|
|
|
|||||||
|
|||||||||
From the The neurotrophins represent a family of survival and
differentiation factors that exert profound effects in the central and
peripheral nervous systems(1) . Five neurotrophins, NGF, ( Responsiveness of neurons to a given neurotrophin is governed by the
expression of two classes of cell surface receptors(7) . For
NGF, these are p75 We focus here
on recent progress in defining 1) neurotrophin protein structure; 2)
the molecular determinants of neurotrophins important for receptor
interaction; and 3) functional properties of neurotrophins determined
from gene targeting experiments. For more comprehensive and detailed
reviews of these and other aspects of neurotrophin function the reader
is referred elsewhere (7, 23, 24, 25) .
Neurotrophin Structure and the Cystine Knot Superfamily The first view of a neurotrophin molecule has been provided
by the structure of NGF. The dimeric NGF possesses a novel tertiary
fold that results in a rather asymmetric molecule with dimensions of 60
Figure 1:
Three-dimensional representation of
NGF. A, the NGF dimer with each subunit colored differently
for clarity. B, the tertiary fold for the NGF subunit.
Toward the opposite end of the protomer, the three disulfide bridges
are clustered and may provide rigidity to this portion of the
structure. The topological arrangement of the disulfides is quite
unusual (Fig. 1B). Two of the disulfide
bridges and their connecting residues form a ring structure
(Cys Each neurotrophin gene encodes a mature sequence
of approximately 120 amino acids, rich in basic
residues(3, 30, 31, 32, 33, 34, 35) .
Of the 24 neurotrophin sequences currently available, only 28 residues
are invariant(24) . From the structure of NGF it is evident
that the majority of these residues play key structural roles either
within the subunit core or the dimer interface, while the remainder
display positive Quite unexpectedly, the NGF tertiary fold
and ``cystine knot'' motif have been identified in structures
of transforming growth factor
Each Neurotrophin-Receptor Interaction Is Distinctive As the earliest event in signal transduction by the
neurotrophins is the interaction with p75 Three vertebrate trk receptor genes have been isolated,
including numerous variants of trk structure(48) .
Each neurotrophin binds preferentially to a specific trk but
also possesses some ability to interact with other trk family
members(7) . BDNF and NT-4/5, the most closely related
neurotrophins from phylogenetic analysis, are preferred ligands for trkB(49) , but NT-3 and NT-4/5 can also bind to trkA receptors (8, 31) . Two important
questions are raised. What are the amino acid requirements for
neurotrophin binding to each receptor? Second, how are the apparent trk specificities encoded within the structure of each
neurotrophin? To address these questions, chemical and genetic
modification approaches have been used to probe specific amino acid
residues, either through the replacement of contiguous stretches of
amino acids or by altering groups of residues that are chemically or
spatially related (24) . The biochemical and biological
consequences of these changes to NGF have been assessed by measuring
receptor binding affinities, trk autophosphorylation, neurite
outgrowth, and neuronal survival. While some losses in bioactivity have
been detected, other mutations have led to new
specificities(16, 50, 51) . These approaches
have led to an emerging picture of key receptor binding determinants on
the NGF molecule. Mutation of three lysine side chains
(Lys It is noteworthy that
these three lysine residues in NGF are not conserved among all
neurotrophins. However, modeling of each neurotrophin suggests the
preservation of a positively charged surface in a topologically similar
region throughout the family (52) (Fig. 1A).
Substitution of residues contributing to each of these positive patches
to alanine has indicated a role in binding each neurotrophin to
p75(51, 52) . Small local structural differences could
account for the distinct association and dissociation rates exhibited
by NGF, BDNF, and NT-3 for p75(10) . Alternatively, it is
possible that additional neurotrophin-specific residues may also make
contributions (51) . Intriguingly, very few single point
mutations to alanine within NGF perturb binding to trk A,
suggesting that this interaction involves a large number of
contacts(16, 24, 51) . Analysis of N-terminal
truncations of NGF(53) , site-specific NGF
mutants(54) , and novel chimeric neurotrophins (16, 55) has established the importance of the N
terminus of NGF in binding to trkA. Also, the C-terminal
residues 112-118 of NGF may play a less specific role in binding trkA(16, 56) . In contrast, analogous
experiments using chimeric BDNF and NT-3 neurotrophins demonstrate that
their N termini are not essential for binding to their respective trks(16, 51) . Other contacts between NGF
and trkA are present in the Based upon the common structural
features of neurotrophins, the trk receptor kinase family, and
the interrelatedness of p75 and the p55 TNF receptor(58) ,
speculative models for p75 and trkA receptors can be proposed (Fig. 2) from the known three-dimensional structures of related
modular sequences. It is generally assumed that the intrinsic symmetry
of NGF is used to cluster individual receptors leading to receptor
dimerization and, in the case of trk, subsequent activation
via an autophosphorylation mechanism(13, 15) .
