| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 42, 39684-39695, October 18, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the
Received for publication, March 22, 2002, and in revised form, July 20, 2002
The insulin receptor (IR) and the
insulin-like growth factor I receptor (IGF-IR) have a highly
homologous structure, but different biological effects. Insulin and
IGF-I half-receptors can heterodimerize, leading to the
formation of insulin/IGF-I hybrid receptors (Hybrid-Rs) that bind IGF-I
with high affinity. As the IR exists in two isoforms (IR-A and IR-B),
we evaluated whether the assembly of the IGF-IR with either IR-A or
IR-B moieties may differently affect Hybrid-R signaling and biological
role. Three different models were studied: (a) 3T3-like
mouse fibroblasts with a disrupted IGF-IR gene (R The insulin receptor (IR)1 and the insulin-like growth
factor (IGF) I receptor (IGF-IR) are
tetrameric glycoproteins composed of two extracellular Given the high degree of homology, the insulin and IGF-I half-receptors
(composed of one The human IR exists in two isoforms (IR-A and IR-B), generated
by alternative splicing of the insulin receptor gene that either excludes or includes 12 amino acid residues encoded by a small exon
(exon 11) at the carboxyl terminus of the IR To investigate these issues, we used three different cellular models.
First, we used R We found that each of the IR isoforms is equally able to form hybrids
with the IGF-IR. Hybrid-RsA and Hybrid-RsB,
however, have different functional characteristics.
Hybrid-RsB have a high affinity only for IGF-I.
Hybrid-RsA have an even higher affinity for IGF-I and bind
also IGF-II and insulin. Insulin binding to Hybrid-RsA
phosphorylates the IGF-IR These data therefore suggest that the relative abundance of IR isoforms
modulates the activation of the IGF system by regulating both binding
and signaling characteristics of Hybrid-Rs. They also provide clues to
the mechanism by which insulin may activate the IGF-IR phosphorylation
cascade and biological effects in a tissue-specific manner. These
findings may have important implications for cell biological responses
to insulin, IGF-I, and IGF-II.
Materials
The pNTK2 expression vectors containing the cDNAs for the A
(Ex11 The following materials were purchased from the indicated
manufacturers: fetal calf serum, glutamine, LipofectAMINE, and DNase I
from Invitrogen (Paisley, UK); RPMI 1640 medium, Dulbecco's modified
Eagle's medium, minimum essential medium, Ham's nutrient mixture
F-12, bovine serum albumin (BSA; radioimmunoassay grade), bacitracin,
phenylmethylsulfonyl fluoride (PMSF), puromycin, bromodeoxyuridine (BrdUrd), and porcine insulin from Sigma; protein G-Sepharose from Amersham Biosciences (Uppsala, Sweden); and
125I-labeled IGF-I (specific activity of 11.1 MBq/µg)
from PerkinElmer Life Sciences (Zaventem, Belgium). IGF-I and IGF-II
were obtained from Calbiochem, and FuGENE 6 transfection reagent was
obtained from Roche Molecular Biochemicals (Mannheim, Germany).
The following anti-IR antibodies were employed: monoclonal antibodies
MA-10 and MA-20 (which recognize the IR The following anti-IGF-IR antibodies were employed: monoclonal antibody
Cells
ARO cells were kindly provided by Dr. A. Pontecorvi
(Regina Elena Cancer Institute, Rome, Italy). A549, IM-9,
HepG2, MDA-MB157, and PC-3 cells were obtained from
American Type Culture Collection. R Transfection Experiments
R Preparation of Cell Lysate
Cells were grown until 80% confluent and serum-starved 24 h before stimulation with the various ligands. For receptor and ERK/Akt activation, cells were stimulated with 10 nM
insulin, IGF-I, or IGF-II for 10 min. For in vitro
Crk phosphorylation, cells were stimulated with 50 nM
insulin, IGF-I, or IGF-II for 5 min. After three washes with
ice-cold PBS, cells were lysed in cold radioimmune precipitation assay
buffer containing 50 mM Tris (pH 7.4), 150 mM
NaCl, 0.5% Nonidet P-40, 0.5% Triton X-100, 0.25% sodium
deoxycholate, 10 mM sodium pyrophosphate, 1 mM
NaF, 1 mM sodium orthovanadate, 2 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml pepstatin, and 10 µg/ml leupeptin.
After being scraped, samples were rotated for 15 min at 4 °C.
Insoluble material was separated from the soluble extract by
microcentrifugation at 10,000 × g for 10 min at
4 °C. Protein concentration was determined by the Bradford assay.
