kd close to 10-1°M (Ref. 51). Klein et al. ~ suggested that the higher-affinity binding sites might be p140trkA dimers. While this suggestion requires formal proof, it is worth noting that several of the other single-peptide-chain tyrosine kinase receptors form dimers in a step that is essential for receptor activation by transphosphorylation, signal transduction and biological activity. In each instance a truncated receptor lacking a cytoplasmic domain was used to make non-functional heterodimers. The receptors include the epidermal growth factor receptorss6, the platelet-derived growth factor receptors57 and at least three of the fibroblast growth factor receptors~8. If p140tr~ fits with the same pattern then it could act, by itself, as the biologically active NGF receptor. Why dirnerization is not observed with the rat and mouse p140trkA is not yet clear. Furthermore, if only 2% of p140trkA receptors form high-affinity binding dimers sl, this would provide only a very low number of such receptors (2% of 230-11 000) on sensory neurons or PC12 cells. Crosslinking suggests that the potenTINS, Vol. 15, No. 9, 1992

cys-rich C ~ leu-rich

~, r,o,,O >

cys-nch

IgGC2

IgG'C2 0

IgGC2

IgGC2

cysr~ch ~ leu-rich

cys-nchu-nch~ le

cysrich

cys rich ~

'go-c~ o

,goc~ ~

IgG-C,,

D

C IgGV @ IgGv loG.C2

rl

Tyr kJnase

Tyr klnase

7yr kmase

kmase

p140

p145

p95

p145

p140

trk A

trk B

trk B

trk C

D trk

Fig. 3. The trk family of receptors. The various domains are described in the Figure. (Modified by Dr Moses Chao from Ref. 64.) tial for NGF-occupied p140tr~ to form dimers or higher oligomers is actually much higher than this29. Yet another finding which supports the idea that p140trkA is able to mediate NGF's actions by itself is the identification of the alkaloid-like compound K-252a, not only as an inhibitor of NGF-mediated neurite outgrowth ~9 and fibroblast proliferation 6°'61, but also as a potent and highly selective inhibitor of the protein kinase activity of p140trkA (and of the other members of this subfamily of receptors) at concentrations that do not inhibit other protein kinases in the receptor-mediated pathways 6°-62.

The trk family of receptors The p140try' receptor is, like p75 NGFR, a heavily glycosylated peptide chain with a single membrane. spanning domain 63 (Fig. 1). It is larger than p75 NGFR,containing some 790 amino acid residues (Fig. 3), and its cytoplasmic domain contains the tyrosine kinase activity that puts it in the larger family of tyrosine kinase receptors, which contains the insulin, epidermal growth factor (EGF) and fibroblast growth factor (FGF) receptors. Other members of the trk family are the products of the trkB and trkC genes, p145~rkB(Ref. 65) and p145 trkc (Ref. 66), respectively (Fig. 3). A number of differentially expressed trkB transcripts are found, one of which encodes a truncated form of the receptor, p95trkB, lacking a significant cytoplasmic domain 67. The p140trkA, p145trkB and p145trkC receptors show sequence similarities, especially in the tyrosine kinase domains. For example, p140trkA, shows 87% sequence identity to p145trkB in this domain and 57% sequence identity in the extracellular domain. The latter domains share several distinctive regions; the cysteine- or leucine-rich regions and the IgG-C2 regions as indicated in Fig. 3. A related gene, Dtrk, is found in Drosophila that is also,referentially expressed in the nervous system~. The encoded product, p140 turk, shares features with the other 327

