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Tumor Biology |
Department of Oncology/Lombardi Cancer Center, Georgetown University Medical Center, Washington, D.C. 20007 [W. L., A. Y.], and Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892 [T. H., M. H., L. F., J. H. P., S. R.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although biochemical lesions such as ras gene activation or p53 mutation have been associated with a number of forms of cancer, very little is known about such alterations in B lineage tumors. An activated c-myc gene appears essential for development of most murine plasma cell tumor (15) but is less common in human myeloma. Activation of myc results most frequently from chromosomal translocation but can also be achieved by inclusion of the myc gene in a retroviral construct or other undefined mechanisms (16) . Although myc rearrangements are rare in multiple myeloma, a variety of other translocations are routinely observed, most involving one of the immunoglobulin loci and a second locus, such as fibroblast growth factor receptor 3 (17) or cyclin D1 (18) . In addition, myc activating translocations are frequently observed in Burkitt’s lymphoma (19, 20) , suggesting that gene activation by translocation may be a common mechanism for deregulation in B lineage neoplasia in both species. Biochemical pathways affected by such translocations have not been defined in terms of association with tumor development. The identification of deregulated pathways is of obvious importance to both further an understanding of the biology of these diseases and to identify potential targets in specific pathways for therapeutic intervention.
One of the emerging themes in loss of growth arrest involves
either overexpression or aberrant activation of growth factor
receptors, leading to deregulation of signaling pathways affecting cell
growth, differentiation, and/or apoptosis. Among such receptors is the
type II tyrosine kinase receptor family, which includes the
insulin receptor,
IGF-IR,2
and ros oncogene (21, 22)
. Insulin and IGF-I
are the physiological ligands for the corresponding receptors that
share a number of properties. Both contain two 
subunits responsible
for ligand binding and two ß subunits containing intracellular
tyrosine kinase domains. Ligand binding induces receptor
clusterization, autophosphorylation, and internalization. The activated
receptor phosphorylates many important intracellular molecules on
tyrosine, including SHC and IRS, leading to activation of MAPK
and PI 3'K pathways, respectively (23)
. Whereas activation
of the insulin receptor is generally implicated in glucose metabolism,
stimulation of the IGF-IR pathway is strongly associated with cell
proliferation, malignant transformation, and antiapoptotic effects
(24)
.
Up-regulation of IGF-IR has been noted in several human cancers, and a role for this receptor in neoplastic development has been strongly suggested by the modulation of tumor growth after introduction of antisense oligonucleotides, antireceptor antibody, or dominant-negative mutants (23, 25) . Furthermore, embryonic fibroblasts from IGF-IR knockout mice are resistant to transformation by a variety of highly transforming proteins, including oncogenes, activated tyrosine kinase receptors, and viral proteins (23) . Taken together, these findings suggest that IGF-IR may play an important role in a number of neoplastic conditions. It was, therefore, of interest to determine whether this receptor might contribute to lymphoid malignancies and, if so, to define associated signaling pathways. In the present study, we describe a critical role for IGF-IR in plasma cell tumor development and identify such a downstream signaling pathway involving IRS-2, PI 3'K, and p70S6K.
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MATERIALS AND METHODS |
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Cell Lysates, Immunoprecipitation, and Immunoblot Analysis.
Prior to lysis, cells were serum starved for 2 h in Dulbecco’s
modified essential medium containing 25 µM
Na3VO4. They were either
not treated or were stimulated with murine IL-4 (100 ng/ml) or human
IGF-I (100 ng/ml; Intergen) for 10 min at 37°C. Cell pellets were
lysed in a Triton X-100-containing buffer as reported
(26)
. Insoluble proteins were removed by centrifugation,
and protein concentration was determined using a Bio-Rad protein assay
kit. Equivalent amounts of cell lysates (0.5–2 mg/sample) were
immunoprecipitated with anti-IGF-IR serum (Santa Cruz Biotechnology; 5
µl/sample), anti-IRS-2 (Ref. 27
; 5 µl/sample),
anti-SHC (Transduction Laboratories, Inc.; 5 µl/sample), or
anti-p70S6K (Upstate Biotechnology, Inc., 5 µl/sample). Washed
immunoprecipitates were electrophoresed on 8% SDS-PAGE gels,
transferred to Immobilon membrane (Millipore), and immunoblotted with
antiphosphotyrosine (anti-pTyr, Upstate Biotechnology, Inc., 2
µg/ml), anti-PI 3'K (Upstate Biotechnology, Inc., 1:1000 dilution),
or anti-p70S6K (1:1000). For direct Western analysis, anti-phospho-MAPK
(1:1000, New England Biolabs) was used. Proteins were detected using an
ECL system from Amersham Pharmacia Biotech.