Ligand Binding Assay for the Hybrid-RA or
Hybrid-RB
Either the Hybrid-RA or Hybrid-RB was
captured by incubating cell lysates for 22 h in Maxisorp
Break-Apart immunoplates (Nunc, Roskilde, Denmark) precoated with 2 µg/ml antibody 83-7. After washing, the immunocaptured receptors were
incubated with 125I-labeled IGF-I (10 pM in 50 mM HEPES-buffered saline (pH 7.6) containing 0.05% Tween
20, 1% BSA, 2 mM sodium orthovanadate, 1 mg/ml bacitracin,
and 1 mM PMSF) in the presence or absence of increasing
concentrations of various unlabeled ligands (insulin, IGF-I, and
IGF-II). After 2 h at room temperature, the plates were washed,
and the radioactivity in each well was counted in a IR, IGF-IR, and Hybrid-R Measurements
Cell lysates were prepared as described above and used for
receptor measurement both by ELISA and Western blot analysis.
ELISA--
The characteristics and specificity of these ELISAs
have been previously described (18). Receptors were captured by
incubating lysates (0.5-60 µg/well) in Maxisorp immunoplates
precoated with the specific monoclonal antibody (2 µg/ml) indicated
below. After washing, the immunocaptured receptors were incubated with
the specific biotinylated monoclonal antibody indicated below (0.3 µg/ml in 50 mM HEPES-buffered saline (pH 7.6) containing
0.05% Tween 20, 1% BSA, 2 mM sodium orthovanadate, 1 mg/ml bacitracin, and 1 mM PMSF) and then with
peroxidase-conjugated streptavidin. The peroxidase activity was
determined colorimetrically by adding 100 µl of
3,3',5,5'-tetramethylbenzidine (0.4 mg/ml in 0.1 M
citrate/phosphate buffer (pH 5.0) with 0.4 µl/ml 30%
H2O2). The reaction was stopped by the addition
of 1.0 M H3PO4, and the absorbance
was measured at 450 nm.
IRs were captured with anti-IR antibody MA-20 and detected with
biotinylated anti-IR antibody CT-1 (30, 33). IGF-IRs were captured with
anti-IGF-IR antibody
The minimal detectable amount of hybrids was 0.125 ng/well (1.25 ng/ml). The assay was linear from 0.125 to 1.0 ng/well. There was no
interference from either 1 ng/well purified IR (from human IR
cDNA-transfected NIH/3T3 cells) or 1 ng/well purified IGF-IR (from
human IGF-IR cDNA-transfected Chinese hamster ovary cells). Multiple dilutions of cells and tissues containing either
Hybrid-RsA or Hybrid-RsB produced
dose-response curves parallel to those obtained with the purified
IR/IGF-IR hybrid standard (Ref. 18 and data not shown). Intra-assay
coefficients of variation were <7% at 0.5 ng/tube and <8%
at 1.0 ng/tube. Inter-assay coefficients of variation were <8 and
<10%, respectively (18).
The ELISAs for the IR and IGF-IR had similar characteristics of
sensitivity and specificity, as previously described (18). Purified
IGF-IR or Hybrid-R (up 1 ng/well) did not interfere in the IR assay,
and purified IR or Hybrid-R did not interfere in the IGF-IR assay. The
minimal detectable amounts were 0.05 ng/tube for the IR and 0.0625 ng/tube for the IGF-IR. Intra-assay coefficients of variation were
<8%, and inter-assay coefficients of variation were <10% for
both assays (18).
Western Blotting--
To confirm data obtained by ELISA,
aliquots of the same lysates were subjected to Western blot analysis.
Cell lysates were incubated at 4 °C under constant rotation for
2 h with 4 µg of the specific anti-receptor antibody and then
for 2 h with protein G-Sepharose. Immunoprecipitates were eluted
and subjected to SDS-PAGE and then immunoblotted (1 µg/ml) as
described below. IRs were immunoprecipitated with anti-IR antibody
MA-20 and blotted with the rabbit anti-IR polyclonal antibody. IGF-IRs
were immunoprecipitated with anti-IGF-IR antibody Hybrid-R Autophosphorylation
Western Blotting--
Cell lysates were incubated at 4 °C
under constant rotation for 1 h with protein G-Sepharose to
eliminate antibody MA-10 bound to the IR. After centrifugation, the
supernatant was incubated at 4 °C under constant rotation for 2 h with 4 µg of anti-Hybrid-R antibody 83-7 coated with protein
G-Sepharose. Immunoprecipitates were eluted and subjected to SDS-PAGE.
The resolved proteins were transferred to nitrocellulose membranes,
immunoblotted with anti-phosphotyrosine monoclonal antibody 4G10, and
revealed by an ECL method. The nitrocellulose membrane was then
stripped with Restore stripping buffer (Pierce) for 30 min at room
temperature and subsequently reprobed with the chicken anti-IGF-IR
polyclonal antibody.