:~::~ i!ii~!!~:!i:!!i~i:~;~; !~;;!i~i~ii;ii!i;~;;~!i~¸¸!!i;;!i~ ;; ~ J ~ J ~ U ~ f ~ 2 ~ ~ f ~ ; ~ ~ : ; ~ ; ~ ~ ~ ; ~ ; ~ mammalian receptors but also has regions characteristic of cell adhesion molecules (the four IgG-C2 and two IgG-V regions). The ligand-binding properties of these receptors have largely been defined with ectopically expressed mammalian (mouse, rat, human) receptors in non-neuronal cells. The assays involved measurement of growth (cell numbers or incorporation of labeled thymidine) or of transformation of several transfected NIH3T3 cell lines. NGF, for example, stimulates thymidine incorporation into a 3T3 cell line expressing p140 trkA, with a half-maximal effect at approximately 4 x 10-1°M, and reversible transformation of these cells in a semisolid medium 69. Displacement of 50% of the binding of labeled NGF from p140 trkA was observed at approximately 10-9M. NT-3 (but not BDNF) also binds to p140 trkA under these conditions with 50% displacement of labeled NGF at a concentration of approximately 4 × 10-9M. However, the displacement is not complete, suggesting overlapping but not identical binding sites for the two neurotrophins. The transforming activity of NT-3 is about 100-fold lower than that of NGF. The mitogenic response mediated by p140 trkA in these experiments occurs in the absence of p75 NGFR. Rat p145 trkB in heterologous 3T3 cells binds both BDNF and NT-3 with dissociation constants of 1.8 × 10-9M and 1.3 × 10-9M, respectively 7°. Half-maximal autophosphorylation of p140 trkB was observed with both ligands at concentrations of 2-4 × 10-9M. In contrast, Glass et a/.71 observed significant survival and proliferation of rat p145 trkBcontaining fibroblasts at 5 × 10-'~M BDNF, a concentration similar to that which promotes halfmaximal survival of BDNF-responsive chick embryonic sensory neurons. Interestingly, BDNFinduced phosphorylation of these same p145 trkB receptors required 10- to 100-fold higher BDNF concentrations. In the survival and proliferation assays NT-3 was about 10-fold less active 46'71, and in a transformation assay about 100-fold less active, than BDNF72. Xenopus and human NT-4 (Refs 72, 73) and NT-5 (Ref. 73) also bind to p145 trkB, and in a 3T3 cell transformation assay with cotransfected p140 trkB and Xenopus NT-4, the latter is only fivefold less active than BDNF. This suggests that mammalian NT-4 may be comparable to mammalian BDNF in its activity on p145 trkB . Mutation of a single cysteine residue in p140 trkB (residue 345 in the second IgG-C2 domain) to serine abolishes the transformation activity of NT-3 and NT-4 but not of BDNF, suggesting at least two different ligandbinding domains may exist in this receptor 72. NT-3 appears to be the major ligand for p145 trkc (Ref. 66). By either steady-state binding or displacement binding, mouse p145 trkC expressedin 3T3 cells shows a Kd of 2-4 X 10-9M. AS was observed for p~/~v A~trkA , about 2% of the steady-state binding of p145 trkc is of higher affinity (2.6 x 10 -11 M, 3000 receptors per celt). Whether this high affinity binding results from the oligomerization of p145 trkc remains to be seen. NT-3 also induces proliferation and transformation of 3T3 cells in the absence of p75 NGFR(Ref. 66). 328