In Vitro IGF-IR Immune Complex Assay.
Cell lines were treated as described above, and equivalent amounts of
cell lysates were immunoprecipitated with anti-IGF-IR serum. An immune
complex assay to determine receptor kinase activity was performed as
described previously (26)
.
The p70S6K Activity Assay.
Various cell lines were similarly treated as stated above and
lysed. Equivalent amounts of cell lysates were immunoprecipitated with
anti-p70S6K (Santa Cruz Biotechnology; SC230; 2 µg/sample) or
anti-Grb2 (Santa Cruz Biotechnology; SC255; negative control). Washed
immunoprecipitates were subjected to a S6K activity assay using a kit
from Upstate Biotechnology, Inc., according to the manufacturer’s
instructions. The peptide AKRRRLSSLRA was used as a substrate in the
activity assay. The results were the mean value from two independent
experiments.
Mitogenic Assay.
Cultured cells were washed twice with PBS and seeded at 2 x 105 cells/well in 24-well plates in RPMI
1640 containing 0.5% BSA (Sigma) with or without IGF-I (100 ng/ml) for
44 h. Cells were pulsed with [3H]thymidine
(Amersham Pharmacia Biotech; 1 µCi/well) for an additional 4 h
prior to harvest. [3H]Thymidine incorporation
was measured in triplicate samples, and the mean value was calculated.
Fold increase of [3H]thymidine incorporation
was calculated by dividing the mean value of untreated wells by that of
corresponding IGF-I-stimulated wells.
Transfection and in Vivo Tumorigenesis.
Electroporation for gene transfer into suspension cells was
performed as reported (28)
. Cell lines were transfected
with 10 µg of pBPV vector containing a lysine to arginine mutation at
the ATP binding site (pBPV-IGF-IRKR, kindly provided by Dr.
Renato Baserga) together with 1 µg of pZipneo as carrier DNA.
Neomycin-resistant cells were selected by growth in the presence of 750
µg/ml of G418 (Life Technologies, Inc.). Parental or IGF-IRKR
transfectants were injected i.p. (2 x 106 cells/mouse) into female BALB/c mice 24 h after pristane priming. Tumor development was monitored twice per
week.
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RESULTS |
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). Whereas abl/myc plasma cell tumors (121.1 and
128.3) expressed only the basal levels of IGF-IR, the levels increased
by 2- and 5-fold in chemical-induced (S107 and X24) and
raf/myc-induced (12.2 and 7.2) plasma cell tumors,
respectively. B cell lymphomas (WEHI231 and NFS-1) only expressed basal
levels of IGF-IR when compared to raf/myc and chemical
plasma cell tumors. IGF-IR up-regulation was reproducibly detected in
five raf/myc lines (data not shown), indicating that
increase in IGF-IR is a common phenomenon in plasma cell tumors induced
by raf/myc oncogenes.
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). The lower level of
autophosphorylation in S107 correlates with lower protein expression
(Fig. 1A
). Although IGF-IR autophosphorylation was not
observed in the chemical-induced X24 line, which may be
attributable to the lower expression of IGF-IR, a 180-kDa
tyrosine-phosphorylated protein was detected in IGF-IR
immunoprecipitates in response to IGF-I stimulation. This protein was
subsequently identified as IRS-2, a substrate of IGF-IR (see Fig. 2
). Because only phosphorylated IGF-IR is able to associate with IRS
molecules in vivo, detection of IRS-2 in the IGF-IR
immunoprecipitates indicates that IGF-IR in X24 line is also
functionally active. In abl/myc plasma cell lines, many
phosphorylated proteins spanning a large size range were
co-immunoprecipitated with anti-IGF-IR indicating that constitutively
activated abl tyrosine kinase phosphorylates numerous
proteins in vivo, some of which associate with IGF-IR. In
striking contrast to the plasma cells, no constitutive or
IGF-I-dependent IGF-IR autophosphorylation was detected in the two B
cell lymphoma lines, WEHI231 and NFS-1. Although IL-4 is known to be a
growth and differentiation factor for B lymphocytes, stimulation
of all lines tested with IL-4 did not induce IGF-IR activation. Taken
together, these results demonstrate that IGF-IR is up-regulated in both
raf/myc- and chemical-induced plasma cell tumors
and that the receptor is functionally active in response to IGF-I.