ELISA--
As previously described (38), 100 µl of the cell
lysates prepared as described above were immunocaptured in Maxisorp
plates coated with antibodies 83-7 (which recognizes both the IR and Hybrid-R) and MA-20 (which recognizes the IR only) at a
concentration of 2 µg/ml in 50 mM sodium bicarbonate (pH
9.0) overnight at 4 °C. After washing, the captured
phosphorylated proteins were incubated with biotin-conjugated
anti-phosphotyrosine antibody 4G10 (0.3 µg/ml in 50 mM
HEPES (pH 7.6), 150 mM NaCl, 0.05% Tween 20, 1% BSA, 2 mM sodium orthovanadate, 1 mg/ml bacitracin, and 1 mM PMSF) for 2 h at 22 °C and then with
peroxidase-conjugated streptavidin. The peroxidase activity was
determined colorimetrically by adding 100 µl of
3,3',5,5'-tetramethylbenzidine (0.4 mg/ml in 0.1 M
citrate/phosphate buffer (pH 5.0) with 0.4 µl/ml 30%
H2O2). The reaction was stopped by the
addition of 1.0 M H3PO4, and the
absorbance was measured at 450 nm.
In Vitro CrkII Phosphorylation
In vitro receptor tyrosine kinase activity for CrkII
was measured as previously described (9) with modifications. 500 µg of proteins were immunoprecipitated with either anti-IR monoclonal antibody MA-20 or anti-Hybrid-R antibody 83-7 coupled to
protein G-Sepharose. Pellets were washed twice with radioimmune
precipitation assay buffer and twice with kinase buffer without ATP and
resuspended in 100 µl of kinase buffer containing 50 mM
HEPES (pH 7.4), 150 mM NaCl, 0.1% Triton X-100, 10 mM MgCl2, 2 mM MnCl2,
0.05% BSA, 50 µM ATP, and 1 µg of glutathione
S-transferase-Crk (provided by Dr. Raymond Birge,
Rockefeller University). Reaction mixtures were incubated at room
temperature for 20 min under continuous agitation. After rapid
centrifugation at 14,000 rpm, supernatants were collected, and 4×
sample buffer was added. Samples were boiled for 3 min; subjected to
SDS-PAGE; and transferred to nitrocellulose membranes, which were
blotted with anti-phosphotyrosine antibody 4G10. Membranes were
stripped and reprobed with anti-CrkII polyclonal antibody (Santa Cruz
Biotechnology Inc.) where required.
ERK1/2 and Akt Phosphorylation in Response to
Insulin, IGF-I, or IGF-II
After the addition of 5× sample buffer, samples were heated at
95-100 °C for 5 min and subjected to reducing SDS-PAGE on 10% polyacrylamide gel. After electrophoresis, the resolved proteins were
transferred to nitrocellulose membranes and subjected to immunoblot
analysis. For ERK1/2 activation studies, the blots were probed with the
phospho-specific ERK1/2 polyclonal antibody. For Akt phosphorylation
studies, the blots were probed with anti-phospho-Akt polyclonal
antibody. The nitrocellulose membranes were then stripped with
stripping buffer for 30 min at room temperature and subsequently reprobed with either anti-ERK1/2 polyclonal antibody or anti-Akt polyclonal antibody. All immunoblots were revealed by the ECL method,
autoradiographed, and subjected to densitometric analysis.
IR Isoform RT-PCR
RT-PCR for IR isoforms was carried out as previously described
(39) using oligonucleotide primers spanning nucleotides
2230-2251 (5'-AAC-CAG-AGT-GAG-TAT-GAG-GAT-3') and 2846-2867
(accession M10051) (5'-CCG-TTC-CAG-AGC-GAA-GTG-CTT-3') of the human IR.
PCR amplification was carried out for 30 cycles of 20 s at
96 °C, 30 s at 58 °C, and 1.5 min at 72 °C using a DNA
thermal cycler (PerkinElmer Life Sciences). After electrophoresis of
the PCR products, the 600- and 636-bp DNA fragments representing the
Ex11 Migration Assays
Cell migration assays were performed as previously described
(40, 41) with minor modifications using modified Boyden chambers (6.5-mm diameter, 10-µm thickness, 8-µm pores; Transwell, Costar Corp., Cambridge, MA) containing polycarbonate membranes coated with 10 µg/ml collagen type IV. 36 h after transfection, HepG2 cells
were serum-starved for 12 h. Cells were then removed from the
plates with Hanks' balanced salt solution containing 5 mM EDTA, 25 mM HEPES (pH 7.2), and 0.01% trypsin; resuspended
at 106 cells/ml; and added to the top of each migration
chamber. Cells were allowed to migrate to the underside of the top
chamber for 6 h in the presence or absence of 10 nM
insulin, IGF-I, or IGF-II, which had been added to the lower chamber.
Filters containing migrated and non-migrated cells were incubated with
X-gal (Promega) as substrate according to the manufacturer's
recommendations. Total cells stained with X-gal were scored using a
×40 objective. The non-migrated cells on the upper membrane surface
were removed with a cotton swab, and the migrated cells attached to the
bottom surface of the membrane stained with X-gal were counted as
described above. Cell migration was expressed as the percent of
migrated cells over total cells. Each determination was performed in triplicate.
