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Volume 17, Number 4,
Issue of February 15, 1997
pp. 1447-1459
Copyright ©1997 Society for Neuroscience
Expression and Distribution of IGF-1 Receptors Containing a
-Subunit Variant (gc) in Developing Neurons
Faustino Mascotti1,
Alfredo Cáceres1,
Karl H. Pfenninger2, and
Santiago Quiroga3
1 Instituto Investigación Médica Mercedes y
Martín Ferreya, Córdoba, Argentina,
2 Department of Cellular and Structural Biology, University
of Colorado School of Medicine, Denver, Colorado, and
3 Departamento Química Biológica, Facultad
Ciencias Químicas, Universidad Nacional de
Córdoba/Consejo Nacional de Investigaciónes
Científicas y Técnicas, Córdoba, Argentina
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
gc is a -subunit variant of the insulin-like
growth factor-1 (IGF-1) receptor highly enriched in growth cone
membranes prepared by subcellular fractionation of fetal rat brain
(Quiroga et al., 1995
). The present study is focused on the expression
and on the cellular and subcellular distribution of gc
in developing neurons and differentiating PC12 cells. In the developing
cerebral cortex and, at least at early stages, in cultured primary
neurons, gc expression was found to be correlated with
neurite outgrowth. In PC12 cells gc expression was nerve
growth factor (NGF)-dependent and also paralleled neurite outgrowth. In
contrast, -subunits of the insulin receptor and/or of other IGF-1
receptors ("P5"; detected with antibody AbP5) were
downregulated as gc expression increased.
Immunofluorescence studies confirmed the enrichment of
gc at growth cones and demonstrated morphologically its
spatial separation from P5, which is confined to the
perikaryon. At the growth cone, gc colocalizes and
associates in a proximal region with microtubules, but it seems
independent of the more peripheral microfilaments. Some
gc immunoreactivity is detected in the perinuclear region of PC12 cells, most likely the Golgi complex and its vicinity. gc seems to emerge from the periphery of this structure
in an apparently vesicular compartment distinct from that carrying
synaptophysin to the growth cones. The facts that (1) gc
expression is correlated closely with neurite outgrowth, that (2) it is
regulated in PC12 cells by a neurotrophin, NGF, and that (3)
gc is concentrated in the proximal growth cone region
raise new questions regarding a possible role of IGF-1 receptors
containing gc in the regulation of neurite growth.
Key words:
IGF-1 receptor;
-subunits;
gc;
growth cones;
neurons;
neurite outgrowth;
neurotrophins;
PC12 cells;
development;
tissue culture
INTRODUCTION
Insulin-like growth factor-1 (IGF-1) is involved
in the regulation of animal growth and tissue differentiation (Froesch
et al., 1985; Daughaday and Rotwein, 1989), including that of the brain. Expression of the IGF-1 gene and its transcript is high in the
developing brain but decreases in the adult (Rotwein et al., 1988;
LeRoith et al., 1992). IGF-1 stimulates the growth and differentiation
of fetal neurons in culture (DiCiccio-Bloom and Black, 1988), increases
neuronal sprouting and outgrowth (Aizenman and De Vellis, 1987; Caroni
and Grandes, 1990; Beck et al., 1993; Ishii et al., 1993), enhances
neuronal protein synthesis (Heidenreich and Toledo, 1989), and
regulates neuronal and glial function (Sara and Hall, 1990). In
addition, IGF-1 has been implicated in the modulation of synaptic
transmission (Schwartz et al., 1992). The receptor for IGF-1 resembles
the insulin receptor and is a disulfide-linked heterotetrameric
(
22) transmembrane glycoprotein with
extracellular ligand-binding () and intracellular tyrosine kinase
() domains (Ullrich et al., 1986). The expression of this receptor
in the CNS is high at late embryonic and early postnatal stages and
declines significantly afterward (Ullrich et al., 1986; Werner et al., 1991), again suggesting an important role for this ligand-receptor system in brain development. This notion is supported further by the
following observations: IGF-1 and its receptor are expressed permanently in the olfactory bulb, where neuronal remodeling and synaptogenesis continue throughout adult life (Bondy, 1991), and transgenic mice lacking IGF-1 receptors exhibit serious defects in
CNS development (Liu et al., 1993).
We have reported previously the biochemical characterization of a
-subunit of the IGF-1 receptor, designated gc. It is
immunochemically distinct from the described forms of this polypeptide,
highly enriched in a growth cone fraction prepared from developing rat brain, and, within the neuron, spatially segregated from the insulin receptor (Quiroga et al., 1995). In the present study we provide new
evidence on the cellular and subcellular localization of
gc as well as on the regulation of its expression.
Three different systems were analyzed: developing rat cerebral cortex,
primary neurons in culture, and PC12 cells differentiating in
vitro. The rat pheochromocytoma cell line, PC12, is an excellent model system for studying growth cone formation, neurite outgrowth, and
the expression of structural and membrane proteins involved in nerve
cell morphogenesis (Greene and Tischler, 1976; Drubin et al., 1985;
Greene et al., 1987; Bearer, 1992; Esmaeli-Azad et al., 1994). In the
absence of nerve growth factor (NGF), the cells are round or polygonal.
On stimulation with NGF, they extend several neurites tipped by well
defined growth cones. To study the relationship between
gc expression and nerve cell development, we took
advantage of this cell system. We analyzed the levels and distribution
patterns of gc during NGF-induced neurite outgrowth. Our
results indicate that gc expression is controlled
developmentally, increasing in parallel with process extension, and
that it is regulated by NGF in PC12 cells. Our studies also provide
morphological evidence that gc is a prominent growth
cone component
in contrast to other -subunits of the insulin or
other IGF-1 receptors (Garofalo and Rosen, 1989), which are essentially
restricted to cell perikarya. In addition, we show that, within growth
cones, gc is associated spatially with microtubules, but
not microfilaments, and segregated from synaptophysin.