Dimerization of trk may require the extracellular
immunoglobulin-like domains, which are also necessary for NGF
binding(59, 60) . From the known biochemical and
binding properties, it is plausible that homo- and heterodimeric
complexes may be formed by neurotrophin factors or by either receptor
type.
Figure 2:
Hypothetical model for neurotrophin
receptor structures, illustrating both the relative size of the ligand
and receptor components using ``borrowed'' structures for the
various receptor domains(75) . Both receptors are shown as
monomers in their ligand-free state but form specific homodimers in the
presence of ligand(13, 15) . The p75 model was built
based on the structure of the TNF p55 receptor (58) with the
four repeated domains highlighted. Sequence analysis of the trk ectodomain has revealed a modular mosaic extracellular
portion(76) . Known structures of relatives of these modules
were used to construct a model of trkA, including 1) the
leucine-rich repeat, LLR(79) ; 2) two consecutive C2
IgG domains; and 3) the intracellular domain of the insulin receptor
tyrosine kinase domain (78) . This figure was generated using
SETOR(79) .
Interactions between trk and p75 Receptors Receptor-mediated signal transduction for the neurotrophins
is unique among polypeptide growth factors since two different
trans-membrane proteins exist for each neurotrophin. In addition to trk family members, the p75 receptor is a member of the family
of receptors represented by TNF, CD40, Fas, and CD40. From Scatchard
analyses, two classes of NGF binding sites exist on the surface of
responsive neurons(61) . These sites differ 100-fold in
equilibrium binding constants, which can be further distinguished by
the rates of ligand dissociation(62) . The proteins responsible
for the high affinity NGF binding site have been the subject of
considerable debate, since p75 and trkA each exhibit
predominately low affinity binding and a small percentage of high
affinity sites has been detected for trkA(13) . The
contribution of each receptor type to the high affinity NGF binding
site has been clarified by kinetic analysis(63) . Whereas p75
displays fast rates of association and dissociation with NGF, trkA interacts with much slower on and off rates. Due to its unusually
slow on rate, NGF binding to trkA results in a K When trkA and
p75 receptors are co-expressed, the on rate is accelerated 25-fold,
creating a new kinetic site whose features are consistent with the high
affinity NGF binding site (K In addition to binding,
signal transduction by trkA can be influenced by
p75(20, 21, 22, 66) . Cell culture
experiments indicate that p75 is capable of enhancing trkA autophosphorylation(21, 22) . A potential
function of the p75 receptor may be to increase the effective
concentration of neurotrophin at the cell surface in order to enhance trk binding(21) . Another model is that an altered
conformation of trk may be formed in the presence of p75,
which facilitates ligand binding and subsequent signaling functions (65) . Direct interactions between p75 and trk receptors have been difficult to document biochemically. However,
immunoprecipitation experiments carried out in cross-linked spinal cord
and brain tissues with NGF binding to p75 can cause the
hydrolysis of sphingomyelin to ceramide(18) , raising the
possibility that signaling through p75 could occur independently of trk. Changes in lipid composition may be generated through p75
to create a different membrane environment for trk receptors.
Finally, apoptosis may be promoted by action of a ligand-independent
form of p75 (19) during specific developmental and
physiological conditions. This activity is supported by similarities in
the C-terminal domains of p75 with the Fas antigen and the p55 TNF
receptor(69) , which are both capable of mediating cell death.
Consequences of Neurotrophin Ablation A powerful genetic approach to the study of ligand-receptor
interactions has been to analyze mouse mutations created by homologous
recombination. Although germ line mutation of many vertebrate genetic
loci does not necessarily lead to a detectable phenotype, inactivation
of the three mammalian trk genes and their ligands results in
considerable neuronal cell loss in mutant mice (Table 2). These
significant cell losses provide a striking verification of the
biological effects of each member of the NT family and the binding
specificities of each neurotrophin.