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A, top panel), irrespective of the
tumor-inducing agent. Weak phosphorylation was observed in one B cell
line, A20, with two others remaining largely negative. The marked
difference in IRS-2 phosphorylation between plasma and B cells could
not be attributed to IRS-2 protein levels, because the highest
expression was observed in the WEHI231 B cell line, and the X24 plasma
cell tumor was one of the lowest (Fig. 2
A, bottom panel).
After normalizing for IRS-2 expression levels, constitutive
phosphorylation of IRS-2 was shown to be 2–5-fold higher in plasma
cell tumors than in B cell lymphomas (Fig. 2B
).
Potential ligands known to be upstream activators of the IRS-2 pathway
include IGF-I and IL-4 (27, 29)
. We therefore assessed
the effects of these two factors on plasma and B cell lines
after serum starvation. Constitutive tyrosine phosphorylation of IRS-2
was still observed in the two raf/myc (12.2 and 7.2) and the
two abl/myc (121.1 and 128.3) lines (Fig. 2C
) but not in chemical-induced tumors (S107 and X24) or B
cell lines (WEHI231 and NFS-1). IRS-2 phosphorylation was readily
induced in the chemical tumors by IGF-I, but not IL-4. IGF-I also
enhanced IRS-2 phosphorylation in the raf/myc lines. In
contrast, this phosphorylation was constitutive in
abl/myc tumors. The IRS-2 response to IGF-I thus
correlates with IGF-IR levels and autophosphorylation described in Fig. 1
. B cell lines also responded to IGF-I, although maximal stimulation
was observed with IL-4. These results indicate that normal serum levels
of IGF-I in conjunction with up-regulated IGF-IR may be sufficient to
constitutively activate this pathway in both chemical- and
raf/myc-induced tumors. Phosphorylation of IRS-2 in the
abl/myc lines may be attributable to the kinase activity of
v-abl, consistent with the results described above
demonstrating numerous phosphorylated proteins associated with the
IGF-IR in these lines (Fig. 1B
). We therefore conclude that
IRS-2 is a major downstream substrate of IGF-IR in tumors induced by
chemicals and raf/myc retrovirus. It may also be
directly phosphorylated by v-abl in abl/myc
lines. Constitutive phosphorylation of IRS-2 occurs under
normal growth conditions in all plasma cell tumors but not B cell
lymphomas.
PI 3'K Is Associated with Tyrosine-phosphorylated IRS-2 in
all Plasma Cell Lines.
Tyrosine-phosphorylated IRS molecules have been shown to
associate with PI 3'K by binding to the src homology 2
domain of the p85 subunit resulting in subsequent PI 3'K activation
(30)
. To assess signal transduction from IRS-2 to PI 3'K
in plasma cell tumors, we analyzed the association of IRS-2 with the
p85 subunit of PI 3'K in co-immunoprecipitation experiments. As seen in
Fig. 3
A, constitutive association of p85 with IRS-2 was detected in
all plasma cell lines, irrespective of inducing agent, and this
association could be further enhanced in the chemical and
raf/myc lines upon IGF-I, but not IL-4, stimulation. The
highest level of association was observed in the two abl/myc
lines and could not be further enhanced with ligands, likely indicating
that IRS-2 is already maximally phosphorylated after abl
transformation as suggested above (Fig. 2)
. In contrast, none of the B
cell lines evidenced this constitutive activation. WEHI231 and NFS-1
demonstrated maximal association in response to IL-4, consistent with
the preferential phosphorylation of IRS-2 after IL-4 stimulation (Fig. 2)
. Activation of PI 3'K was also detected by measuring
phosphatidylinositol phosphate production in all plasma cell tumors
either IGF-I dependently (chemical-induced and
raf/myc-induced) or constitutively
(abl/myc-induced; data not shown). Thus, the constitutive
association of IRS-2 with PI 3'K in plasma but not B cell tumors
strongly suggests that activation of the PI 3'K pathway through IRS-2
may be critical for plasma cell tumor development.