MATERIALS AND METHODS
Cell cultures. PC12 cells (obtained from Dr. A. Ferreira, Harvard Medical School, Cambridge, MA) were grown in DMEM
supplemented with 10% horse serum and incubated at 37°C in a
humidified 5% CO2 atmosphere. Cells were plated onto
poly-L-lysine-coated glass coverslips at densities ranging
from 5000-10,000 cells/cm2. After plating they were
maintained for 2-3 d in serum-free medium supplemented with the
additives of Bottenstein and Sato (1979). When appropriate, NGF
(Boehringer Mannheim, Indianapolis, IN) was added at a concentration of
50 ng/ml. For some experiments PC12 cells were treated with
cytochalasin D (5 µM) for 20 min or nocodazole (10 µg/ml) for 30 or 60 min. Primary neuronal cultures were prepared from
the hippocampi or cerebral cortex of rat fetus (17-20 d gestation) as
previously described (Cáceres et al., 1986, 1992; Kosik and
Finch, 1987). Briefly, neurons were dissociated with trypsin in Ca- and
Mg-free medium and plated onto poly-L-lysine-coated coverslips. Hippocampal neurons then were incubated for 2-3 hr in
Eagle's Minimum Essential Medium (MEM) containing 10% horse serum to
allow for attachment. Subsequently, these coverslips were transferred
into dishes containing astroglial cells. The cultures were maintained
in MEM supplemented with 0.1% ovalbumin and the additives of
Bottenstein and Sato (1979).
Immunofluorescence. Cells were fixed before or after mild
detergent extraction under microtubule-stabilizing conditions and processed for immunofluorescence as previously described (Cáceres et al., 1992; DiTella et al., 1994) (see also Black et al., 1994). The
primary antibodies used were a monoclonal antibody (mAb) against tyrosinated -tubulin (clone TUB-1A2, mouse IgG, Sigma, St. Louis, MO) diluted 1:1000; a mAb against acetylated -tubulin (clone 6-11B-1; Ferreira and Cáceres, 1989); an affinity-purified rabbit antiserum against gc (Quiroga et al., 1995) diluted 1:50
or 1:100; and a rabbit antiserum designated AbP5, which recognizes
previously described -subunits of both the insulin and the IGF-1
receptors (Garofalo and Rosen, 1989). The double-antibody staining
protocol consisted of labeling with a first primary antibody, washing
with PBS, staining with fluorophore-labeled secondary antibody, washing again with PBS, and then repeating this procedure for the second primary antibody. Incubations with primary antibodies were for 1 or 3 hr at room temperature, whereas incubations with secondary antibodies
(FITC or rhodamine-labeled, generated in the goat and obtained from
Boehringer Mannheim) were performed for 1 hr at 37°C. The cells were
observed with an inverted microscope (Zeiss Axiovert 35M) equipped with
epifluorescence optics and photographed with 40× or 100× objectives
(Zeiss, Oberkochen, Germany) and Tri X-Pan (400 ASA) or Kodak Gold Plus
(400 ASA) film (Eastman Kodak, Rochester, NY).
In some experiments, the distribution of gc and
synaptophysin was evaluated with high-resolution video microscopy and
image processing as described (DiTella et al., 1994, 1996). So that images of labeled cells could be made, the epifluorescence illumination was attenuated with glass neutral density filters. Images were formed
on the faceplate of a Silicon Intensified Target camera (SIT;
Hamamatsu, Middlesex, NJ), set for manual high voltage, gain, and black
level. They were digitized directly into a Metamorph/Metafluor image
processor (Universal Imaging, West Chester, PA) controlled by a host
IBM-AT computer. After digitization, images were corrected for shading
distortion by dividing by a low-pass-filtered image of a featureless
field and normalizing to the maximum intensity within that image. In
some cases pseudocolor images were generated with the red/green overlay
menu of the Metamorph/Metafluor system. For the purpose of presentation
fluorescent images were photographed directly from a high-resolution
video monitor with a 35 mm camera (automatic exposure setting). Film
negatives were printed with equal exposure times.
Morphometric analysis. For some experiments the neurite
lengths of NGF-treated PC12 cells were measured with the morphometric menu of the Metamorph system as described (Cáceres et al.,
1992).
Polyacrylamide gel electrophoresis and Western blotting.
Whole-cell homogenates from brain tissue (cerebral cortex) or from cultured cells were prepared as described previously (Cáceres et
al., 1988, 1992), and polypeptides were resolved by polyacrylamide gel
electrophoresis (7.5% acrylamide; Laemmli, 1970). Polypeptides were
electro-transferred to nitrocellulose and then probed with the
gc or the AbP5 antibodies using an alkaline phosphatase
detection kit (Promega ProtoBlot Detection Kits, Madison, WI) or
iodinated protein A (see also Quiroga et al., 1995). For some
experiments gc protein levels were quantitated in
whole-cell homogenates from PC12 cells with a dot immunobinding assay
as previously described (Cáceres et al., 1992).
Autophosphorylation and immunoprecipitation of gc.
Membranes from PC12 cells cultured for 72 hr in the presence of
NGF were resuspended in phosphorylation buffer (50 mM
HEPES, pH 7.8, and 2.5 mM MnCl2) with or
without 10 nM IGF-1 (final reaction volume, 50 µl), and
kinase reactions were performed as described (Garofalo and Rosen,
1989). Then gc antibody was added and allowed to bind overnight at 4°C. Protein G-agarose was added, and the incubation was
continued for 120 min at room temperature. Immune complexes were
collected by centrifugation, and the beads were washed as described
(Garofalo and Rosen, 1989). Autophosphorylated receptors were eluted by
addition of Laemmli sample buffer (Laemmli, 1970). They were analyzed
by electrophoresis in 7.5% polyacrylamide gels and
autoradiography.
