Cell, Vol. 65, 915-916, June
14, 1991,
Copyright 0
1991 by Cell Press
Keeping Track of Neurotrophin Receptors Mark Bothwell Department of Physiology and Biophysics University of Washington Seattle, Washington 98195
In the manner of many other growth factors, the prototypical neurotrophic factor, nerve growth factor (NGF), has recently been found to be a member of a small gene family encoding structurally and functionally related proteins, collectively referred to as neurotrophins. Individual neurotrophins exert similar functional effects, but on different neuronal populations. Each of the neurotrophins described to date-NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin3 (NT-3)-interacts with cellsurface receptors that are heterogeneous with regard to binding affinity. For NGF and BDNF, analysis of equilibrium binding data indicates the existence of two receptor populations with dissociation constants of about IO-” M (“high affinity”) and 10e9 M (“low affinity”) (Sutter et al., 1979; Rodriguez-TBbar and Barde, 1988). The low affinity receptor binds NGF, BDNF, and NT-3 with similar affinity, while high affinity receptors exist that bind either NGF or BDNF selectively (Rodriguez-TBbar et al., 1990; Squint0 et al., 1991). Functional response to aneurotrophin apparently is mediated specifically by the high affinity receptors. Rodriguez-TBbar et al. have proposed that there is a shared component of neurotrophin receptors, known as ~75, with the specificity of each receptor determined by a second component. A variety of studies have shown that low and high affinity NGF receptor forms are interconvertible. The interrelationshipsof thevarious neurotrophin receptors and of their high and low affinity forms have now been described. The emerging story has profound implications for neurobiology and quite probably for the biology of nonneural tissues also. The first neurotrophin receptor molecular clones isolated (Johnson et al., 1986; Radeke et al., 1987) encode a 75-80 kd intrinsic membrane protein (~75) that binds NGF, BDNF, and NT-3 with comparable low affinity (Kd of 10d9 M) when expressed in fibroblastic cells (RodriguezT&bar et al., 1990; Squintoet al., 1991). ~75 has a relatively small cytoplasmic domain containing none of the structural motifs known to function in signal transduction in other receptors. Yet, transfection of ~75 cDNA clones into appropriate neuronal cell lines generates both high and low affinity NGF binding and renders these cells functionally responsive to NGF with regard to gene induction and enhanced neurite outgrowth (e.g., Hempstead et al., 1989). The response of such cell lines is weak in comparison with the response of primary neuronal cultures and the rat PC12 neuronal cell line, which expresses endogenous high affinity NGF receptors. The meager cellular response of transfected neuronal cell lines expressing ~7.5 may be
Minireview
ascribed, at least in part, to the shortcomings of the cell lines. This interpretation is supported by experiments employing acDNAencoding achimeric receptor consisting of the ligand-binding domain of the epidermal growth factor (EGF) receptor linked to the juxtamembrane domain and cytoplasmic domain of ~75. While endogenously encoded EGF receptors promote proliferation of normal PC1 2 cells, cells transfected with the chimeric receptor construct respond to EGF by neuronal differentiation in a manner that is nearly identical to their response to NGF (Yan et al., 1991). However, while ~75 may be capable of mediating responses to NGF, it is not clear that it is essential for NGF response. Weskamp and Reichardt (1991) demonstrated that polyclonal antibodies against ~75 have no effect on NGF responsiveness of neuronal cells, yet these antibodies effectively block all NGF binding on low affinity (& of 10e9 M) sites as well as a portion of binding to high affinity sites. These investigators concluded that NGF response is mediated by a subpopulation of high affinity receptors that are antigenically distinct from ~75. Earlier studies provided the first clues concerning the identity of another NGF receptor. Carbodiimide-induced chemical cross-linking of “51-labeled NGF to receptors on PC12 cells reveals a 100 kd complex comprised of ~75 linked to NGF (Grob and Bothwell, 1983). Evidence has been presented that indicates that this 100 kd complex is derived from high affinity as well as low affinity binding sites (Green and Greene, 