BrdUrd Incorporation
HepG2 cells were seeded onto coverslips in six-well plates in
complete medium. 24 h later, they were transfected with empty vector-IR-A/IR-B + histone H2B-GFP in triplicates as described above. 12 h later, the medium was replaced with Dulbecco's
modified Eagle's medium and 0.1% BSA, and the cells were
serum-starved for 24 h. Then, 10 nM insulin, IGF-I, or
IGF-II was added, and the cells were further incubated for 36 h.
Cells were incubated with 10 µM BrdUrd for 1 h,
fixed in 3.7% paraformaldehyde in PBS for 15 min at room temperature,
and incubated with 50 nM NH4Cl in PBS. Cells
were then permeabilized with 0.3% Triton X-100 in PBS; incubated with
blocking solution containing 10% normal goat serum in PBS for 45 min
at room temperature; and exposed to a mixture containing anti-BrdUrd
antibody (diluted 1:200 in PBS plus 10% normal goat serum), 20 mM MgCl2, 0.5% Nonidet P-40, and DNase I
(1:500) for 1 h at room temperature. Coverslips were washed three
times with PBS and incubated with Texas Red-conjugated goat anti-mouse
antibody (1:200) in PBS plus 10% normal goat serum for 45 min at room
temperature. Cells were counterstained with Hoechst 33258, and
coverslips were mounted onto glass slides with gel/ mount
(Biomeda). Coverslips were scored at ×40 magnification under an
Olympus microscope, and images were randomly acquired with an ORCA
digital camera (Hamamatsu) and superimposed with ImageProPlus software.
Numbers were calculated as the percent of BrdUrd-incorporating cells
among GFP-positive cells, and the increases induced by growth factors
were calculated as the percent over basal levels.
IR-A and IR-B Moieties Can Form Hybrid-Rs with the Same
Efficiency
Transfected R Established Human Cell Lines--
To study native Hybrid-R
functional characteristics in non-transfected cells, we studied a panel
of established human cell lines (IM-9 lymphoblasts, ARO thyroid cancer
cells, MDA-MB157 breast cancer cells, PC-3 prostate cancer cells, A549
lung cancer cells, and HepG2 hepatoblastoma cells). In these cells, we
measured the IR isoform relative abundance and the IR, IGF-IR, and
Hybrid-R content. With the exception of IM-9 cells, which expressed
only IR-A, the remaining cell lines expressed both IR-A and IR-B. In these cell lines, IR-A content ranged from 24 to 82% of the total IR
content. All these cells also expressed IGF-IRs and Hybrid-Rs. Hybrid-R
content was in all cases in accordance with the random assembly model
(Table II), confirming data obtained in transfected cells.
We also evaluated Hybrid-R content in HepG2 hepatoblastoma cells before
and after exposure to dexamethasone, which causes cell differentiation
and a change in the IR isoform relative abundance (29). In agreement
with previous reports, IR-A decreased from 82 to 14% of the total cell
IR content after dexamethasone-induced differentiation (Fig.
2 and Table
III). Undifferentiated HepG2 cells
therefore predominantly expressed Hybrid-RsA, whereas
differentiated HepG2 cells predominantly expressed
Hybrid-RsB.
Insulin/Insulin-like Growth Factor I Hybrid Receptors Have
Different Biological Characteristics Depending on the Insulin Receptor
Isoform Involved*
§¶,
,,,§, and
Istituto di Medicina Interna, Malattie
Endocrine e del Metabolismo, University of Catania, Ospedale Garibaldi,
95123 Catania, Italy, the § Istituto Mediterraneo di
Oncologia, 95100 Catania, Italy, and the
** Dipartimento di Medicina Clinica e Sperimentale,
University of Catanzaro, Policlinico Mater Domini, via T. Campanella
115, 88100 Catanzaro, Italy
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells)
cotransfected with the human IGF-IR and with either the IR-A or IR-B
cDNA; (b) a panel of human cell lines variably
expressing the two IR isoforms; and (c) HepG2 human
hepatoblastoma cells predominantly expressing either IR-A or IR-B,
depending on their differentiation state. We found that Hybrid-Rs
containing IR-A (Hybrid-RsA) bound to and were activated by
IGF-I, IGF-II, and insulin. By binding to Hybrid-RsA,
insulin activated the IGF-I half-receptor 
-subunit and the IGF-IR-specific substrate CrkII. In contrast, Hybrid-RsB
bound to and were activated with high affinity by IGF-I, with low
affinity by IGF-II, and insignificantly by insulin. As a consequence, cell proliferation and migration in response to both insulin and IGFs
were more effectively stimulated in Hybrid-RA-containing
cells than in Hybrid-RB-containing cells. The relative
abundance of IR isoforms therefore affects IGF system activation
through Hybrid-Rs, with important consequences for tissue-specific
responses to both insulin and IGFs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and two
transmembrane -subunits linked by disulfide bonds. Each -subunit,
containing the ligand-binding site, is ~130 kDa, whereas each
-subunit, containing the tyrosine kinase domain, is ~95-97 kDa.