RESULTS
Expression of gc in neurons from
developing brain
Our previous study (Quiroga et al., 1995) showed that growth cone
membranes isolated from fetal brain are enriched in gc, suggesting that the expression of this protein is correlated with process formation in developing neurons. To test this hypothesis, we
have examined the time course of expression and the relative levels of
gc during postnatal development of rat cerebral cortex. In addition, we analyzed its expression and distribution in primary cultures of neurons from fetal rat cerebral cortex (Kosik and Finch,
1987) and hippocampus (Cáceres et al., 1986; Dotti et al., 1988).
For the first type of experiment, whole homogenates of cerebral cortex
prepared on postnatal days 1, 3, 6, and 13 (P1-P13) and from adult
rats were probed with the rabbit antiserum against gc by
Western blotting. The gc antibody recognized a single,
somewhat broad and heterogenous, immunoreactive band of ~97 kDa in
the tissue extracts obtained at P1, P3, P6 and P13, whereas a second
and faster migrating band (90 kDa) was present in the homogenates
prepared from adult cerebral cortex (Fig. 1). gc expression was high at early postnatal stages but
declined gradually and considerably with increasing age. In the adult
brain the lowest levels were detected and the 97 kDa band appeared
sharpened, as compared with earlier stages.
Fig. 1.
The expression of gc in the
developing rat cerebral cortex as revealed by immunoblot analysis of
whole-tissue extracts. P1-P13, Postnatal days 1-13; Ad, adult. gc is
highly expressed in the developing cerebral cortex. A second and faster
migrating band is present in the homogenates prepared from the adult
cerebral cortex. Protein (20 µg) was loaded in each lane; the
immunoblots were revealed by a rabbit ProtoBlot staining kit. The blots
shown are representative of three independent experiments, all of which generated essentially identical results.
[View Larger Version of this Image (63K GIF file)]
We next studied the distribution of gc in primary
neurons sprouting in culture. After 12 hr in culture, most cortical and hippocampal pyramidal neurons had extended several short
undifferentiated neurites, designated as minor processes; at this stage
gc immunolabeling was concentrated heavily in the growth
cones (Fig. 2A,B). Several hours
later, the neurons differentiated one of their minor processes into an
axon. In this type of culture any neurite that exceeded the other
processes of the same neuron in length by 10 µm or more was
considered to be an axon (Craig and Banker, 1994). The growth cones of
these axons also displayed heavy staining for gc. An example of such a growth cone, labeled with the gc
antibody, is shown in Figure 2D (tubulin staining of
the same axon is shown in Fig. 2C). This particular neurite
was over 100 µm long, exceeding in length the minor neurites from the
same cell by >70 µm. Comparisons of gc fluorescence
intensities of axonal growth cones versus those of minor processes
revealed no significant differences. Our results also showed that the
expression of gc in cortical and hippocampal pyramidal
neurons was transient, declining significantly when cells began
dendritic differentiation. Thus, in older cultures (>4 d in
vitro) when the (nonaxonal) minor processes had become dendrites
and were elongating, gc immunolabeling decreased
considerably, disappeared eventually from dendritic growth cones, and
became diffusely distributed throughout the cell (data not shown).
Fig. 2.
The distribution of gc in primary
neurons. Double-immunofluorescence micrographs of a hippocampal
pyramidal neuron maintained in culture for 12 hr show the
distribution of tyrosinated -tubulin (A) and
gc (B). Note the intense and selective
labeling of neurite tips with the gc antiserum.
Double-immunofluorescence micrographs also show the distribution of
microtubules (C) and gc
(D) in an axon-like process from a hippocampal
pyramidal neuron maintained in culture for 24 hr. Note that
gc immunolabeling is restricted to the axonal growth
cone. Calibration bar, 10 µm.
[View Larger Version of this Image (71K GIF file)]
Expression and distribution of gc in PC12 cells
In whole PC12 cell extracts the anti-gc antibody
recognized a single polypeptide of ~105 kDa (Fig.
3A, lane 1). Thus, the gc-immunoreactive polypeptide from PC12 cells exhibited
a higher apparent molecular mass than the single
gc-immunoreactive protein species detected in tissue
extracts obtained from the developing rat cerebral cortex (~97 kDa;
Fig. 3A, lane 3). For the Western blot shown in
Figure 3A, lane 1, extracts were prepared from
PC12 cells cultured for 48 hr in the presence of NGF, i.e., from
differentiating PC12 cells that had begun to form neurites. However,
the gc antigen was not or only very weakly detectable by
Western blot in undifferentiated cells grown without NGF, even when
two- to threefold higher protein amounts were loaded on the gels (Fig.
3A, lane 2).
Fig. 3.
The expression of gc and
P5 in PC12 cells. A, Immunoblot analysis
of whole-cell extracts from NGF-treated (lane 1) or
nontreated (lane 2) PC12 cells reacted with the
gc antiserum. NGF induces the expression of a single
gc-immunoreactive protein species with an apparent
molecular mass of 105 kDa. gc-immunoreactive polypeptides were not detected in undifferentiated PC12 cells under
these conditions. A single gc-immunoreactive species of 97 kDa (see also Fig. 1 and Quiroga et al., 1995) is detected in tissue
extracts obtained from 3-d-old rat cerebral cortex (lane 3) or in whole-cell extracts from hippocampal pyramidal cells differentiated in culture for 48 hr (lane 4).