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The general conclusion from these studies in non~v and p145 trkB, but neuronal cells is that p_4A~trkA probably not p145 trkc, show overlapping specificities in the binding of the neurotrophins. Moreover, where quantitative binding studies have been carried out, all three receptors display dissociation constants around 10 -9 M. As noted before, by itself this information does not allow a distinction between high-affinity (biologically active) and lowaffinity receptors. The exceptions are the finding of a small percentage of a high-affinity binding component (approximately 10-1°-10 -11 M) in the Scatchard analysis of p140 trkA and p145 trkc binding s1'72, and the very low concentration of BDNF (5 x 10 -12 M) required to promote survival of the p145trkB-expressing FGF-dependent 3T3 cells71. When these data are compared to the binding characteristics of chick embryonic sensory neurons the biggest difference is seen in the specificity of binding. As noted before, the actual high-affinity binding sites for NGF, BDNF and NT-3 in these neurons are highly specific for the particular ligand, requiring two to three orders of magnitude higher concentration of either of the other two ligands to displace the specific ligand. It is reasonable to assume from the fact that NGF, BDNF and NT-3 are retrogradely transported to the sensory neurons in the dorsal root ganglia TM that the three receptors are expressed on the neurons (perhaps on overlapping but also different populations of neurons) and mediate the high-affinity binding. This means that p140 trkA , p145 trkB and p145 trkC show a much higher specificity of binding in sensory neurons than in fibroblasts. An obvious difference between these two cell types is the presence of p75 NGFR in the neurons, suggesting that p75 NGFRis responsible, in some way, for creating this higher specificity. While the tyrosine kinase signal transduction mechanisms of the trk receptors may be sufficient to mediate the biological effects of the neurotrophins, p75 NGFRmay directly or indirectly influence this mechanism. What role does p75 NGFRplay in NGF's actions? One other fact is also clear from the study of the trk receptors in fibroblasts. The p140 trkA , p145 trkB and p145 trkc receptors can mediate growth and transformation of these cells in the absence of p75 NGFR. Does this also apply to neurons or neuronal cell lines? PC12 cells do not normally respond to BDNF and it is now clear that they lack p145 trkB. Occupation of p75 NGFRby BDNF on these cells, therefore, is not sufficient to induce differentiation. Transient transfection of PC12 cells with p145 trkB makes them responsive to BDNF and NT-3 (Ref. 46). Similarly, transfection of p140 trkA into a variant non-NGFresponsive PC12 cell lacking p140 trkA restores its ability to survive and initiate neurite outgrowth when treated with NGF (Ref. 75). While these last two experiments implicate the trk receptors in the biological response they do not preclude a role for p75 NGFR. A different approach using mutants of NGF has been taken to remove this qualification. The tryptophan at amino acid residue 21 in NGF can be replaced by either leucine or phenylalanine TINS, Vol. 15, No. 9, 1992

.......................................................................................................................................................................... .......................................................................................... •......................................................................... without any effect on specific biological activity, as measured by neurite outgrowth 76. However, the affinity of binding of these two mutant NGFs to p75 NGFR on either PC12 cells or the L cell line expressing only p75 NGFRis reduced 16-fold for the leucine mutant and 30-fold for the phenylalanine mutant. Binding of NGF to p75 NGFR does not, therefore, contribute to biological activity. The same conclusion was reached in an even more definitive manner with a triple mutant of NGF in which three adjacent residues (lysine 32, lysine 34 and glutamic acid 35) were mutated to alanine residues77. This mutant displays less than 1% of the binding to p75 NGFR, compared to wild type NGF, but still retains 65% of the specific biological activity of NGF and, importantly, binds to p140 trkA (expressed in NIH 3T3 cells) with only a twofold reduction in affinity. If p75 NGFRis involved in the biological activity of NGF it would probably have to interact with p140 trkA in some way. Several lines of evidence suggest that such an interaction is unlikely to be at the level of the receptors themselves. For example, three different antibodies against p75 NGFR, which recognize three different epitopes, immunoprecipitate p75 NGFR but do not co-precipitate p140 trkA from PC12 cells29 . Correspondingly, anti-p140 trkA antibodies do not co-precipitate p75 NGFR(Refs 29, 49). Although both p75 NGFR and p140 trkA form dimers or higher oligomers that can be precipitated by anti-p75 NGFR and anti-p140 trkA antibodies, respectively, none of these higher molecular weight complexes is recognized by both antibodies 28. Another antibody against the extracellular domain of p75 NGFR inhibited NGF binding to p75 NGFR but still permitted NGF-induced neurite outgrowth 78. Furthermore, under conditions where NGF binding is restricted to the biologically active HNGFR, as defined pharmacologically, only an NGF-p140 trkA complex is observed after crosslinking with a variety of agents26 '27 . If p75 NGFR and p140 trkA interact physically in the mechanism of action of NGF, then a crosslinked NGF-p75 NGFR complex should be observed under these conditions even if the NGFp140trkA--p75 NGFR complex is tOO rare to observe. The failure to observe this complex argues against the interaction of the two receptors, although it could be argued that NGF crosslinks more efficiently to p140 trkA. On the other hand, there are a number of observations which suggest that p75 NGFR and p140 trkA can influence each other. The NR18 cell line, a variant of PC12, lacks p75 NGFR and shows one component of NGF binding by Scatchard analysis. Transfection of p75 NGFR into these cells generates a second component of binding of higher affinity than the original binding, and NGF now induces c-fos expression in these cells, a reasonable indication of an NGF-incluced biological response 79. In an extension of this experiment it was observed that when NR18 cell membranes are fused with fibroblast membranes expressing p75 NGFR, several hundred receptors with a Kd of 2.5 X 10 -11M are generated 8 0 . Both experiments suggest that p75 NGFR interacts with a second NGF receptor, TINS, Vol. 15, No. 9, 1992