1986). However, application of other cross-linkers yields, in addition to the 100 kd crosslinked species, a 160 kd complex derived exclusively from the high affinity component of NGF binding (Hosang and Shooter, 1985). While some investigators concluded that the 160 kd NGF-receptor complex might consist of a ternary complex of NGF, ~75, and a third protein of 60-70 kd, Radeke and Feinstein (1991) utilized a reversible cross-linker to demonstrate that the 160 kd complex consists of NGF and a 135 kd protein that is unrelated to ~75. ~75 lacks tyrosine kinase activity, the hallmark of many other growth factor receptors; nevertheless, NGF stimulates protein tyrosine phosphorylation in responsive cells (Maher, 1988), and the 160 kd cross-linked NGF-receptor complex contains phosphotyrosine (Meakin and Shooter, 1991a). Furthermore, following incubation of PC12 cells with NGF, antibodies against NGF immunoprecipitate a 135 kd protein with tyrosine kinase activity (Meakin and Shooter, 1991 b). The identity of this 135 kd protein did not remain obscure for very long. Martin-Zanca et al. (1989) reported that the human trk proto-oncogene encodes a 140 kd tyrosine kinase (pl 40trk) with the structural characteristics of a growth factor receptor. In situ hybridization in mouse embryos revealed that the trk gene is expressed prominently in sensory neurons of spinal ganglia and in the portion of sensory neurons of cranial ganglia that originates from the neural crest (Martin-Zanca et al., 1990). As these cells are principal targets
Cell 916
of NGF action, several laboratories were led to investigate whether ~140”~ represents the 140 kd NGF receptor protein, with affirmative results. NGF specifically stimulates tyrosine autophosphorylation of ~140”~ in PC1 2 cells, in spinal ganglia sensory neurons, and in NIH 3T3 cells transfected with trk cDNA. In addition, antibodies against ~140”~ immunoprecipitate the 160 kd cross-linked NGF-receptor complex from a variety of cell types including PC12 cells, human neuroblastoma ceil lines, and NIH 3T3 cells expressing ~140”~ (Kaplan et al., 1991a, 1991 b; Klein et al., 1991). These results indicate that ~140” is a component of the high affinity NGF receptor. While there is agreement among various investigators on these points, there is disagreement concerning the manner in which high affinity receptors are generated from ~140”. One group of investigators (Kaplan et al., 1991 b; Hempstead et al., 1991) found that expression of either pl 40wkor ~75 individually in NIH 3T3 cells yields cell membranes that bind NGF with only low affinity (Kd of 10eg M), while fusion of membranes containing ~140” with membranes containing ~75 generated a binding profile with both high and low affinity components (Kds of 3 x 10-l’ M and 1 x 10eg M), such as is normally seen in membranes of neuronal cells and cell lines. Also, transient coexpression of ~75 and ~140’~~ in several different cell lines yields both high and low affinity binding, while expression of ~75 or ~140’~~ individually gives only low affinity binding. Furthermore, the NR18 cell line, which is capable of a functional response to NGF only after transfection with p75 cDNA (Hempstead et al., 1989), expresses endogenous frk mRNA, yet possesses high affinity NGF binding only after transfection with ~75. Thus, they concluded that the functional high affinity NGF receptor represents a complex of ~75 and p140”, while the individual components each bind NGF with low affinity. However, Klein et al. (1991) found that expression of pl 40trk in NIH 3T3 cells, in the absence of ~75 expression, is sufficient to generate both higher and lower affinity NGF binding. Employing binding to whole cells (at reduced temperatures to block endocytosis), between 4% and 8% of receptor sites have a Kd of 1 x lo-lo M (compared with 4 x lo-” M for PC12 cells in parallel experiments); the majority of sites have a Kd in the range of 2.5 x 1 O-9 M to 7 x 10m9 M (compared with 1 x 10mg M for PC12 cells). They concluded that ~140’~~ by itself constitutes the high affinity NGF receptor with no requirement for ~75. In this model it is not clear why only a few percent of ~140’~~ sites bind NGF with high affinity, since cross-linking experiments in PC12 cells indicate that NGF binding to this protein is exclusively of high affinity (Hosang and Shooter, 1985). Klein et al. suggested that the affinity of ~140’~~ in NIH 3T3 cells is reduced by some form of posttranslational modification. Observation of transcripts hybridizing weakly to trk cDNA probes led to the isolation of cDNA clones of trk5. trkt3 encodes a transmembrane tyrosine kinase protein (~140~~~~) closely related in structure to ~140” and also expressed predominantly in the nervous system (Klein et al., 1990; Middlemas et al., 1991). Spurred by the reason-