Several general conclusions can
be made regarding null mice in which the expression of neurotrophins or
their receptors has been eliminated. First, there is a striking
agreement in the phenotypes displayed by NGF, BDNF, and NT-3 mutant
mice and homozygous mutations in trkA, trkB, and trkC (Table 2). The defects in each case correlate with
the cellular populations known to express each neurotrophin and its
cognate receptors(25, 48) . Second, targeted
disruptions of the trk receptor genes lead to a slightly more
severe phenotype than ligand mutations, measured by the extent of cell
death and the target populations that are affected. This difference in
phenotype suggests that individual neurotrophin ligands may be more
dispensable than individual receptors and emphasizes the ability of
multiple neurotrophins to interact with a single receptor. Finally,
survival of cells in the central nervous system (CNS) in null mutant
mice appears to be relatively unaffected compared with the peripheral
nervous system (PNS). Despite overall structural similarities,
neurotrophins do not display the same effects with each trk receptor or with the p75 receptor. NT-3, for example, can bind to
all three trk receptors, but gives different trophic
responses(8, 70) . This behavior may reflect the
ability of neurotrophins to bind multiple trk receptors by
using different receptor binding sites. The divergent dependency of
p75-deficient neurons toward NGF, BDNF, and NT-3 in culture (71) may be attributed to the subtle differences in the binding
properties of each neurotrophin for p75, largely governed by their
electrostatic surfaces (Fig. 1C). The rates of
dissociation are known to be markedly different, with NGF > NT-3
> BDNF(10) . These differences may also account for the
differential transport of NGF, BDNF, and NT-4/5 in sensory
neurons(72) . Another important criteria is the ratio of p75 to trk family members, which may dictate the degree of
responsiveness to individual
neurotrophins(22, 65, 70) . It is conceivable
that the binding properties exhibited for p75 and trk receptors may result in distinctive consequences in downstream
biological responses. Five decades of research on NGF have led to the discovery of
a small family of evolutionarily conserved proteins, which have vital
functions in the survival and neuronal development of specific neuronal
populations. The generation of mice lacking neurotrophin expression has
recapitulated classic experiments using anti-NGF antibodies to dissect
the physiological effects of trophic factor deprivation(73) .
Very similar outcomes resulted from both the NGF immunodepletion
experiments and the transgenic mouse experiments. The genetic results
also verify the structural predictions made from binding results in
heterologous cells. The findings in cell culture and animal
experiments clearly indicate the efficacy of neurotrophic factors for
promoting the survival of prominent neuronal populations such as
sensory and motor neurons. The high degree of conservation of
neurotrophin structure is accompanied by a surprising variation in the
amino acid contacts used by each neurotrophin with p75 and the trk receptor family members. It is this variation that may provide
specificity for each ligand-receptor complex. The future challenge will
be to make use of this knowledge to design effective therapeutic
strategies to treat neurodegeneration and nerve injury.
Volume 270,
Number 34,
Issue of August 25, pp. 19669-19672, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
INTRODUCTION
Neurotrophin Structure and the Cystine Knot Superfamily
Each Neurotrophin-Receptor Interaction Is Distinctive
Interactions between trk and p75 Receptors
Consequences of Neurotrophin Ablation
Summary
FOOTNOTES
REFERENCES
)BDNF, NT-3, NT-4/NT-5, and NT-6, associate as non-covalent
homodimers in their biologically active form (2, 3) .
These target-derived trophic factors are active on distinct sets of
embryonic neurons, whose dependence is in some cases restricted in
duration(4) . The neurotrophins are currently under
investigation as therapeutic agents for the treatment of
neurodegenerative disorders and nerve injury, either individually or in
combination with other trophic factors such as ciliary neurotrophic
factor or fibroblast growth factor (5, 6) . (p75) and p140
(referred to as trk or trkA) receptor
tyrosine kinase. The related trkB receptor tyrosine kinase
binds both BDNF and NT-4/5(8) , and the trkC receptor
binds only NT-3(9) , while p75 is able to interact with each
neurotrophin with equal affinity, though with different rate
constants(10) . After binding ligand, the neurotrophin-receptor
complex is internalized and retrogradely transported in the axon to the
soma(11) . Both receptors undergo ligand-induced dimerization (12, 13) that activates multiple signal transduction
pathways. These include the ras-dependent pathway utilized by trk(14, 15) to mediate neurotrophin effects
such as survival and differentiation(16, 17) , while
p75 activates less well understood pathways (18, 19) and can modulate trk activity(20, 21, 22) .