These receptors share >50% overall amino acid sequence homology and
84% homology in the tyrosine kinase domains. After ligand binding,
activated receptors recruit and phosphorylate docking proteins,
including the insulin receptor substrate-1 family proteins Gab1
and Shc (1-5), leading to the activation of many intracellular
mediators, including phosphatidylinositol 3-kinase, Akt, and
ERK1/2, involved in the regulation of cell metabolism, proliferation,
and survival. Although both the IR and IGF-IR similarly activate major
signaling pathways, subtle differences exist in the recruitment of
certain docking proteins and intracellular mediators between the two
receptors (6-9). These differences are the basis for the predominant
metabolic effect elicited by IR activation and the predominant
mitogenic, transforming, and anti-apoptotic effect elicited by IGF-IR
activation (10-13). According to the classical view, insulin binds
with high affinity to the IR (100-fold higher than to the IGF-IR),
whereas both insulin-like growth factors (IGF-I and IGF-II) bind to the IGF-IR (with 100-fold higher affinity than to the IR).
- and one -subunit) can heterodimerize, leading
to the formation of insulin/IGF-I hybrid receptors (Hybrid-Rs) (14-16). In many tissues, Hybrid-Rs are the most represented receptor subtype (17). Hybrid-Rs may also be overexpressed in a variety of human
malignancies as a result of both IR and IGF-IR overexpression (18-21).
However, the biological role of these Hybrid-Rs is still unclear.
Functional studies have indicated that Hybrid-Rs behave more like
IGF-IRs than IRs because they bind to and are activated by IGF-I with
an affinity similar to that of the typical IGF-IR. In contrast,
Hybrid-R activation in response to insulin occurs with much lower
affinity (22, 23). Hybrid-Rs are therefore believed to provide
additional binding sites to IGF-I and to increase cell sensitivity to
this growth factor (17-19). These studies have not, however, taken
into account the different IR isoform contribution to Hybrid-R
formation and function.
-subunit (see Table I).
The relative abundance of IR isoforms is regulated by tissue-specific
and unknown factors (24, 25). Recently, we found that IR-A (but not
IR-B) binds IGF-II with high affinity and behaves as a second
physiological receptor for IGF-II in fetal and dedifferentiated
(malignant) cells (26-28). We therefore hypothesized that the relative
abundance of the two isoforms may affect the functional properties of
Hybrid-Rs and modulate, in this way, the activation of the IGF system.
fibroblasts, which are 3T3-like cells
derived from IGF-IR knockout mice. These cells also have low levels of
endogenous IR. We cotransfected these cells with both the human IGF-IR
gene and a construct encoding either IR-A or IR-B to obtain cells
expressing either Hybrid-RsA or Hybrid-RsB,
respectively (see Table I). Second, we employed a panel of human cell
lines that express the two IR isoforms in variable amounts. Third, we
used HepG2 hepatoblastoma cells that express predominantly either IR-A
or IR-B depending on the culture conditions (29).
-subunit and activates CrkII, an
IGF-IR-specific substrate. Accordingly, cell transfection with IR-A
cDNA (but not with IR-B cDNA) markedly increases cell motility
in response not only to IGF-I, but also to insulin and IGF-II.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) and B (Ex11+) isoforms of the human IR
were kindly provided by Dr. Axel Ullrich (Max Planck Institute
of Biochemistry, Martinsried, Germany). The pECE expression vector
containing the cDNA encoding the human IGF-IR was a gift of Dr. R. Roth (Department of Molecular Pharmacology, Stanford University,
Stanford, CA). The pCH110 expression vector for -galactosidase was
kindly provided by Dr. F. Tatò (Universitá di Roma
"La Sapienza," Rome, Italy). The expression vector for pBOS-H2B-GFP
was kindly provided by Dr. J. Y. Wang (University of
California at San Diego, San Diego, CA).
-subunit, but only poorly
recognize the Hybrid-R) (Dr. I. D. Goldfine, University of
California at San Francisco, San Francisco, CA) (30, 31); monoclonal
antibody CT-1 (which recognizes the IR -subunit) and monoclonal
antibody 83-7 (which recognizes the -subunits of both the IR and
Hybrid-R) (Dr. K. Siddle, University of Cambridge, Cambridge,
UK) (32, 33); a rabbit polyclonal antibody that recognizes the IR
-subunit (Transduction Laboratories, Lexington, KY); and polyclonal
antibody 29B4 (which recognizes the IR -subunit) (Santa Cruz
Biotechnology Inc., Santa Cruz, CA).