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presumably p140 trkA, in the NR18 cell to generate a high-affinity NGF binding component. The interesting point about these results is that not only are p140 tr~ levels in NR18 cells very low 8°, but so is the number of high-affinity binding sites that are generated. However, as noted earlier, NGF needs only to interact with a small number of high-affinity receptors to mediate its biological activities. The cotransfection of p75 NGFRand p140 trkA in COS cells also results in the generation of NGF receptors (approximately 3000 per cell) with a Kd of 3 x 10-11M against a background of p75 NGFR receptors with a Kd of 5 X 10-9M. Transfection of either receptor alone results in receptors with a Kd of approximately 10-9M (Ref. 80). Finally, transfection of p140 trkA into a human melanoma cell line expressing very high numbers of p75 NGFR also results in approximately 3000 receptors per cell, with a Kd of 3 x 10 -11M (Ref. 80). This number of receptors is again similar to the number of highaffinity receptors found on neurons. The ability of p75 NGFRto create a higher-affinity NGF binding component suggests that this receptor does participate in a signal transduction mechanism. This has been confirmed in a key experiment using a chimeric receptor81. PC12 cells possess EGF receptors and respond to EGF by dividing. When these cells are transfected with a chimeric receptor comprising the extracellular domain of the EGF receptor and the membrane-spanning and cytoplasmic domain of p75 NGFR they now respond to EGF with neurite outgrowth and the induction of transin mRNA, an NGF-responsive gene. Clearly, the activation of the cytoplasmic domain of p75 NGFR activates a signalling mechanism which is dependent on an intact membrane-spanning domain from p75 NGFR.Similar conclusions can be drawn from the fact. that p75 NGFR introduced into the NR18 cell line re-establishes NGF-induced phosphorylation of certain intracellular proteins while two cytoplasmic domain deletion mutants of p75 NGFR do not 82 . One potential signal transduction mechanism for p75 NGFR was mentioned above, namely activation of a G protein. If this, or some other mechanism, is activated by the binding of NGF to p75 NGFRthen it is possible that it could lead to post-translational modification of p140 trkA (or of p75 NGFRitself) and to a modification of its ability to bind NGF. The latter may arise from small conformational changes in the receptor or from changes in its ability to form what may be the biologically active p140 trkA dimer. Finally, it is likely that the receptors alone can mediate functions other than neuronal survival or differentiation. In this regard, two examples can be mentioned. Schwann cells in peripheral nerve, when activated after injury, express relatively large numbers of p75 NGFR(the presence of one or other of the trk receptors is not yet determined) which may serve a direct role in Schwann cell function 83. Also, the repression of p75 NGFRsynthesis in developing kidney with anti-sense oligonucleotides inhibits the differentiation of the kidney tubules 84. The distribution of both p75 NGFRand the trk receptors in nonneuronal tissues suggests that this list is likely to be enlarged in the near future. 329