25
30 Å (26) (Fig. 1A).
Each NGF subunit is dominated by two pairs of anti-parallel
-strands that contribute to the molecule's flat, elongated
shape (Fig. 1B,
-
). These -strands are
connected at one end of the protomer by three short loops. These loops
are known to be highly flexible from the variability of their
conformations observed within independently determined structures of
NGF in different crystalline environments(26, 27) .
-Strands are labeled according to (26) . The three
disulfide bridges are drawn as whitesticks with green indicating the sulfur- atom. C,
electrostatic potential at the surface of NGF and various modeled
neurotrophins (adapted from (74) ). Blue indicates
positive potential and red indicates negative. This figure was
made using GRASP(75) .
-Cys
,
Cys
-Cys
) through which the third
disulfide bridge (Cys
-Cys
) passes to
form a tightly packed ``cystine knot'' motif(28) . Fig. 1A shows the parallel orientation of the NGF
subunits. This arrangement allows the two pairs of
-strands from
each protomer to pack against each other, generating an extensive
subunit interface. The interface has a largely hydrophobic character
comprised primarily of aromatic residues, consistent with the tight
association constant (10M) measured for
NGF(29) .
angles or are involved in hydrogen bonding or
salt bridge interactions. The preservation of these features indicates
that the neurotrophins will adopt similar conformations to that of NGF,
as has been recently demonstrated(36, 37) . Further
indication of the conserved neurotrophin structure, in particular the
dimer interface, is seen by their ability to form mixed heterodimers
with other neurotrophins(38, 39, 40) . It is
not certain at this time whether these heterodimers form and are fully
active in vivo.
(TGF-), platelet-derived
growth factor (PDGF), and more recently in human chorionic
gonadotrophin
(hCG)(41, 42, 43, 44, 45) .
Members of this diverse structural superfamily of ligands typically
form homo- or heterodimeric species. However, each representative of
known structure shown in Table 1has a different subunit
arrangement and intersubunit bonding pattern within its respective
dimer(46) . This diversity is also seen functionally since each
ligand activates quite different receptor classes and signaling
pathways. Other putative members of the cystine knot superfamily
include the Norrie disease protein, mucin, and von Willebrand
factor(47) .
or trk receptors, these molecular recognition events have been the focus
of intensive investigation. The availability of purified ligand and
receptor components and cell lines expressing individual receptor types
has allowed for affinity cross-linking, equilibrium binding
measurements, and site-specific mutagenesis of neurotrophin sequence.
, Lys
, and Lys
) to alanine
within NGF resulted in a complete loss of binding to p75 but maintained
binding to trkA and produced a survival response(50) .
These data confirmed earlier predictions of the role of electrostatic
interactions between NGF and p75
, made based on their
opposite overall electrostatic charge and the clustering of positive
charge on the surface of the NGF(26) .
-hairpin loops, including
residues 31 and 40-49 (in particular Glu and
Asn
) and residues 91-97(16, 57) .
The intrinsic flexibility of these loops and the N and C termini of NGF
and their apparent importance for trkA interaction(26, 27) suggest conformational
changes may occur on formation of a productive NGF-trk complex. Similarly positioned residues within these hairpin loops
for BDNF contribute to trkB interaction(16, 57) . Less flexible regions of
structure are also important, in particular several surface accessible
residues within
-strands 3 and 4 of the main framework of
NGF(16) . A similar conclusion was reached for NT-3, suggesting
that this surface may be used by all neurotrophins to engage their
respective trk receptors(s)(16, 51) . It is
clear from these data that despite some notable differences in the
manner in which various neurotrophin-trk receptor pairs interact, in
each case the binding determinants extend over a large surface area
that takes advantage of the neurotrophin's elongated
shape(16) . There is however, a conspicuous absence of a
conserved set of residues on the surface of each neurotrophin that
could define a common functional trk receptor interaction
site. This is curious in light of the extraordinarily high conservation
found for species variants of certain neurotrophins, such as NT-3. One
rationalization is the parallel evolution of distinctive
neurotrophin-trk specificities. Structurally, the ideal
candidates for determining specificity are the variable hairpin loops
and the N and C termini. These regions are solvent-accessible and can
accommodate sequence variation without perturbing the three-dimensional
structure. They would therefore be capable of adapting functionally to
interact with a specific trk and could allow for optimization
of individual neurotrophin-trk contacts, thus explaining the
apparent differences between trk receptor interactions for
different neurotrophins(16, 51, 57) . Insight
into neurotrophin specificity for the trk receptors will come
from structural studies of complexes of neurotrophins bound to their
preferred trk ectodomains.