IR-3 (which recognizes the IGF-IR -subunit and only poorly
recognizes the Hybrid-R) (Oncogene Research, Cambridge, MA) (34);
monoclonal antibody 17-69 (which recognizes the -subunits of both
the IGF-IR and Hybrid-R) (Dr. K. Siddle) (35); and a chicken polyclonal
antibody that recognizes the IGF-IR -subunit (Upstate Biotechnology,
Inc., Lake Placid, NY). Anti-phospho-ERK1/2 and anti-phospho-Akt
antibodies were purchased from New England Biolabs (Beverly, MA);
anti-phosphotyrosine monoclonal antibody 4G10 was from Upstate
Biotechnology, Inc.; and anti-BrdUrd antibody was from BD PharMingen
(Erembodegem, Belgium).
mouse
fibroblasts (3T3-like mouse cells derived from animals with a targeted
disruption of the IGF-IR gene, expressing ~5 × 103
insulin receptors/cell) were kindly provided by Dr. R. Baserga (Kimmel Cancer Center, Jefferson University, Philadelphia, PA) (Table I). HepG2 and MDA-MB157 cells were
routinely grown in minimum essential medium supplemented with 10%
fetal bovine serum. A549, PC-3, IM-9 and ARO cells were routinely grown
in RPMI 1640 medium supplemented with 10% fetal bovine serum. The
R mouse fibroblasts were routinely grown in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum.
Description of receptors and tranfected cells studied
cells were grown in 35-mm plates until 60-70%
confluent. They were first transfected with 2 µg of pECE expression
vector containing the cDNA encoding the IGF-IR (36) and
cotransfected with 0.2 µg of pSV2 plasmid encoding the hygromycin
resistance gene by the LipofectAMINE method according to the
manufacturer's protocol. Cells were then subjected to antibiotic
selection in medium supplemented with 400 µg/ml hygromycin for 3 weeks. Stably transfected clones were tested for receptor content by
ELISA. Cell clones were further transfected with the pNTK2 expression vector containing the cDNA for either the A (Ex11) or
B (Ex11+) isoform of the human IR (37) and cotransfected
with the pPDV6+ plasmid encoding the puromycin resistance
gene. Cells were subsequently subjected to antibiotic selection in
medium supplemented with 400 µg/ml hygromycin and 2.4 µg/ml
puromycin for 3 weeks. Receptor content was evaluated in selected
clones by ELISA. Cell clones expressing similar amounts of either IR-A
or IR-B, IGF-IR, and Hybrid-R (either the Hybrid-RA or
Hybrid-RB) were selected for subsequent studies. For
migration studies, HepG2 cells were transiently transfected by the
FuGENE 6 method according to the manufacturer's protocol. Briefly,
4 × 105 cells were seeded in six-well plates and
grown for 24 h in complete medium (minimum essential medium
with 10% fetal bovine serum). Thereafter, a transfection mixture
containing 2 µg of pNTK2-IR-A/IR-B + 0.2 µg of -galactosidase or
histone H2B-GFP + 12 µl of FuGENE 6 in 100 µl of minimal
essential medium without serum or antibiotics was added to each well.
Cells were grown in complete medium; and after 48 h, they were
assayed for -galactosidase activity or scored under a fluorescence
microscope for GFP expression.
-counter.
IR-3 and detected with biotinylated antibody
17-69 (34, 35). Hybrid-Rs were captured with anti-IR antibody 83-7, which recognizes both the Hybrid-R and IR, and detected with
biotinylated anti-IGF-IR antibody 17-69 (32, 35). The receptor content
was evaluated by comparing each sample with a standard curve, as
previously described (18).
IR-3 and blotted
with the chicken anti-IGF-IR polyclonal antibody. Hybrid-Rs were
immunoprecipitated with anti-IR antibody 83-7 and blotted with the
chicken anti-IGF-IR polyclonal antibody. Western blot
specificity was evaluated by examining the interference of 200 ng of
purified receptor of each subtype added to a cell lysate containing
~200 ng of IR, IGF-IR, or Hybrid-R.
and Ex11+ IR isoforms were analyzed by
scanning densitometry and compared with the standards. Standard
preparation was carried out using mRNA from NIH/3T3 cells
transfected with both IR isoform cDNAs mixed at various ratios and
co-amplified by RT-PCR. To verify that the larger cDNA was really
IR-B, RT-PCR products were subjected to BanI
digestion. Only cDNA containing exon 11, the restriction site for
the enzyme, was digested.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Cells--
R cells,
which do not express endogenous IGF-IR and have low levels of
endogenous IR (which are not recognized by the anti-human IR
antibodies used), were first transfected with a plasmid
containing the cDNA of the human IGF-IR and then with a plasmid
containing either the IR-A or IR-B cDNA. The stable transfectants
obtained were evaluated for IR, IGF-IR, and Hybrid-R content, as
described under "Experimental Procedures." In these cotransfected
cell clones (IGF-IR + IR-A or IGF-IR + IR-B), Hybrid-R content was in
close accordance with the value predicted by the random assembly model, indicating that each of the two IR isoforms can form Hybrid-Rs with the
same efficiency (Table II). Western blot
analyses, carried out as described under "Experimental Procedures,"
proved to be specific for each receptor measured (Fig.
1A) and confirmed ELISA data
(Fig. 1B and Table II).