able prediction that receptors for BDNF and NT-3 should be structurally related to the receptor for NGF, two groups of investigators have shown that ~140”~ constitutes a receptor for both BDNF and NT-3 (Squint0 et al., 1991; Soppet et al., 1991). p140fks expressed in COS cells or NIH 3T3 cells binds both BDNF and NT-3, but did not bind NGF, as assessed both by chemical cross-linking studies and by equilibrium binding analysis. Furthermore, BDNF and NT-3 stimulate ~140 frkBtyrosine phosphorylation, while NGF does not. Competition binding experiments led Squint0 et al. to suggest that binding of BDNF and NT-3 to p140” may be of higher affinity than binding to ~75. However, more extensive equilibrium binding studies of Soppet et al. indicated that binding of BDNF and NT-3 to p140tika is exclusively low affinity (Kd of about 2 x lo-’ M). Thus, the manner in which high affinity receptors for NGF, BDNF, or NT-3 might be generated from ~140” and pl 4otrkB is controversial. Several studies have examined the functional consequences of expression of p140trk and ~140’~~~. Squint0 et al. employed transient transfection to express p140f”‘@ in PC12 cells that do not express endogenous p140Vks and do not respond to BDNF or NT-3. Following transfection with ~140’~‘~, a subpopulation of PC12 cells extended neurites in response to BDNF and NT-3 in a manner that was similar to, but more robust than, that obtained with NGF. Thus, it seems clear that pl 40tfk8can function as a receptor for either BDNF or NT-3. These experiments do not shed light on the question of whether p75 is required as a component of functional receptors for BDNF and NT-3, since endogenous ~75 is present in the PC12 cells employed. Injection of frk mRNA causes Xenopus oocytes to undergo meotic maturation in response to nanomolar concentrations of NGF (Nebreda et al., 1991). Since it is not known whether Xenopus oocytes express a homolog of ~75, again these results do not reveal whether ~75 is required for a functional response. Expression of ~75 mRNA alone in Xenopus oocytes does not induce maturation but does promote the induction of maturation by progesterone (Sehgal et al., 1988), supporting the concept that the functions of ~75 and ~140”~ are somehow related. The ability of ~140”~~~ to mediate responses to both BDNF and NT-3 is surprising, since several neuronal populations are differentially responsive to BDNF and NT-3. It is possible that specific cellular environments or, possibly, interaction with ~75 alters the specificity of pl 40wm. Alternatively, additional neurotrophin receptors may exist that have greater specificity; it is reasonable to expect that these may also be structurally related to ~140” and pl 40trkB. In summary, while it is now clear that rrkand f&B encode functional receptors for neurotrophins, the role of ~75 with regard to trk and trkB remains unclear. Two alternative models still seem viable. In the first model, high affinity neurotrophin receptors consist of ~140” or ~140~~~~acting alone or in conjunction with cell proteins that remain to be identified. In a second model depicted in the figure, ~75, p140”, and p140VkB individually give low affinity receptors, while association of p75 with pl 40Nk or pl 40irkB generates a high affinity receptor.
discriminate between BDNF and NT-3? No matter what the ultimate resolution of these issues may be, the identication of members of the trk gene family as neurotrophin receptors opens many new avenues for investigation. One wonders how many neurotrophins and how may trk-related neurotrophin receptors there may be, and how many neuronal populations may be subject to regulation by this system. Furthermore, there have been a handful of reports indicating that NGF is a mitogen for some cell types. The discovery that neurotrophin receptors are tyrosine kinases with potential for stimulating cell proliferation, as well as neuronal differentiation, suggests that the possible function of neurotrophins as physiological regulators of proliferation of nonneuronal cells should be more thoroughly explored.
3.NGF 0.