B, Autophosphorylation of IGF-1 receptor in PC12
membranes immunoprecipitated with the gc antibody. The
addition of 10 nM IGF-1 (lane 2)
dramatically stimulates the phosphorylation of the immunoprecipitated
~105 kDa polypeptide, as compared with the control experiment
(lane 1). C, Immunoblot analysis of
whole-cell extracts from NGF-treated (lane 1) or
nontreated (lane 2) PC12 cells reacted with the AbP5 antiserum (P5). The 102 kDa
P5-immunoreactive band is considerably less abundant in
NGF-treated than in nontreated PC12 cells (NGF treatment was for 3 d). Protein (20 µg) was loaded in each lane, and the
immunoblots were processed with a rabbit ProtoBlot staining kit. The
blots and autoradiogram shown are representative of at last three
independent experiments, all of which generated essentially identical
results.
[View Larger Version of this Image (13K GIF file)]
To ascertain that the 105 kDa immunoreactive band detected in PC12
cells was indeed the gc subunit of the IGF-1 receptor, we performed an autophosphorylation experiment with membranes of PC12
cells (grown with NGF) incubated in the presence or absence of IGF-1.
Figure 3B shows autoradiograms of the immunoprecipitates obtained with the gc antibody and resolved by
SDS-polyacrylamide gel electrophoresis. A single radiolabeled band was
detected at 105 kDa, and its intensity was greatly enhanced when the
autophosphorylation was performed in the presence of IGF-1 (Fig.
3B, lane 2). This established the identity of the
105 kDa band as gc.
The expression of gc in differentiating PC12 cells was
compared with that of other -subunits of the insulin and IGF-1
receptors with antibody AbP5. This polyclonal antibody was raised
against a synthetic peptide representing part of the human insulin
receptor -subunit (amino acids 1328-1343; Garofalo and Rosen,
1989). The antibody cross-reacts with the -subunit of the rat
insulin receptor as well as with -subunits of the IGF-1 receptor
that are distinct from the gc-immunoreactive species
(Garofalo and Rosen, 1989; S. Quiroga, unpublished observations).
Figure 3C shows that AbP5 recognizes a single polypeptide at
102 kDa in PC12 cells. In contrast to gc, the
AbP5-immunoreactive band (termed P5) is relatively faint
in extracts of NGF-differentiated PC12 cells and much stronger in the
nondifferentiated cells (Fig. 3C, lane 1 vs
lane 2, respectively).
To correlate gc and P5 immunoreactivity
quantitatively with differentiation, we determined average neurite
lengths and -subunit expression (by dot immunobinding assay of
whole-cell extracts; Cáceres et al., 1988) in PC12 cells treated
for up to 4 d with NGF. Table 1 shows the results.
The dramatic increase of NGF-induced neurite length observed over
4 d was paralleled by a more than sixfold increase of
gc, whereas expression of P5 decreased to less than one-half the control value in the presence of NGF. If NGF-differentiated (4 d) PC12 cells were deprived of the neurotrophin for 6 hr, neurite length decreased dramatically, and so did
gc expression. P5 expression did not
change during this short deprivation period, however. These results
showed in PC12 cells that gc expression was tightly
controlled by NGF, together with differentiation, but that
P5 obeyed an inverse regulation pattern.
Immunolocalization of gc in PC12 cells
In the next series of experiments the spatial distribution of
gc was studied in PC12 cells by double immunolabeling
with anti-gc and a mAb that recognized tyrosinated
-tubulin (clone TUA 1.2). PC12 cells cultured in the absence of NGF
had a round or polygonal morphology, as expected (Fig.
4A, tubulin antibody), and exhibited
very weak immunofluorescence when incubated with the gc
antibody (Fig. 4B). When used at very high
concentrations (dilution 1:5-1:10), the gc antibody
labeled a small perinuclear area of the cell cytoplasm that resembled
the Golgi complex (data not shown). As expected, a dramatic increase in
gc immunofluorescence was evident when PC12 cells were
cultured in the presence of NGF. This phenomenon was detected as early
as 24 hr after the addition of NGF, when PC12 cells began to acquire a
neuron-like morphology. At that stage the cells had several short
neurites tipped with small growth cones. gc
immunofluorescence was localized preferentially to the perinuclear
region and to the growth cones (Fig. 4D; compare with
tubulin staining in 4C), whereas most of the cell body and of the neuritic shafts was devoid of gc immunolabeling.
An exception was occasional short neurites that contained a continuous
band of granular staining between the perinuclear region and the growth cones (see arrow, Fig. 4D). After 72 hr in
the presence of NGF, PC12 cells had extended several long neurites that
ended in prominent growth cones. At this stage gc
immunostaining had become very intense within the growth cone area but
had disappeared completely from neuritic shafts (Fig.
4F). A similar pattern was detected in PC12 cells
cultured with NGF for longer periods of time (3-7 d).
Fig. 4.
gc becomes localized to growth
cones in differentiated PC12 cells. Double-immunofluorescence
micrographs show the distribution of tyrosinated -tubulin
(A, C, E) and
gc (B, D,
F) in PC12 cells. PC12 cells cultured in the
absence of NGF display low positive immunofluorescence for the
gc antibody (A, B). A
dramatic increase in gc immunofluorescence is detected
in PC12 cells treated with NGF for 2 (C,
D) or 4 (E, F) d.
In these cells gc is localized preferentially in the
perinuclear region and at neurite tips. In young NGF-treated PC12
cells, a few neurites display a granular type of staining that extends
from the cell body to the tip (arrow in
D); however, in further differentiated PC12 cells, the
cell body and neuritic shafts are devoid of labeling (E,
F). Calibration bar, 10 µm.