IR, IGF-IR, and Hybrid-R content in cell clones obtained from
R fibroblasts transfected with the IGF-IR and with either
the IR-A (clones A28, A25, and A48) or IR-B (clones B15, B22, and B3)
cDNA
View larger version (52K):
[in a new window]
Fig. 1.
A, specificity of Western blot analysis.
To cell lysates from transfected fibroblast cell clones containing the
IR (upper panel), the IGF-IR (middle panel), or
the Hybrid-R (lower panel) were added 200 ng of purified IR
(lane 2), Hybrid-R (lane 3), or IGF-IR
(lane 4). In measurements of each receptor, no interference
by the other two related receptors was observed. B,
expression of the IR, IGF-IR, and Hybrid-R in stably transfected
R cell clones. R cells were transfected
either with IGF-IR and IR-A cDNAs (clones R+A28,
R+A25, and R+A48) or with IGF-IR and IR-B
cDNAs (clones R+B15, R+B22, and
R+B3). Receptors were immunoprecipitated (IP)
and detected by Western blot analysis as described under
"Experimental Procedures." Clones R+A25 and
R+B22 had a similar receptor content and were selected for
functional studies. IB, immunoblot.
View larger version (28K):
[in a new window]
Fig. 2.
Time course of IR isoform expression in HepG2
cells during differentiation. HepG2 cells were cultured in
the absence (time 0) or in the presence of dexamethasone for the
indicated times, and IR isoform expression was measured by RT-PCR.
Numbers on the bottom indicate the relative abundance of IR
isoform expression (%) calculated from densitometric analysis. The
results are representative of three separate experiments. M,
MARKER 600 bp.
IR-A relative abundance and IR, IGF-IR, and Hybrid-R content in a panel
of human malignant cells and in human hepatoma HepG2 cells before and
after differentiation
Hybrid-RsA and Hybrid-RsB Have Different Binding and Activation Properties with Regard to Insulin and IGFs
To study the different binding characteristics of Hybrid-RsA and Hybrid-RsB, we used two double-transfected cell clones (R+A25 and R+B22) expressing similar amounts of either Hybrid-RsA or Hybrid-RsB (Fig. 1 and Table II). Cells were solubilized, and Hybrid-Rs were immunopurified with monoclonal antibody 83-7, which does not recognize the IGF-IR. 125I-Labeled IGF-I was then allowed to bind to immunocaptured receptors in the absence or presence of increasing concentrations of various unlabeled ligands (insulin, IGF-I, and IGF-II).
The displacement curves indicate that Hybrid-RsA bound
IGF-I with high affinity, ~8-fold higher compared with
Hybrid-RsB (Fig. 3).
Moreover, Hybrid-RsA also bound insulin and IGF-II with an
affinity ~30-fold higher than that of Hybrid-RsB. In
contrast, Hybrid-RsB bound only IGF-I with high affinity
(Fig. 3). Half-maximal inhibition of 125I-labeled IGF-I
(EC50) by the three ligands in both Hybrid-RsA
and Hybrid-RsB is given in Table
IV.
|
|
To compare the ligand affinity of Hybrid-Rs with that of homodimeric
receptors, R cells were stably transfected with cDNAs
for IGF-IR, IR-A, or IR-B. Binding studies were carried out on
immunopurified receptors from these cells by displacing either
125I-labeled IGF-I or 125I-labeled insulin with
increasing concentrations of unlabeled ligands (insulin, IGF-I, and
IGF-II). As previously reported (26), the IGF-IR bound both
IGFs (but not insulin) with high affinity, and both IR isoforms bound
insulin with high affinity and IGF-I poorly. However, only IR-A bound
IGF-II with high affinity. EC50 values are given in Table
IV.
Data consistent with those obtained in stable transfectants of
R cells were also obtained in Hybrid-Rs immunopurified
from HepG2 cells (Fig. 3). In undifferentiated HepG2 cells (which
predominantly express IR-A and Hybrid-RsA), IGF-I, IGF-II,
or insulin displaced 125I-labeled IGF-I with an affinity in
the physiological concentration range (EC50 = 0.4, 0.6, and
4.5 nM, respectively). In contrast, in differentiated HepG2
cells (which predominantly express IR-B and Hybrid-RsB),
the EC50 values were 1.8 for IGF-I, 4.0 for IGF-II, and 20 nM for insulin (Fig. 3).
The binding characteristics of Hybrid-Rs were also studied in a variety of established human cell lines (Table II). In Hybrid-Rs immunopurified from IM-9 cells (which express only IR-A and Hybrid-RsA) or from PC-3, MDA-MB157, and ARO cells (all which predominantly express Hybrid-RsA), both IGFs and insulin efficiently displaced 125I-labeled IGF-I. EC50 values ranged 0.2 to 0.6 nM for IGF-I, 0.3 to 0.7 nM for IGF-II, and 1.8 to 3.2 nM for insulin. In contrast, in A549 cells (which predominantly express IR-B (76%) and Hybrid-RsB), the EC50 values were 1.5 nM for IGF-I, 10 nM for IGF-II, and >100 nM for insulin.