BDNF, NT-3
KD = 1 O-’ M
Ml
f!;:
KD= IO-”
M
References Green, 15326. Model for the Generation of High Affinity Association of p75 with ~140”” or p140”8
Neurotrophin
Binding
by
S. H., and Greene,
Grob, P., and Bothwell, 6819-6828. Hempstead, 243,373-375.
It may be argued that the failure of antibodies that block NGF binding to p75 to eliminate high affinity binding of NGF (Weskamp and Reichart, 1991) is inconsistent with the second model. However, if high affinity neurotrophin binding results from occupation of a site in a sterically crowded cleft lying between p75 and ~140, as shown in the figure, then immunoglobulins recognizing an epitope near the neurotrophin binding site of ~75 might be prevented from reacting when p75 is associated with ~140”~. The involvement of p75 in some capacity in neurotrophin signal transduction seems certain, since p75 is expressed in every neuronal cell type that is known to respond to either NGF, BDNF, or NT-3. However, if p75 does interact with ~140, the association must be quite labile, since immunoprecipitation of p75 from detergent extracts of PC12 cells does not coprecipitate ~140” (Meakin and Shooter, 1991 b). What will be necessary to settle these issues definitively? First, it must be noted that stable transfected cell lines coexpressing p75 and pl 40Rk or pl 40pkB cDNAs have not yet been generated. Careful comparison of the neurotrophin-binding properties of such cells with those of cells expressing p75 and ~140 individually would be useful. Also, it must be noted that Scatchard analysis of equilibrium binding data is a black art, subject to a host of artifacts. The two classes of neurotrophin receptor affinity are more easily distinguished on the basis of differing kinetic rate constants than they are from analysis of equilibrium binding data. Kinetic analysis comparing p75 and ~140 binding properties has not yet been reported. Finally, these questions can only be definitively resolved by examining a functional end point. Does coexpression of p75 with trk or trk6 proteins reduce the concentration of neurotrophin required to generate a functional response? Does expression of p75 enhance the ability of trk to discriminate between NGF and NT-3, or the ability of trkB to
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Squinto, S. P., Stitt, T. N., Aldrich, T. H., Davis, S., Bianco, S. M., Radziewski, C., Glass, D. J., Masiakowski, P., Furth, M. E., Valenzuela, D. M., DiStefano, P. S., and Yancopoulos, G. D. (1991). Cell 6.5, 885-893. Sutter, (1979).
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25.2,561-
14, 1991,
Copyright 0
1991 by Cell Press
Keeping Track of Neurotrophin Receptors Mark Bothwell Department of Physiology and Biophysics University of Washington Seattle, Washington 98195
In the manner of many other growth factors, the prototypical neurotrophic factor, nerve growth factor (NGF), has recently been found to be a member of a small gene family encoding structurally and functionally related proteins, collectively referred to as neurotrophins. Individual neurotrophins exert similar functional effects, but on different neuronal populations. Each of the neurotrophins described to date-NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin3 (NT-3)-interacts with cellsurface receptors that are heterogeneous with regard to binding affinity. For NGF and BDNF, analysis of equilibrium binding data indicates the existence of two receptor populations with dissociation constants of about IO-” M (“high affinity”) and 10e9 M (“low affinity”) (Sutter et al., 1979; Rodriguez-TBbar and Barde, 1988). The low affinity receptor binds NGF, BDNF, and NT-3 with similar affinity, while high affinity receptors exist that bind either NGF or BDNF selectively (Rodriguez-TBbar et al., 1990; Squint0 et al., 1991). Functional response to aneurotrophin apparently is mediated specifically by the high affinity receptors. Rodriguez-TBbar et al. have proposed that there is a shared component of neurotrophin receptors, known as ~75, with the specificity of each receptor determined by a second component. A variety of studies have shown that low and high affinity NGF receptor forms are interconvertible. The interrelationshipsof thevarious neurotrophin receptors and of their high and low affinity forms have now been described. The emerging story has profound implications for neurobiology and quite probably for the biology of nonneural tissues also. The first neurotrophin receptor molecular clones isolated (Johnson et al., 1986; Radeke et al., 1987) encode a 