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Concludingremarks The evidence to date shows that p140 trkA can mediate the biological activities of NGF in fibroblasts (cell growth and transformation) and the PC12 cell line (cell survival and neurite outgrowth) in the absence of p75 NGFR. This conclusion extends to the other neurotrophins in their interactions with p140 trkA, p145 trkB and p145 trkc. Furthermore, halfmaximal biological responses require only relatively low occupancy of a p140 trkA receptor (or other trk receptors) with a Kd of approximately 10-9M. The binding studies on chick embryonic sensory neurons also lead to the same conclusion, although this time the high-affinity receptors have dissociation constants of approximately 10 -11 M. Does this mean that chick p140 trkA, unlike rat p140 trkA, has a Kd of 10 -11 M? This measurement has not yet been made, but if it turned out to be true it would support the idea that p140 trkA is the biologically active NGF receptor on chick sensory neurons, and also would explain why the dose-response curve for NGF actions on PC12 cells lies at significantly higher concentrations than that for sensory neurons. A second point that needs to be cleared up is whether p140 trkA forms dimers in order to initiate the signal transduction mechanism and whether this changes the affinity of binding. This can readily be determined using a truncated p140 trkA receptor as a dominant-negative mutation, as was done for the other tyrosine kinase receptors. It should, however, be stressed that the appropriate system for these measurements is a cell that expresses the low number of high-affinity p140 trkA receptors shown in Table I; clearly embryonic chick sensory neurons themselves would be an ideal system. The alternative viewpoint, that both p140 trkA and p75 NGFRare needed to produce a biologically active NGF receptor, is based largely on Scatchard analysis of NGF binding to membranes or cells expressing one or both receptors. The original paper in this seriesTM showed that a high-affinity binding site of Kd approximately 10-11M could be created in rat PC12 cell membranes. The puzzle about this conclusion is that one would expect PC12 cells to show the same NGF dose-response curve as chick sensory neurons and they don't. On the other hand, if chick p140 trkA also has a Kd of 10-9M then there would be evidence, in both chick and rat, that the interaction of two receptors, both with dissociation constants of 10-9M, generates a binding component with a Kd of 10 -11M. To resolve this issue will require either a demonstration that the two receptors interact (and the evidence against this event is summarized above) or the characterization of the putative post-translational modifications of the two receptors induced by each other that could change receptor affinities. The evidence that crosstalk between the receptors determines the specificity of neurotrophin-binding in neurons is rather compelling at the moment. This idea could be tested in 3T3 cells by seeing if the broad specificity of p145 trks, for example, is narrowed when it is coexpressed with p75 NGFR . However, such an experiment may fail in 3T3 cells if the tyrosine kinase substrates are different, or react differently, in 3T3 330