[View Larger Version of this Image (74K GIF file)]
Our subcellular fractionation data published earlier (Quiroga et al.,
1995) indicated spatial separation of gc from other insulin and IGF-1 receptor -subunits. Therefore, we performed immunolocalization studies with the AbP5 antibody on PC12 cells. Uniformly distributed AbP5 immunofluorescence was readily detectable in
undifferentiated PC12 cells (data not shown). To demonstrate the
localization of P5 versus gc in
differentiated PC12 cells, we performed double immunofluorescence. All
cells were labeled with the tubulin antibody (secondarily tagged with
the green FITC) and then with either AbP5 or anti-gc
(secondarily tagged with the red rhodamine). Because permeabilization
was necessary to reveal the gc antigen (presumably
because the epitope is intracellular) and, of course, tubulin, we could
not decide whether the gc staining was on the cell
surface and/or in the cellular interior. However, at least part of the
perinuclear label almost certainly was associated with sites of
synthesis, especially distal regions of the Golgi complex (compare Fig.
7). At the growth cone, however, at least some of the gc
label must have been associated with the cell surface, because the
receptor could be activated by externally applied IGF-1 (Quiroga et
al., 1995). Figure 5A shows the expected pattern for gc, i.e., intensely red-yellow staining of
growth cones (primarily gc), green neurites (mostly
tubulin), and yellow perikarya (overlap of tubulin and
gc). Figure 5B illustrates the contrasting
pattern for AbP5: absence of obvious growth cone labeling, green
neurites stained with anti-tubulin only, and yellow perikarya doubly
labeled with anti-tubulin and AbP5. (As before, these samples had to be
permeabilized for immunolabeling so that it was difficult to
discriminate between -subunits exposed on the cell surface and those
within the cell). These experiments indicate strikingly the
differential distribution of gc versus the other
-subunit(s) (P5) in differentiated PC12 cells.
Fig. 7.
gc and synaptophysin appear
to emerge from the perinuclear region in distinct vesicle-like
structures. Double-immunofluorescence micrographs show the distribution
of synaptophysin (A) and gc (B) in the perinuclear region of a PC12 cell treated with NGF for
3 d. The arrows in A point at
apparent strands of small fluorescent dots.
C, Overlay of digitized synaptophysin
(green) and gc
(red) images shown in A and
B, respectively. The open arrow points at a thin region of the perikaryon where there is no overlap between labeled compartments. Calibration bar, 10 µm.
[View Larger Version of this Image (55K GIF file)]
Fig. 5.
Differential distribution of gc and
AbP5-immunoreactive insulin/IGF-1 -subunits. A,
Double-immunofluorescence micrograph shows the distribution of
tyrosinated -tubulin (green) and of gc (red). gc is highly
enriched at neurite tips. B, Double-immunofluorescence micrograph shows the distribution of tyrosinated -tubulin
(green) and AbP5 immunofluorescence
(red) in a differentiated PC12 cell. Overlapping label
appears yellow. Note the absence of AbP5 immunolabeling within the neurites and growth cones. Calibration bar, 10 µm.
[View Larger Version of this Image (51K GIF file)]
To investigate gc further, we used fluorescence
microscopy combined with image processing to analyze its subcellular
distribution in differentiating PC12 cells and to compare it with that
of synaptophysin. Synaptophysin is a membrane protein associated with
synaptic vesicles and enriched in the growth cones of developing
neurons (Fletcher et al., 1990). Although the antibodies specific for
gc and synaptophysin labeled essentially the same areas
of the cell, namely the perinuclear region and the growth cones (Fig.
6A,B), we detected clear differences in their staining patterns. For example, as shown in Figure 6, the
distribution of gc immunofluorescence (Fig.
6D,F) in growth cones exhibited in some cases
little overlap with that of synaptophysin (Fig. 6C,E). Also,
during the initial stages of neurite extension (24-48 hr after the
addition of NGF) growth cones highly immunoreactive for
gc commonly were stained only faintly or not at all with the synaptophysin antibody, although prominent immunolabeling was
detected in the perinuclear region (Fig.
6E,F). Within the perikarya of differentiating
PC12 cells, we were able to observe punctate fluorescent structures of
variable size, vesicle-like structures clearly located in the cell
interior and apparently emerging from the perinuclear region. With the
synaptophysin antibody (Fig. 7A), these
fluorescent dots were quite small and numerous and tended to form
strands radiating from the perinuclear region (arrows). The
gc antibody (Fig. 7B), however, labeled a
heterogeneously sized, generally much larger and sparser, compartment.
A pseudocolor superimposition image is shown in Figure 7C.
There was substantial overlap between the staining patterns in the
thicker region of the cell near the nucleus (near the top left-hand
corner), as expected. However, in the thinner, more peripheral parts of
the perikaryon (arrow) there was very little, if any,
overlap between the staining patterns.
Fig. 6.
gc and synaptophysin exhibit
similar, but not identical, distributions within growth cones.
Double-immunofluorescence micrographs show the distribution of
synaptophysin (A, C, E)
and gc (B, D, F) in PC12 cells treated with NGF for 3 d.
Both antigens are localized preferentially in the perinuclear region of
the cell bodies and in growth cones (arrows), but there
are clear differences in their staining patterns (see text).
Calibration bar, 10 µm.