75-80 kd intrinsic membrane protein (~75) that binds NGF, BDNF, and NT-3 with comparable low affinity (Kd of 10d9 M) when expressed in fibroblastic cells (RodriguezT&bar et al., 1990; Squintoet al., 1991). ~75 has a relatively small cytoplasmic domain containing none of the structural motifs known to function in signal transduction in other receptors. Yet, transfection of ~75 cDNA clones into appropriate neuronal cell lines generates both high and low affinity NGF binding and renders these cells functionally responsive to NGF with regard to gene induction and enhanced neurite outgrowth (e.g., Hempstead et al., 1989). The response of such cell lines is weak in comparison with the response of primary neuronal cultures and the rat PC12 neuronal cell line, which expresses endogenous high affinity NGF receptors. The meager cellular response of transfected neuronal cell lines expressing ~7.5 may be
Minireview
ascribed, at least in part, to the shortcomings of the cell lines. This interpretation is supported by experiments employing acDNAencoding achimeric receptor consisting of the ligand-binding domain of the epidermal growth factor (EGF) receptor linked to the juxtamembrane domain and cytoplasmic domain of ~75. While endogenously encoded EGF receptors promote proliferation of normal PC1 2 cells, cells transfected with the chimeric receptor construct respond to EGF by neuronal differentiation in a manner that is nearly identical to their response to NGF (Yan et al., 1991). However, while ~75 may be capable of mediating responses to NGF, it is not clear that it is essential for NGF response. Weskamp and Reichardt (1991) demonstrated that polyclonal antibodies against ~75 have no effect on NGF responsiveness of neuronal cells, yet these antibodies effectively block all NGF binding on low affinity (& of 10e9 M) sites as well as a portion of binding to high affinity sites. These investigators concluded that NGF response is mediated by a subpopulation of high affinity receptors that are antigenically distinct from ~75. Earlier studies provided the first clues concerning the identity of another NGF receptor. Carbodiimide-induced chemical cross-linking of “51-labeled NGF to receptors on PC12 cells reveals a 100 kd complex comprised of ~75 linked to NGF (Grob and Bothwell, 1983). Evidence has been presented that indicates that this 100 kd complex is derived from high affinity as well as low affinity binding sites (Green and Greene, 1986). However, application of other cross-linkers yields, in addition to the 100 kd crosslinked species, a 160 kd complex derived exclusively from the high affinity component of NGF binding (Hosang and Shooter, 1985). While some investigators concluded that the 160 kd NGF-receptor complex might consist of a ternary complex of NGF, ~75, and a third protein of 60-70 kd, Radeke and Feinstein (1991) utilized a reversible cross-linker to demonstrate that the 160 kd complex consists of NGF and a 135 kd protein that is unrelated to ~75. ~75 lacks tyrosine kinase activity, the hallmark of many other growth factor receptors; nevertheless, NGF stimulates protein tyrosine phosphorylation in responsive cells (Maher, 1988), and the 160 kd cross-linked NGF-receptor complex contains phosphotyrosine (Meakin and Shooter, 1991a). Furthermore, following incubation of PC12 cells with NGF, antibodies against NGF immunoprecipitate a 135 kd protein with tyrosine kinase activity (Meakin and Shooter, 1991 b). The identity of this 135 kd protein did not remain obscure for very long. Martin-Zanca et al. (1989) reported that the human trk proto-oncogene encodes a 140 kd tyrosine kinase (pl 40trk) with the structural characteristics of a growth factor receptor. In situ hybridization in mouse embryos revealed that the trk gene is expressed prominently in sensory neurons of spinal ganglia and in the portion of sensory neurons of cranial ganglia that originates from the neural crest (Martin-Zanca et al., 1990). As these cells are principal targets
Cell 916