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) and
kinase activity assay (Fig. 3C
). IGF-I stimulation of
chemical (S107) and raf/myc (7.2 and 12.2) lines
resulted in the slower migrated bands as detected by a p70S6K specific
antibody. On the other hand, the constitutive shifting bands were
detected from both abl/myc lines (121.1 and
128.3), consistent with constitutive activation of PI 3'K (see Fig. 3A
). In WEHI231 B cell lymphoma line, only the two faster
migrating bands were detected in the presence of IGF-I. Induction of
p70S6K activation by IGF-I in FDC-P2 myeloid progenitor line was
reported previously (32)
and confirmed by the mobility
shifting assay (Fig. 3B
).
To directly prove that p70S6K is activated in plasma cell tumor
lines, its activity was measured by immunoprecipitating equivalent
amounts of cell lysates with anti-p70S6K followed by an in
vitro phosphorylation assay using peptide AKRRRLSSLRA as
substrate. As shown in Fig. 3
C, all of the plasma cell tumor
lines responded to IGF-I for p70S6K induction, ranging from 1.6- to
5.2-fold. The specific detection of p70S6K activity was demonstrated by
using an isotype-matched anti-Grb2 antibody for immunoprecipitation as
a negative control. Dependency on PI 3'K for p70S6K activity was
established by including wortmannin, a known inhibitor of PI 3'K, which
completely reversed IGF-I-induced p70S6K activities in several lines
analyzed. We reproducibly observed that IGF-I was able to weakly induce
p70S6K activity in the two abl/myc lines, suggesting some
other pathways other than IRS-2 may be induced by IGF-I to stimulate
extra PI 3'K activity in abl-transformed lines. Together,
the p70S6K results substantiate the observation of activation of
IRS-2/PI 3'K among the all plasma cell tumors.
The SHC/MAPK Pathway Is Constitutively Activated in
abl/myc Plasma Cell Tumors.
SHC can be tyrosine phosphorylated by the activated IGF-IR
through the interaction of phosphotyrosine binding domain from SHC with
the NPXpY motif located at the juxtamembrane domain of
IGF-IR (33)
. The phosphorylated SHC interacts with the
src homology 2 domain of Grb2, thus activating
SOS/Ras/Raf/MAPK cascade. To understand whether this cascade is also
involved in the tumor development of mouse plasma cells, we tested SHC
phosphorylation. As shown in Fig. 4
A, although the p52 SHC phosphorylation was only detected in
12.2 line in response to IGF-I among the all
raf/myc and chemical tumors, constitutive
phosphorylation of SHC on tyrosine was detected in both 121.1 and 128.3
abl/myc lines. Phosphorylation of SHC was also
observed in WEHI231 B lymphoma in response to IL-4. Similar expression
levels of p52 SHC were detected from all lines (Fig. 4B
).
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; the lower level of phosphorylation presented in the 128.3
line in resting condition was not reproducible). In contrast, we were
not able to detect any MAPK activation in both raf/myc and
chemical plasma cell tumors. Constitutive activation of MAPK was also
found in the two B cell lymphomas (Fig. 5B
). Again, weak
induction of MAPK phosphorylation was observed in the WEHI231 B cell
line in response to IL-4, consistent with SHC phosphorylation (see Fig. 4A
). Similar levels of p42 Erk2 expression were detected
among the all lines (Fig. 5
, bottom panels).
Together, these results indicate that SHC/MAPK pathway is mainly active
in abl/myc-induced plasma cell tumors.
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, addition of IGF-I in the absence of serum led to a 4.3- and 8-fold
increase in proliferation of the 7.2 and 12.2
raf/myc lines, respectively, compared to
non-IGF-I-treated controls. IGF-I was also able to induce a 2-fold
increase in mitogenesis in the S107 chemical-induced line. In contrast,
neither abl/myc lines (121.1 and 128.3) nor B
cell lines (WEHI231 and A20) responded to IGF-I in vitro,
correlating with the absence of IGF-IR up-regulation or
IGF-I-dependent activation of the IGF-IR/IRS-2/PI 3'K cascade in these
cell lines. These results demonstrate that IGF-I can be an important
mitogen-inducing in vitro cell proliferation of
raf/myc and chemical-induced plasma cell tumors
but not earlier stage B lymphomas. The lack of proliferation to IGF-I
in abl/myc tumors suggests that abl
may bypass any requirement for IGF-IR, possibly by direct activation of
the IRS-2/PI 3'K/p70S6K and SHC/MAPK pathways.