of
10
-10
M. A
similar K
has also been determined for
BDNF binding to trkB(64) . These affinities are
distinct from the high affinity binding site, K
= 10
M, measured in
sensory neurons(61, 64) .
=
10
M). This site requires an excess ratio
of p75 to trk(65) . Hence, one function of p75 is to
increase the binding affinity of NGF. It should be emphasized that the
interactions of BDNF, NT-3, and NT-4/5 with cognate trk receptors or with p75 may be different from those exhibited by
NGF, as noted by mutagenesis experiments.
I-NGF suggest that an association
between the trkA and p75 may take place(67) .
Photobleaching experiments following a fluorescently tagged p75
receptor have also revealed a potential physical interaction with trkA(68) .
), transforming growth factor ;
PDGF, platelet-derived growth factor; hCG, human chorionic
gonadotrophin; trk, tropomyosin receptor kinase; CNS, central
nervous system; PNS, peripheral nervous system.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  A. M. Colangelo, M. R. Bianco, L. Vitagliano, C. Cavaliere, G. Cirillo, L. De Gioia, D. Diana, D. Colombo, C. Redaelli, L. Zaccaro, et al. A New Nerve Growth Factor-Mimetic Peptide Active on Neuropathic Pain in Rats J. Neurosci., March 12, 2008; 28(11): 2698 - 2709. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Qian, J. Zhang, X.-Y. Tan, A. Wood, D. Gill, and S. Cho Novel Agonist Monoclonal Antibodies Activate TrkB Receptors and Demonstrate Potent Neurotrophic Activities. J. Neurosci., September 13, 2006; 26(37): 9394 - 9403. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Chen and C. S. von Bartheld Role of Exogenous and Endogenous Trophic Factors in the Regulation of Extraocular Muscle Strength during Development Invest. Ophthalmol. Vis. Sci., October 1, 2004; 45(10): 3538 - 3545. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. B. Nguyen, L. Harris, E. Szönyi, S. A. Baughman, V. G. Hale, N. O. Dybdal, M. D. Sadick, and E. Escandón Tissue Disposition and Pharmacokinetics of Recombinant Human Nerve Growth Factor after Acute and Chronic Subcutaneous Administration in Monkeys Drug Metab. Dispos., May 1, 2000; 28(5): 598 - 607. [Abstract] [Full Text]
|
|
|
|
 |
|
 H. Moreno, M. Nadal, E. Leznik, M. Sugimori, I. Lax, J. Schlessinger, and R. Llinas Nerve growth factor acutely reduces chemical transmission by means of postsynaptic TrkA-like receptors in squid giant synapse PNAS, December 8, 1998; 95(25): 14997 - 15002. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. C. Edsall, G. G. Pirianov, and S. Spiegel Involvement of Sphingosine 1-Phosphate in Nerve Growth Factor-Mediated Neuronal Survival and Differentiation J. Neurosci., September 15, 1997; 17(18): 6952 - 6960. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Kullander, D. Kaplan, and T. Ebendal Two Restricted Sites on the Surface of the Nerve Growth Factor Molecule Independently Determine Specific TrkA Receptor Binding and Activation J. Biol. Chem., April 4, 1997; 272(14): 9300 - 9307. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Wu, S. Butz, Y.-s. Ying, and R. G.W. Anderson Tyrosine Kinase Receptors Concentrated in Caveolae-like Domains from Neuronal Plasma Membrane J. Biol. Chem., February 7, 1997; 272(6): 3554 - 3559. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Xie, M. A. Tisi, T. T. Yeo, and F. M. Longo Nerve Growth Factor (NGF) Loop 4 Dimeric Mimetics Activate ERK and AKT and Promote NGF-like Neurotrophic Effects J. Biol. Chem., September 15, 2000; 275(38): 29868 - 29874. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |