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FIGURE 7.14 The pattern of NT-3 expression is revealed by a lacZ reporter gene, and nerves are coun-
terstained with a neurofilament antibody. A. A transverse section through the thoracic region of an E11 mouse
embryo shows DRGs (drg), and peripheral nerves (n). NT-3 expression (blue) is prominently around the DRG
and sensory-motor projection. B. Peripheral axons within the forelimb growing through mesenchyme (ms)
are surrounded by NT-3 expression. There is almost no NT-3 expression at the end of the limb (asterisk),
which is not yet innervated. Lmc = lateral motor column, s = skin. (Reprinted from Farinas et al., 1996).
survival of neurons in the sensory, trigeminal, and
nodose ganglia.
Neurotrophins may promote survival even before
an axon has reached its target. The development of NT-
3 expression can be followed with a lacZ reporter gene
during the embryonic period when DRG neurons first
extend their axons. NT-3 is expressed heavily along the
path of growth (Figure 7.14), and this may explain why
loss of NT-3 expression can affect neuron survival
before target innervation has occurred (Farinas et al.,
contain a putative transmembrane protein containing
a tyrosine kinase on its cytoplasmic tail (Martin-Zanca
et al., 1986). The oncogene apparently results from a
genetic rearrangement that fuses a tyrosine kinase
with part of a nonmuscle tropomyosin sequence,
leading to its name: tropomyosin related kinase or Trk
(later called TrkA). The trk proto-oncogene was cloned,
and the distribution of its mRNA was examined in
vivo. The highest levels of expression are confined to
the cranial sensory, dorsal root, and sympathetic
ganglia (Martin-Zanca et al., 1990). Most importantly,
the TrkA protein (also called p140) is a high-affinity
binding site for NGF, and the binding event induces
tyrosine kinase activity (Kaplan et al., 1991a).
Three high-affinity neurotrophin Trk receptors have
now been isolated: TrkA, TrkB, and TrkC (Figure 7.13).
The latter two were discovered by taking the trk
sequence and performing low-stringency binding
screens with cDNA libraries. In this manner, two
sequences were isolated that encoded for 145 kD recep-
tor tyrosine kinases, named TrkB and TrkC (Barbacid,
1994). Each Trk receptor has two immunoglobulin-like
repeats in the extracellular domain and a tyrosine
kinase with autophosphorylation sites in the cytoplas-
mic domain. The extracellular domains are about 50%
homologous, but each Trk displays a specific affinity
for one or two of the neurotrophins: TrkA is specifi-
cally activated by NGF, Trk B is specifically activated
by BDNF or NT-4, and TrkC is specifically activated
by NT-3. Trk receptors may also be activated in
the absence of neurotrophins from within the neurons,
a process called transactivation. For example, the small
neurotransmitter, adenosine, can produce Trk phos-
phorylation through its G protein-coupled receptor.
The actived Trk is then able to promote survival of
PC12 cells and hippocampal neurons in vitro (Lee and
Chao, 2001).
Even before a receptor for NGF was discovered, it
was known that NGF binds to a site on the axon ter-
minal with very high affinity. The b subunit of NGF
can be labeled with 125 I, and used to perform binding
studies on freshly dissociated chick sensory neurons.
These experiments reveal two types of binding sites
(Sutter et al., 1979). The first displays a lower affinity
for NGF (e.g., nanomolar concentrations saturate the
binding sites), while the second displays a higher affin-
ity for NGF (e.g., picomolar concentrations saturate
the binding sites). In fact, there are two different types
of NGF receptor that are associated with these binding
kinetics, and each one has now been isolated.
The high-affinity receptor was discovered through
a series of interesting observations (Figure 7.15). Ini-
tially, it was found that NGF exposure induces rapid
phosphorylation of proteins on their tyrosine residues,
suggesting that the receptor might be a kinase (Maher,
1988). Soon after, an oncogene was discovered in
human colon carcinoma cells, and this turned out to
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