[View Larger Version of this Image (86K GIF file)]
Interactions between gc and the cytoskeleton of
growth cones
The distribution of several growth cone-associated surface
antigens seems to depend on interactions with microtubules and/or microfilaments (Goslin et al., 1989; Bearer, 1992; DiTella et al.,
1994). The highly polarized distribution of gc raised
the question of whether this membrane protein also interacted with the
cytoskeleton. To study such relationships, we examined first whether
and where gc was detectable in association with the
cytoskeletons remaining after detergent extraction of differentiated
PC12 cells (prepared under microtubule-stabilizing conditions; see
Material and Methods). The cytoskeletons were double-labeled with the
antibody to tyrosinated -tubulin and the gc antiserum
and then analyzed by fluorescence microscopy. On detergent extraction
most gc immunolabeling was lost from the perinuclear
region. However, it remained unaffected within growth
cones, where it colocalized extensively with tyrosinated microtubules
(Fig. 8A,B). It is unlikely that this
was the result of incomplete solubilization and extraction of membrane
proteins because, under the same extraction conditions, the staining
for synaptophysin was abolished completely (Fig. 8C,
synaptophysin label, and 8D, gc label
of the same cell). Therefore, our observation indicated that a
significant proportion of gc of the growth cone was
linked somehow to the cytoskeleton.
Fig. 8.
gc and dynamic microtubules
colocalize within growth cones. Double-immunofluorescence micrographs
show the distribution of tyrosinated -tubulin
(A) and gc (B) in a
detergent-extracted cytoskeletal preparation from a differentiated PC12
cell. Note the colocalization of gc immunolabeling with
tyrosinated microtubules at the neurite tip. Double-immunofluorescence
micrographs also show the distribution of synaptophysin
(C) and gc (D) in a
detergent-extracted cytoskeletal preparation from a differentiated PC12
cell. Synaptophysin has disappeared completely from growth cones
(arrows), although gc immunolabeling
remains unaffected. p, Perinuclear region. Calibration
bar, 10 µm.
[View Larger Version of this Image (76K GIF file)]
Next we investigated whether gc was linked to
microtubules and/or to actin filaments. Differentiated PC12 cells were
treated with the microtubule toxin, nocodazole, for 30 or 60 min before fixation, and the distribution of gc and microtubules
was analyzed by immunofluorescence. As shown in Figure
9, this treatment depolymerized throughout the cell most
of the microtubules containing tyrosinated -tubulin (Fig.
9B), but it preserved the tubules enriched in acetylated
-tubulin (a marker of stable polymer); these were localized
preferentially to neuritic shafts but absent from growth cones (Fig.
9A; Ferreira and Cáceres, 1989; Arregui et al., 1991). The distribution of gc (Fig. 9D) was altered
dramatically in nocodazole-treated cells; the immunostaining
disappeared from the growth cones while punctate and disperse labeling
appeared along neuritic shafts (compare Fig. 9C,
detyrosinated microtubules of the cell seen in 9D). Growth
cones, however, remained attached, and their gross morphology
essentially was unchanged in these experiments (see also
Gonzalez-Agosti and Solomon, 1996). On removal of nocodazole,
tyrosinated microtubules rapidly reassembled proximo-distally from the
cell body toward the neurites as well as within growth cones.
gc immunolabeling at neuritic tips closely paralleled the reappearance of tyrosinated microtubules within the growth cones
(data not shown).
Fig. 9.
The growth cone localization of
gc depends on the integrity of dynamic microtubules.
Double-immunofluorescence micrographs show the distribution of
detyrosinated (A) and tyrosinated
(B) -tubulin in a detergent-extracted cytoskeletal
preparation from a differentiated PC12 cell treated with
nocodazole for 30 min. Note the complete disappearance of
tyrosinated microtubules. Double-immunofluorescence micrographs
also show the distribution of detyrosinated microtubules (C) and gc (D) in a
detergent-extracted cytoskeletal preparation from a differentiated
PC12 cell treated with nocodazole for 30 min. Note the retraction of
gc immunolabeling from neurite tips and the presence of
immunostaining along neurite shafts. n, Nucleus. Calibration bar, 10 µm.
[View Larger Version of this Image (64K GIF file)]
It is now well established that the organization of microfilaments is
altered considerably after microtubule depolymerization (Goslin et al.,
1989; DiTella et al., 1994) so that the observed nocodazole-induced
redistribution of gc could be a secondary phenomenon
dependent on actin. Therefore, we sought to determine whether
gc distribution was dependent on the integrity of actin filaments. Initially, we compared the patterns of gc and
actin filaments in normal growth cones and then analyzed the
distribution of these proteins in cells treated with cytochalasin D. Phalloidin staining of differentiated PC12 cells revealed actin
filaments primarily within the peripheral growth cone structures, as
expected, and in some cases, in veils and filopodia along neuritic
shafts (Fig. 10A). These F-actin-rich
peripheral growth cone structures were almost devoid of microtubules
(Fig. 10B; cf. Forscher and Smith, 1988; Letourneau
and Shattuck, 1989). Comparing the distributions of gc
(Fig. 10D), F-actin (Fig. 10A,C),
and tubulin (Fig. 10B) revealed that the
gc staining pattern resembled more closely that of
tubulin than that of F-actin (see also Fig. 8A,B).
Although both phalloidin and the gc antiserum stained
growth cones prominently, the F-actin-rich structures, the filopodia
and lamellipodia, were in a position clearly distal to the region
enriched in gc, with little overlap between them (Fig.
10C,D; compare position of arrows). In most cases, F-actin disassembly induced by cytochalasin D (Fig.
10E,F) did not alter growth cone morphology
and did not result in changes in the distribution of gc
immunofluorescence in growth cones. However, sometimes the cytochalasin
treatment caused the collapse and/or detachment of growth cones and the
loss of gc immunolabeling from neuritic tips.