cells compared to neurons. A key piece of information which is still missing is whether or not p75 NGFR takes advantage of its mastoparan-like domain to interact with G proteins to generate a signal transduction mechanism as suggested by the results from the EGFR-p75 NcFR chimeric receptor. Finally, the specific roles of both types of receptors will probably only emerge in their entirety as a result of gene overexpression or knockout experiments in transgenic animals, or with the antisense methodologies. Given the importance of the neurotrophins in nervous system development, such studies should not be too long in forthcoming. Selected references 1 Levi-Montalcini,R. (1987) EMBOJ. 6, 1145-1154 2 Hohn, A., Leibrock, J., Bailey, K. and Barde, Y-A. (1990) Nature 344, 339-341 3 Maisonpierre,P. C. et al. (1990) Science 247, 1446-1451 4 Hallb66k,F., Ib~fiez, C. F. and Persson,H. (1991) Neuron 6, 845-858 5 Thoenen,H. (1991) Trends Neurosci. 14, 165-170 6 Berkemeier,L. R. etal. (1991) Neuron 7, 857-866 7 Bothwell,M. (1991) Ce//65, 915-918 8 MacDonald,N. Q. etaL (1991) Nature 354, 411-414 9 Gage, F. H., Armstrong, D. M., Williams, L. R. and Varon, S. (1988) J. Comp. Neurol. 269, 147-155 10 Heffi, F., Hartikka,J. and Knusel,B. (1989) Neurobiol. Aging 10, 515-533 11 Thoenen, H. and Barde, Y-A. (1980) Physiol. Rev. 60, 1284-1334 12 Hamburger,V., Brunso-Bechtold,J. K. and Yip, J, W. (1981) J. Neurosci. 1, 60-71 13 Barde,Y-A. (1989) Neuron 2, 1525-1524 14 Wyatt, S., Shooter,E. M. and Davies,A. M. (1990) Neuron 4, 421-427 15 Sutter,A., Riopelle,R. J., Harris-Warwick,R. M. and Shooter, E. M. (1979) J. Biol. Chem. 254, 5972-5982 16 Sutter, A. L. (1979)in Transmembrane 5iEnallin& (Bilensky, M., Collier, R. J., Steiner, D. F, and Fox, C. F., eds), pp. 659-667, Alan R. Liss 17 Schechter,A. L. and Bothwell,M. A. (1981) Ce1124,867-874 18 Godfrey, E. W. and Shooter, E, M. (1986) J. Neurosci. 6, 2543-2550 19 Rodriguez-T4bar,A. and Barde, Y-A. (1988) ]. Neurosci. 8, 3337-3342 20 Rodriguez-T~bar,A., Dechant,G., Gotz, R. and Barde, Y-A. (1992) EMBO J. 11, 917-922 21 Greene,L. A. and Tischler,A. S. (1976) Proc. NatlAcad, 5ci. USA 73, 2424-2428 22 Bernd, P. and Greene, L. A. (1984) J. Biol. Chem. 259, 15509-15516 23 Woodruff, N. R, and Neet, K. E. (1986) Biochemistry 25, 7956-7966 24 Cohen, P., Sutter, A., Landreth, G., Zimmerman, A. and Shooter, E. M. (1980)J. Biol. Chem. 255, 2949-2954 25 Layer, P, and Shooter, E. M. (1983) J. Biol. Chem. 258, 3012-3018 26 Hosang, M. and Shooter, E. M. (1985) J. Biol. Chem. 260, 655-662 27 Radeke,M. J. and Feinstein,S. (1991) Neuron 7, 141-150 28 Meakin, S. O., Suter, U., Drinkwater, C. C., Welcher, A. A. and Shooter, E. M. (1992) Proc. Natl Acad. 5ci. USA 89, 2374-2378 29 Meakin,S. O. and Shooter,E. M. (1991) Neuron 6, 153-163 30 Vale, R. D., Ignatius, M. J. and Shooter, E. M. (1985) J, Neurosd. 5, 2762-2770 31 Massague,J., Guillette,B. J., Czech,M. P., Morgan, C. J. and Bradshaw, R. A. (1981) J. Biol. Chem. 256, 9419-9424 32 Hosang, M. and Shooter, E. M. (1987) ENIBO J. 6, 1197-1202 33 Vale, R. D. and Shooter, E. M. (1983) Biochemistry 22, 5022-5028 34 Greene,S. H. and Greene, L. A. (1986) J. Biol. Chem. 261, 15316-15326 35 Johnson,D. etal. (1986) Ce1147, 545-554 TINS, VoL 15, No. 9, 1992

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36 Radeke, M. J., Misko, T. P., Hsu, C., Herzenberg, L. A. and Shooter, E. M. (1987) Nature 325, 593-597 37 Chandler, C. E., Parsons, L. M., Hosang, M. and Shooter, E. M. (1984) J. Biol. Chem. 259, 6882-6889 38 Large, T. H. etal. (1989) Neuron 2, 1123-1134 39 Welcher, A. A., Bitler, C. M., Radeke, M. J. and Shooter, E. M. (1991) Proc. Natl Acad. Sci. USA 88, 159-163 40 Baldwin, A. M., Bitler, C. M., Welcher, A. W. and Shooter, E M. (1992) J. Biol. Chem. 267, 8352-8359 41 Yan, H. and Chao, M. V. (1991) J. Biol. Chem. 266, 12099-121O4 42 Feinstein, D. L. and Larhammer, D. (1990) FEBS Lett. 272, 7-11 43 DiStephano, P. S. and Johnson, E. M., Jr (1988) Proc, Natl Acad. Sci. USA 85, 270--274 44 Itoh, N. etal. (1991) Ce1166, 223-243 45 Rodriguez-T~bar, A., Dechant, G. and Barde, Y-A. (1990) Neuron 4, 487-492 46 Squinto, S. P. etaL (1991) Ce1165, 885--893 47 Ernfors, P., Ib&fiez, C. F., Ebendal, T., Olson, L. and Persson, H. (1990) Proc. Natl Acad. Sci. USA 87, 5454-5458 48 Ernfors, P. et al. (1988) Neuron 1,983-991 49 Kaplan, D. R., Hernpstead, B. L., Martin-Zanca, D., Chao, M. V. and Parada, L. F. (1991) Science 252, 554-558 50 Kaplan, D. R., Martin-Zanca, D. and Parada, L. F. (1991) Nature 350, 158--160 51 Klein, R., Jing, S., Nanduri, V., O'Rourke, E. and Barbacid, M. (1991) Cell 65, 189-197 52 Meakin, S. O. and Shooter, E. M. (1991) Proc. NatlAcad. Sci. USA 88, 5862-5866 53 Martin-Zanca, D., Hughes, S. H. and Barbacid, M. (1986) Nature 319, 743-748 54 Martin-Zanca, D., Barbacid, M. and Parada, L. (1989) Genes Dev. 4, 683-694 55 Barbacid, M., Lambelle, F., Pulido, D. and Klein, R. (1991) Biochim. Biophys. Acta 1072, 115-127 56 Kashles, O., Yarden, Y., Fischer, R., UIIrich, A. and Schlessinger, J. (1991)Mol. Cell. Biol. 11, 1454-1463 57 Ueno, H., Colbert, H., Escobedo, J. A. and Williams, L. T. (1991 ) Science 252, 844-848 58 Ueno, H., Gunn, M., Dell, K., Tseng, A., Jr and Williams, L. (1992) J. Biol. Chem. 267, 1470-1476

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59 Kiozumi, S. et aL (1988)J. Neurosci. 8, 715-721 60 Tapely, P., Lamballe, F. and Barbacid, M. (1992) Onco&ene 7, 371-381 61 Nye, S.H. et aL MoL Ceil. BioL (in press) 62 Berg, M. M., Sternberg, D. W., Parada, L. F. and Chao, M. V. (1991) J. Biol. Chem. 267, 13-16 63 Martin-Zanca, D. R., Oskan, R., Mitra, G., Copeland, T. and Barbacid, M. (1989) MoL Cell. Biol. 9, 24-33 64 Schneider, L. and Schweiger, S. (1991) Oncogene 6, 1807-1817 65 Klein, R., Parada, L. F., Coulier, F. and Barbacid, M. (1989) ENIBO J. 8, 3701-3709 66 Lamballe, F., Klein, R. and Barbacid, M. (1991) Ceil 66, 967-979 67 Klein, R., Conway, D., Parada, L F. and Barbacid, M. (1990) Cell 61,647-656 68 Pulido, D., Campuzano, S., Koda, T., Modollel, J. and Barbacid, M. (1992) EMBO J. 11,391-404 69 Cordon-Cardo, C. et al. (1991) Cell 66, 173-183 70 Soppet, D. et aL (1991) Cell 65, 895-903 71 Glass., D. J. etaL (1991) Ce1166, 405-413 72 Klein, R., Lambelle, F., Bryant, S. and Barbacid, M. (1992) Neuron 8, 1-20 73 Ip, N. Y. et al. (1992) Proc. Natl Acad. Sci. USA 89, 3060-3064 74 DiStephano, P. S. et aL (1992) Neuron 8, 983-993 75 Loeb, D. M. etal. (1991) Ceil66, 961-966 76 Drinkwater, C., Suter, U., Angst, C. and Shooter, E. M. (1992) Proc. R. Soc. Lond. Ser. B 246, 307-313 77 Ib~.hez, C. F. etaL (1992) Cell69, 1-20 78 Weskamp, G. and Reichardt, L. F. (1991) Neuron 6, 649663 79 Hempstead, B. L., Schleifer, L. S. and Chao, M. V. (1989) Science 243,373-375 80 Hempstead, B. L., Martin-Zanca, D., Kaplan, D. R., Parada, L. F. and Chao, M. V. (1991) Nature 350, 678683 81 Yan, H., Schtessinger, J. and Chao, M. V. (1991) Science252, 561-563 82 Berg, M., Sterrnberg, D., Hempstead, B. and Chao, M. (1991) Proc. Natl Acad. Sci. USA 88, 7106-7110 83 Taniuchi, M. H., Clark, H. B. and Johnson, E. M., Jr (1986) Proc. Nat/Acad. Sci. USA 83, 4094-4098 84 Sariola, H. etal. (1991)Science 254, 571-573