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A, an increase in receptor protein levels of more than 2-fold
was observed in both 7.2/IGF-IRKR and 12.2/IGF-IRKR-1 transfectants.
Expression of IGF-IRKR mutant in these lines was also confirmed by flow
cytometric analysis (not shown). IGF-IR tyrosine phosphorylation was
largely eliminated in the IGF-IRKR mutant transfectants in comparison
to that in the parental lines (Fig. 7B
). Correspondingly,
the kinase activity of IGF-IR was similarly reduced in both
transfectants (Fig. 7C
). Thus, the mutant receptor acts in a
dominant negative fashion to trans-inhibit phosphorylation
of endogenous receptor and provides us a tool to analyze the role of
IGF-IR in vivo.
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, mice injected with parental lines began developing tumors by
approximately day 10, with nearly 100% incidence by day 30. In
striking contrast, expression of IGF-IRKR in line 7.2 suppressed
tumorigenesis in all animals for more than 30 days postinjection. Two
independently derived 12.2 transfectants were similarly assayed. No
tumors were observed in 60% of mice receiving the 12.2/IGF-IRKR-1
line. Tumors that did develop were markedly delayed. The second line,
12.2/IGF-IRKR-2, was selected for high IGF-IR expression by
fluorescence activated cell sorting. Inoculation of this line resulted
in 100% of animals remaining tumor free for the duration of the
experiment. These results strongly suggest that IGF-I is not only a
proliferating factor for plasma cell tumor growth in vitro,
but expression and activation of IGF-IR are also likely to be
obligatory events for the development of or the maintenance of these
tumors in vivo.
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DISCUSSION |
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. As a consequence, an IGF-I-responsive downstream cascade
including IRS-2, PI 3'K and p70S6K (Figs. 2 and 3)
is activated in the
former two tumor types. This cascade is also activated in the
abl/myc lines in the absence of IGF-IR up-regulation, most
likely by direct phosphorylation of IRS-2 by v-abl, because
v-abl is co-precipitated with IRS-2 in
vivo.3
When raf/myc and chemical lines are grown under normal
conditions (10% FCS), IRS-2 is constitutively phosphorylated (Fig. 2A
) and associates with PI 3'K (Fig. 3A
). Thus,
the IRS-2/PI 3'K portion of the cascade is constitutively activated in
all plasma cell tumors, irrespective of the inducing agent,
representing identification of a common deregulated biochemical pathway
in plasma cell malignancy.
The signaling pathways used by three kinds of plasma cell tumors
and B cell lymphomas differ greatly. Whereas
raf/myc and chemical tumors respond to IGF-I for
the activation of IRS-2/PI 3'K/p70S6K cascade,
abl/myc plasma cell tumors additionally possess
an activated SHC/MAPK pathway. Activation the SHC/MAPK pathway was
reported previously in abl-transformed pre-B cell lymphomas
(35)
. Although no explanation could be provided to address
how raf-transformed tumors did not activate MAPK pathway,
its sole stimulation by abl/myc tumors may
represent the differences of IGF-IR versus abl
tyrosine kinase in activating this pathway. Furthermore, whether IGF-I
is the only ligand existing in the serum responsible for IRS-2
phosphorylation in both chemical and raf/myc tumors remains
to be determined, because no constitutive activation of the
up-regulated IGF-IR was observed in these two plasma cell tumors (see
Figs. 1A
and 2).
The IRS-2/PI 3'K/p70S6K cascade is not constitutively activated in a
series of B cell lymphomas even in the presence of serum and can only
be activated by exogenous ligand, preferentially IL-4 (see Figs. 2–4
).
Furthermore, constitutive activation of MAPK in the two B cell
lymphomas may indicate that the cooperation between PI 3'K and MAPK may
be obligatory for the B cell lymphoma development. The definitive role
played by IRS-2/PI 3'K/p70S6K cascade in the pathogenesis of B cell and
plasma cell tumor development awaits further determination. We are
currently attempting to express several mutant molecules potentially
blocking either IRS-2/PI 3'K/p70S6K or SHC/MAPK cascade in these two
sets of B lymphoid malignancies and to test tumorigenicity in
vivo.
The biological significance of deregulation of the IGF-IR pathway
was demonstrated both in vitro and in vivo. Lines
with IGF-IR up-regulation responded mitogenically to exogenous IGF-I.