Fig. 10.
gc does not colocalize with
F-actin. Double-immunofluorescence micrographs show the distribution of
F-actin (A) and tyrosinated microtubules
(B) in a differentiated PC12 cell. Note that F-actin, visualized by phalloidin staining, is detected primarily in regions distal or peripheral to, and almost devoid of, tyrosinated
microtubules. Arrows point at corresponding proximal
growth cone structures. Double-immunofluorescence micrographs also show
the distribution of F-actin (C) and gc
(D) in a differentiating PC12 cell. Phalloidin and
anti-gc stain the tips of neurites prominently, but
F-actin localizes to a position distal to that enriched in
gc. Arrows point at corresponding
proximal growth cone structures. n, Nucleus. Finally,
double-immunofluorescence micrographs show the distribution of F-actin
(E) and gc (F) in
differentiating PC12 cells treated with cytochalasin D for 30 min. Note
the complete disappearance of phalloidin staining from neurite tips,
whereas gc immunolabeling remains highly enriched within
growth cones (arrowheads). n, Nucleus. Calibration bars, 10 µm.
[View Larger Version of this Image (95K GIF file)]
DISCUSSION
The molecular identity of gc remains unclear.
We have not been able to establish so far whether it is, e.g., the
product of a separate gene or the result of a specific
post-translational modification of a "conventional" IGF-1 receptor
-subunit. Clear, however, are the distinctive immunochemical
properties of gc (Quiroga et al., 1995).
Our Western blots identified gc in developing cerebral
cortex as a heterogeneous band of 97 kDa, as expected (see Quiroga et
al., 1995). The observed heterogeneity of this band most likely is the
result of different phosphorylation states of the polypeptide, but
differential glycosylation may be involved as well. In PC12 membranes
the gc antibody recognized a larger 105 kDa band.
IGF-1-stimulated autophosphorylation of the immunoprecipitated
polypeptide identified it as gc. The higher
Mr of gc in PC12 cells, as
compared with brain, is consistent with previous studies showing that
IGF-1 receptor -subunits present in cells outside the CNS exhibit a higher apparent molecular weight, a phenomenon resulting apparently from differential glycosylation (Ocrant et al., 1988). That greater glycosylation accounts for the higher Mr of
gc in PC12 cells thus is likely but has not been
established.
Spatial and temporal distribution of gc
in development
The previous fractionation studies (Quiroga et al., 1995)
demonstrated that gc immunoreactivity in fetal brain is
membrane-associated. Our immunofluorescence analysis of primary neuron
cultures presented here shows morphologically that gc is
highly enriched in growth cones, whereas there is little
immunoreactivity associated with neuritic shafts. The
quasi-solid staining of neuronal or PC12 growth cones can be
explained by the abundance of immunoreactivity in the plasma membrane,
combined with the flattened configuration of the growth cone in culture
and the staining of intracellular compartments carrying
gc to the distal neurite (see below). Based on the
staining pattern in the cell body, i.e., its diffuse distribution excluding the nucleus and the enhanced perinuclear labeling,
gc immunoreactivity in the perikaryon most likely can be
attributed to sites of synthesis, especially the Golgi complex (compare
with below), rather than plasmalemmal receptors (cells were
permeabilized before labeling). During the early phase of
differentiation of cultured hippocampal pyramidal cells
gc is present in the growth cones of all types of
neurite, including minor processes as well as axons. However, after
2-3 d in culture, when axons have elongated considerably and minor
processes begin to differentiate into dendrites, gc
immunoreactivity becomes redistributed throughout the cell while
declining significantly overall. It follows that, in cultured neurons,
gc (unlike synaptophysin, synapsin I, and GAP43;
Fletcher et al., 1990) is not targeted specifically to axonal growth
cones, and its expression is transient. The former observation is
surprising considering the high enrichment of gc in the
growth cone fraction from fetal brain, which is predominantly axonal in
origin (Saito et al., 1992; Lohse et al., 1996). However,
gc distribution in the developing neuron may be related
to the potential of all processes to differentiate into axons, at least
during the early stages (Dotti and Banker, 1987). Conceivably, our
observations in culture may reflect the (initial) lack of a mechanism
sorting certain types of membrane protein to the axon during the
establishment of neuronal polarity (Dotti and Simons, 1990). Finally,
the different environmental conditions of the cultured neurons may
explain the apparent discrepancy between these findings and those
obtained by subcellular fractionation of fetal brain.
Growth cone enrichment of gc in the brain, in cultured
neurons, and in PC12 cells suggests a correlation between neurite outgrowth and gc expression. Although this correlation
is less clear in primary neurons in culture (initial increase followed by decline after the first few days in culture), it holds
quantitatively for NGF-stimulated PC12 cells as well as for the
developing brain: the gc polypeptide is abundant during
the late fetal and early postnatal days of development when neurite
formation is prevalent. [Our more recent data indicate that
brain-derived neurotrophic factor (BDNF) also increases
gc expression, concomitant with neurite outgrowth, in
primary cultures of hippocampal neurons; studies in progress.] During
maturation of the brain and in the adult, expression of the 97 kDa
gc subunit is much reduced. However, a clearly
detectable amount of immunoreactivity remains. This may be analogous to
the reduced but continued expression in the adult brain of other
growth-regulated and growth-cone-enriched proteins, such as GAP43; this
phenomenon has been attributed to continued sprouting activity (see
Pfenninger et al., 1991). The appearance after P13 of a second 90 kDa
polypeptide reacting with the gc antibody is not
understood at this time. This polypeptide may be the product of
oligodendrocytes, which appear relatively late in development.
Establishment and maintenance of
gc distribution
Our immunofluorescence data on cultures of primary neurons and
differentiated PC12 cells indicate that gc labeling does
not stain the very periphery of the growth cone, the
microfilament-filled filopodia, and lamellipodia.