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Acknowled&ements The authors thank our colleaguesat Stanford for valuable discussionsand the National Institute of Neurological Disordersand Stroke for support of original work describedin this review. E M. Shooter is Chairmanof the ScientificAdvisory Boardand a Director of Regeneron Pharmaceuticals.

Vive la difference! Karen J. Berkley Hormonal effects are increasingly recognized as important influences on neuronal function and, ultimately, on animal behavior. Such 'hi&her' behavioral effects are well studied, particularly in relation to sexually dimorphic behaviors. Yet, somewhat surprisingly, a si&niflcant proportion of more basic neuroscience research papers fail to specify the sex of the subjects used. In this brief article Karen Berkley argues that knowledge of, and controlling for, the sex of research animals is important. In addition, if females are used, their reproductive,cyde status could provide a deliberate strategy to invesbgate the effects of gonadal steroid hormones on biological functions. Consider a fictitious paper, published in a reputable, peer-reviewed journal, in which the authors present the results of an extensive and elegant study describing the effects of pharmacological manipulations of opioid receptors on the electrophysiological and genetic responses of CNS neurons to noxious cutaneous stimuli. The results are compared with the effects of these same manipulations on the behavioral responses of the conscious animal to similar stimuli. The w o r k was carried out, say the authors, on '63 Sprague-Dawley rats, of 2 2 5 - 3 2 3 g'. TINS, Vol. 15, No. 9, 1992

W h a t ' s w r o n g with this paper? O n the face of it, nothing. But, on reflection, it becomes evident that, although the article contains data potentially important for the treatment and understanding of pain, it is missing something important - information a b o u t the sex of the rats. In a survey of 100 articles, published recently in four reputable, peer-reviewed neuroscience journals from North America and Europe, while virtually all of the articles duly described the species and n u m b e r of subjects used, an astounding 4 5 % failed to state the sex of the subjects (Table I). W h y is this issue important? The reason is simple. With the recent explosion of molecular and cellular biology techniques into the field of neuroscience, it has become increasingly apparent that many, and maybe all, aspects of normal and, importantly, pathological neural functions are s o m e h o w influenced by, or directly involve, gonadal steroid hormones 1. From gene activation to axonal transport 2-4, from membrane receptors to neurotransmitters 3'5-7, from peripheral nerves to

© 1992, ElsevierSciencePublishersLtd,(UK)

KarenJ. Berkleyis at the Dept of Psychology, Florida State University, Tallahassee, FL 323O6-I051, USA.

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The nerve growth factor family of receptors.

The neurotrophins, of which nerve growth factor (NGF) is the best known example, support the survival and differentiation of chick embryo sensory neur...
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