Response was proportional to the level of receptor expression (Fig. 6)
,
indicating that IGF-I is a functional growth factor for these cells. Of
greater significance, in terms of lymphoid tumor biology, are the
results of in vivo experiments with lines transfected with a
kinase inactive form of the IGF-IR. Expression of IGF-IRKR in the two
highest receptor-expressing lines inhibited endogenous IGF-IR
autophosphorylation and kinase activity (Fig. 7)
. Results of in
vivo inoculation of these lines revealed that for the highest
IGF-IR expresser (12.2), 60% of animals were protected from tumor
development, and tumors that did develop were markedly delayed. Using a
second 12.2 transfectant selected for higher mutant receptor levels,
tumor-free incidence was increased to 100% (Fig. 8)
. For the 7.2 line,
tumor-free survival was similarly striking in that 100% of animals
remained negative for tumor development for the course of the
experiment. These studies strongly suggest that activation of the
IGF-IR pathway is obligatory for either the development or survival of
plasma cell tumors in vivo. The pathological importance of
up-regulation and activation of this receptor is readily noted in that
its ligand, IGF-I, is one of the major growth factors present in plasma
and other body fluids. Thus, a potential paracrine loop between IGF-I
and up-regulated receptor may promote deregulated growth. We speculate
that expression of KR mutant will greatly inhibit growth of
chemical-induced tumors, because the level of endogenous IGF-IR
up-regulation was lower than that of raf/myc lines. Another
prediction from these studies is that abl/myc tumors, in
which IGF-IR is not up-regulated, will be unaffected in terms of
in vivo growth by the dominant negative mutant of IGF-IR. To date,
we have been unable to express the mutant in these lines, so that an
experimental test of this hypothesis awaits further studies.
Although IGF-IR up-regulation in both retrovirally and chemically induced tumors further supports the suggestion of a key role in tumorigenesis, mechanisms underlying receptor induction are currently unknown. The highest levels of receptor protein are associated with raf-mediated tumor development, suggesting that raf overexpression and/or cooperation with myc leads to up-regulation. On the other hand, several other proteins, including IRS-2, PI 3'K, and members of the Bcl-2 family, are not up-regulated.4 It will be interesting to test whether raf/myc expression would change the status of p53 and WT1 tumor suppressor genes, because mutations of them leading to inactivation of their tumor suppressing functions greatly enhanced the IGF-IR promoter activity (36, 37) . In addition, N-myc overexpression also led to increased IGF-IR expression and promoter activity (38) . Currently, we are testing whether IGF-IR up-regulation relies on the transcriptional activation and, if so, to dissect the promoter region of IGF-IR.
Although molecular mechanisms associated with human myeloma are largely unknown, the current studies using the murine model raise the possibility that the IGF-I/IGF-IR pathway may also be important for the development of human disease. In fact, it has been demonstrated previously that some myeloma lines undergo a mitogenic response to IGF-I (39) , although subsequent steps in a signaling cascade have not been defined. Preliminary results5 indicate significant IGF-IR expression in myeloma cell lines, and studies are in progress to determine whether the IGF-IR pathway plays a role in the development of these tumors. Pursuit of this line of investigation may eventually lead to a better understanding of molecular mechanisms involved in myeloma and may help identify possible points in biochemical pathways amenable to therapeutic intervention.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Department of Oncology/Lombardi Cancer Center, Georgetown
University Medical Center, NRB, E407, 3970 Reservoir Road, N.W.,
Washington, D.C. 20007. Phone: (202) 687-8387; Fax: (202) 687-6402;
E-mail: wwl{at}gunet.georgetown.edu 
2 The abbreviations used are: IGF-IR,
insulin-like growth factor I receptor; IGF-I, insulin-like growth
factor I; IRS, insulin receptor substrate; PI 3'K, phosphatidylinositol
3'-kinase; MAPK, mitogen-activated protein kinase; SHC, Src
homology and collagen.
3 M. Heller and S. Rudikoff, unpublished
observations.
4 W. Li and S. Rudikoff, unpublished
observations.
5 N. Ge and S. Rudikoff, unpublished
observations.
Received 1/20/00. Accepted 5/11/00.
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, 7.2; 
,
7.2/IGF-IRKR; 
, 12.2; 
, 12.2/IGF-IRKR-1; 
, 12.2/IGF-IRKR-2.