Instead, labeling is coextensive with the distal ends of dynamic
(tyrosinated) microtubules, which stop in the proximal region of the
growth cones (Forscher and Smith, 1988). This suggests that
gc may be anchored to these microtubules to maintain its
spatial distribution. Indeed, a substantial proportion of
gc (unlike synaptophysin) is resistant to detergent extraction and remains codistributed with the distal regions of tyrosinated microtubules. Furthermore, actin depolymerization has
little effect on gc distribution as long as neurites
remain attached; in contrast, depolymerization of dynamic microtubules with nocodazole results in gc spreading to more proximal
segments of neurites, where stable microtubules (detyrosinated or
acetylated -tubulin) are present. Based on these data,
gc falls into a category of proteins, more specifically
receptors, capable of interacting directly or indirectly with
polymerized tubulin. For example, there are two receptors at the
synapse that are linked to microtubules. One of these is the
-aminobutyric acid receptor A, which seems to interact directly with
tubulin (Item and Sieghart, 1994); the other is the glycine receptor,
which is linked to microtubules by gephyrin, a putative
microtubule-associated protein involved in membrane-cytoskeleton
interactions (Prior et al., 1992). The gc-microtubule
association raises the interesting possibility of functional effects of
the gc-containing IGF-1 receptor on the cytoskeleton.
One of the proteins that is known to be phosphorylated by activation of
the IGF-1 receptor is the microtubule-associated protein-2, MAP2
(Pillion et al., 1992). Therefore, gc could be positioned at the growth cone to control neurite outgrowth by modulating the phosphorylation status of proteins known to be actively
involved in cytoskeletal assembly, such as MAP2 or tau (Cáceres
and Kosik, 1990; Cáceres et al., 1992; Harada et al., 1994).
To establish its distal, polarized distribution, gc must
be transported from perikaryal sites of synthesis to the growth cones.
Synaptophysin, although not nearly so abundant in axonal growth cones
as in synaptic endings (Lohse et al., 1996), is an example of a
membrane protein shuttled to, and concentrated in, growth cones. Our
micrographs of PC12 cells show very small synaptophysin-positive dots
in the vicinity of heavily stained perinuclear regions, consistent with
the antigen being sequestered into small (putatively synaptic) vesicles
and about to be exported into the growing neurites. When cells are
labeled with anti-gc, one finds in the same region sparser, irregularly sized, and generally larger fluorescent dots. These images are compatible with the presence of a larger and distinct
vesicular compartment, presumably also destined for export to growth
cones. This is of particular interest because plasmalemmal growth of
the developing neurite is known to occur primarily at the growth cone,
and the plasmalemmal precursor is believed to be the large, clear,
irregularly sized vesicles typically found in growth cones (Pfenninger
and Maylié-Pfenninger, 1981; Lockerbie et al., 1991; Pfenninger
and Friedman, 1993).
Regulation of gc expression and
functional implications
As discussed already, the expression of
gc-containing IGF-1 receptors is correlated, at least at
the early stages, with growth cone formation and neurite outgrowth.
Particularly striking, however, is the effect of NGF on the expression
of gc and of -subunits of other IGF-1 and insulin
receptors (P5, recognized by AbP5) in PC12 cells.
Undifferentiated PC12 cells express P5 almost exclusively, whereas gc is essentially undetectable with
our methods. In contrast, under the influence of NGF P5
expression declines and remains confined to the perikaryon, whereas
that of gc increases several-fold. Furthermore, NGF
withdrawal causes not only neurite retraction but also a precipitous
drop in gc levels. It follows that IGF-1 receptors
containing gc are strictly regulated by NGF and thus are
a differentiation product of PC12 cellsin contrast to
P5-containing receptors, which seem to be more important
for trophic effects in the proliferating cells (they are also abundant
in developing brain). The observed differences in regulation and
distribution between gc and P5 may be
particularly significant because the IGF-1 and insulin receptors do not
seem to have mainly redundant functions in vivo, despite a
high degree of similarity in protein sequence and substrate specificity
(Ullrich et al., 1986; Steele-Perkins et al., 1988; Gronborg et al.,
1993). The predominant physiological actions of insulin seem to involve glucose, protein, and lipid metabolism. In contrast, IGF-1 seems to
function in most cells primarily as a mitogenic peptide and, in the
particular case of the developing nervous system, as a neurotrophic
factor promoting neurite outgrowth (see introductory remarks). Although
the reasons for these functional differences are poorly understood (Le
Roith et al., 1992), the contrasting regulations and distributions of
gc-containing IGF-1 versus P5-containing insulin/IGF-1 receptors may be essential for their apparently different
functional roles in the neuron.
FOOTNOTES
Received Aug. 27, 1996; revised Nov. 12, 1996; accepted Dec. 3, 1996.
This work was supported by grants from Consejo Nacional de
Investigaciónes Científicas y Técnicas, Consejo de
Investigaciónes de la Provincia de Córdoba, Argentina, and
Secretaria de Ciencia y Técnica, Universidad Nacional de
Córdoba, Argentina, to A.C. and S.Q., and by National Institutes
of Health Grant NS-24672 awarded to K.H.P. We express our gratitude to
Drs. R. S. Garofalo and D. Beltramo for providing some of the
antibodies used in the present study and to Gray Grether for expert
assistance with the completion of this manuscript.
Correspondence should be addressed to Dr. Karl H. Pfenninger,
University of Colorado, Health Sciences Center, Department of Cellular
and Structural Biology, Box B-111, 4200 East Ninth Avenue, Denver, CO
80262.
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