of NGF action, several laboratories were led to investigate whether ~140”~ represents the 140 kd NGF receptor protein, with affirmative results. NGF specifically stimulates tyrosine autophosphorylation of ~140”~ in PC1 2 cells, in spinal ganglia sensory neurons, and in NIH 3T3 cells transfected with trk cDNA. In addition, antibodies against ~140”~ immunoprecipitate the 160 kd cross-linked NGF-receptor complex from a variety of cell types including PC12 cells, human neuroblastoma ceil lines, and NIH 3T3 cells expressing ~140”~ (Kaplan et al., 1991a, 1991 b; Klein et al., 1991). These results indicate that ~140” is a component of the high affinity NGF receptor. While there is agreement among various investigators on these points, there is disagreement concerning the manner in which high affinity receptors are generated from ~140”. One group of investigators (Kaplan et al., 1991 b; Hempstead et al., 1991) found that expression of either pl 40wkor ~75 individually in NIH 3T3 cells yields cell membranes that bind NGF with only low affinity (Kd of 10eg M), while fusion of membranes containing ~140” with membranes containing ~75 generated a binding profile with both high and low affinity components (Kds of 3 x 10-l’ M and 1 x 10eg M), such as is normally seen in membranes of neuronal cells and cell lines. Also, transient coexpression of ~75 and ~140’~~ in several different cell lines yields both high and low affinity binding, while expression of ~75 or ~140’~~ individually gives only low affinity binding. Furthermore, the NR18 cell line, which is capable of a functional response to NGF only after transfection with p75 cDNA (Hempstead et al., 1989), expresses endogenous frk mRNA, yet possesses high affinity NGF binding only after transfection with ~75. Thus, they concluded that the functional high affinity NGF receptor represents a complex of ~75 and p140”, while the individual components each bind NGF with low affinity. However, Klein et al. (1991) found that expression of pl 40trk in NIH 3T3 cells, in the absence of ~75 expression, is sufficient to generate both higher and lower affinity NGF binding. Employing binding to whole cells (at reduced temperatures to block endocytosis), between 4% and 8% of receptor sites have a Kd of 1 x lo-lo M (compared with 4 x lo-” M for PC12 cells in parallel experiments); the majority of sites have a Kd in the range of 2.5 x 1 O-9 M to 7 x 10m9 M (compared with 1 x 10mg M for PC12 cells). They concluded that ~140’~~ by itself constitutes the high affinity NGF receptor with no requirement for ~75. In this model it is not clear why only a few percent of ~140’~~ sites bind NGF with high affinity, since cross-linking experiments in PC12 cells indicate that NGF binding to this protein is exclusively of high affinity (Hosang and Shooter, 1985). Klein et al. suggested that the affinity of ~140’~~ in NIH 3T3 cells is reduced by some form of posttranslational modification. Observation of transcripts hybridizing weakly to trk cDNA probes led to the isolation of cDNA clones of trk5. trkt3 encodes a transmembrane tyrosine kinase protein (~140~~~~) closely related in structure to ~140” and also expressed predominantly in the nervous system (Klein et al., 1990; Middlemas et al., 1991). Spurred by the reason-
able prediction that receptors for BDNF and NT-3 should be structurally related to the receptor for NGF, two groups of investigators have shown that ~140”~ constitutes a receptor for both BDNF and NT-3 (Squint0 et al., 1991; Soppet et al., 1991). p140fks expressed in COS cells or NIH 3T3 cells binds both BDNF and NT-3, but did not bind NGF, as assessed both by chemical cross-linking studies and by equilibrium binding analysis. Furthermore, BDNF and NT-3 stimulate ~140 frkBtyrosine phosphorylation, while NGF does not. Competition binding experiments led Squint0 et al. to suggest that binding of BDNF and NT-3 to p140” may be of higher affinity than binding to ~75. However, more extensive equilibrium binding studies of Soppet et al. indicated that binding of BDNF and NT-3 to p140tika is exclusively low affinity (Kd of about 2 x lo-’ M). Thus, the manner in which high affinity receptors for NGF, BDNF, or NT-3 might be generated from ~140” and pl 4otrkB is controversial. Several studies have examined the functional consequences of expression of p140trk and ~140’~~~. Squint0 et al. employed transient transfection to express p140f”‘@ in PC12 cells that do not express endogenous p140Vks and do not respond to BDNF or NT-3. Following transfection with ~140’~‘~, a subpopulation of PC12 cells extended neurites in response to BDNF and NT-3 in a manner that was similar to, but more robust than, that obtained with NGF. Thus, it seems clear that pl 40tfk8can function as a receptor for either BDNF or NT-3. These experiments do not shed light on the question of whether p75 is required as a component of functional receptors for BDNF and NT-3, since endogenous ~75 is present in the PC12 cells employed. Injection of frk mRNA causes Xenopus oocytes to undergo meotic maturation in response to nanomolar concentrations of NGF (Nebreda et al., 1991). Since it is not known whether Xenopus oocytes express a homolog of ~75, again these results do not reveal whether ~75 is required for a functional response. Expression of ~75 mRNA alone in Xenopus oocytes does not induce maturation but does promote the induction of maturation by progesterone (Sehgal et al., 1988), supporting the concept that the functions of ~75 and ~140”~ are somehow related. The ability of ~140”~~~ to mediate responses to both BDNF and NT-3 is surprising, since several neuronal populations are differentially responsive to BDNF and NT-3. It is possible that specific cellular environments or, possibly, interaction with ~75 alters the specificity of pl 40wm. Alternatively, additional neurotrophin receptors may exist that have greater specificity; it is reasonable to expect that these may also be structurally related to ~140” and pl 40trkB. In summary, while it is now clear that rrkand f&B encode functional receptors for neurotrophins, the role of ~75 with regard to trk and trkB remains unclear. Two alternative models still seem viable. In the first model, high affinity neurotrophin receptors consist of ~140” or ~140~~~~acting alone or in conjunction with cell proteins that remain to be identified. In a second model depicted in the figure, ~75, p140”, and p140VkB individually give low affinity receptors, while association of p75 with pl 40Nk or pl 40irkB generates a high affinity receptor.
discriminate between BDNF and NT-3? No matter what the ultimate resolution of these issues may be, the identication of members of the trk gene family as neurotrophin receptors opens many new avenues for investigation. One wonders how many neurotrophins and how may trk-related neurotrophin receptors there may be, and how many neuronal populations may be subject to regulation by this system. Furthermore, there have been a handful of reports indicating that NGF is a mitogen for some cell types. The discovery that neurotrophin receptors are tyrosine kinases with potential for stimulating cell proliferation, as well as neuronal differentiation, suggests that the possible function of neurotrophins as physiological regulators of proliferation of nonneuronal cells should be more thoroughly explored.
3.NGF 0.
BDNF, NT-3
KD = 1 O-’ M
Ml
f!;:
KD= IO-”
M
References Green, 15326. Model for the Generation of High Affinity Association of p75 with ~140”” or p140”8
Neurotrophin
Binding
by
S. H., and Greene,
Grob, P., and Bothwell, 6819-6828. Hempstead, 243,373-375.
It may be argued that the failure of antibodies that block NGF binding to p75 to eliminate high affinity binding of NGF (Weskamp and Reichart, 1991) is inconsistent with the second model. However, if high affinity neurotrophin binding results from occupation of a site in a sterically crowded cleft lying between p75 and ~140, as shown in the figure, then immunoglobulins recognizing an epitope near the neurotrophin binding site of ~75 might be prevented from reacting when p75 is associated with ~140”~. The involvement of p75 in some capacity in neurotrophin signal transduction seems certain, since p75 is expressed in every neuronal cell type that is known to respond to either NGF, BDNF, or NT-3. However, if p75 does interact with ~140, the association must be quite labile, since immunoprecipitation of p75 from detergent extracts of PC12 cells does not coprecipitate ~140” (Meakin and Shooter, 1991 b). What will be necessary to settle these issues definitively? First, it must be noted that stable transfected cell lines coexpressing p75 and pl 40Rk or pl 40pkB cDNAs have not yet been generated. Careful comparison of the neurotrophin-binding properties of such cells with those of cells expressing p75 and ~140 individually would be useful. Also, it must be noted that Scatchard analysis of equilibrium binding data is a black art, subject to a host of artifacts. The two classes of neurotrophin receptor affinity are more easily distinguished on the basis of differing kinetic rate constants than they are from analysis of equilibrium binding data. Kinetic analysis comparing p75 and ~140 binding properties has not yet been reported. Finally, these questions can only be definitively resolved by examining a functional end point. Does coexpression of p75 with trk or trk6 proteins reduce the concentration of neurotrophin required to generate a functional response? Does expression of p75 enhance the ability of trk to discriminate between NGF and NT-3, or the ability of trkB to
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