Receptor autophosphorylation was evaluated in intact cells expressing
either only Hybrid-RsA or Hybrid-RsB after
exposure to either insulin or IGFs in the presence of a molar excess of
the IR-blocking antibody MA-10, which does not recognizes Hybrid-Rs, as
evaluated by immunoprecipitation experiments (data not shown). This
procedure was used to avoid the interference of IRs. Cells were then
solubilized, and receptors were immunopurified with antibody 83-7 (which recognizes the IR and Hybrid-R, but not the IGF-IR).
Autophosphorylation/activation of immunopurified Hybrid-Rs was measured
by Western blotting. As shown in Fig.
4A, IGF-I, IGF-II, and insulin
were all able to efficiently activate Hybrid-RsA, whereas
only IGF-I was able to efficiently activate Hybrid-RsB.
Both IGF-II and insulin were much less effective in
Hybrid-RsB than in Hybrid-RsA. Similar results
were obtained in parallel experiments in which Hybrid-R
autophosphorylation was quantitated by ELISA (Fig. 4B). These autophosphorylation data are therefore in close accordance with
results from binding studies and suggest that Hybrid-RsA
may be regarded as additional receptors for IGF-I, IGF-II, and also
insulin, whereas, in contrast, Hybrid-RsB should be
regarded as selective receptors for IGF-I.
|
Hybrid-RsA (but Not Hybrid-RsB) Shift Insulin to IGF-IR Signaling
Because insulin bound to the Hybrid-RA with an
affinity within the physiological range, we evaluated the ability of
insulin to activate the IGF-IR -subunit of the
Hybrid-RA. For this purpose, either R+A25 or
R+B22 cell clones were stimulated with insulin, IGF-I, or
IGF-II and then solubilized as described under "Experimental
Procedures." Samples were immunoprecipitated with
anti-phosphotyrosine antibody 4G10, subjected to SDS-PAGE, and blotted
with anti-IGF-IR antibody. In R+A25 cells, which express
only Hybrid-RsA, insulin recruited the IGF-IR to the
tyrosine phosphorylation cascade with a potency similar to that of
IGF-II, albeit lower than that of IGF-I (Fig.
5). By contrast, in R+B22
cells, which express only Hybrid-RsB, IGF-IR recruitment by
insulin was very weak and much lower than that induced by IGF-I or
IGF-II (Fig. 5). Reblotting with anti-phosphotyrosine antibody 4G10
showed that, in R+A25 cells, IGF-II stimulated the tyrosine
phosphorylation of the 97-kDa band (containing both the IR and IGF-IR
-subunits) with a higher potency than in R+B22
cells.
|
We then evaluated whether insulin, via the Hybrid-RA, is
able to activate IGF-IR-specific intracellular mediators like the small
adapter protein CrkII, which is phosphorylated by the IGF-IR, but not
by the IR (9, 42, 43). To this purpose, either R+A25 or
R+B22 cell clones were stimulated with insulin or IGF-I,
and immunopurified receptors were incubated with CrkII and ATP in
kinase buffer as described under "Experimental Procedures." When
IRs were immunopurified (with antibody MA-20), no CrkII phosphorylation
was observed (Fig. 6), confirming that
CrkII is not a substrate of the IR. In contrast, when Hybrid-Rs were
immunopurified (with antibody 83-7), Hybrid-RsA (but not
Hybrid-RsB) were able to phosphorylate CrkII in response to
insulin (Fig. 6), a difference that may be explained by the high
affinity of insulin for Hybrid-RsA. Both
Hybrid-RsA and Hybrid-RsB were able to
phosphorylate CrkII in response to IGF-I. Taken together, these data
suggest that insulin may activate IGF-IR-specific intracellular
pathways by interacting with Hybrid-RsA.
|
Hybrid-RA and Hybrid-RB Post-receptor Signaling
Double-transfected R+A25 and R+B22 cell
clones were used to study the ligand ability to activate the
post-receptor signaling pathways in intact cells expressing similar
amounts of the three receptor subtypes (IGF-IR, IR, and Hybrid-R), but
different isoforms. Parallel experiments were also carried out
in cells containing only IR-A (RIR-A cells), IR-B
(RIR-B cells), or IGF-IR (R+ cells). Cells
were exposed to each ligand (10 nM) for 10 min, and
phosphorylation of the intracellular substrates ERK1/2 kinase (p42/p44
mitogen-activated protein kinase) and Akt was subsequently measured by
Western blotting.
Both substrates ERK1/2 and Akt had similar activation patterns in
response to the different ligands. Insulin was the most potent
stimulating factor in both double-transfected cell clones, as expected
by the presence of elevated IR levels (Fig.
7). IGF-II was approximately as potent as
IGF-I in R+A25 cells (Fig. 7) because of its high affinity
for both IR-A and Hybrid-RsA, whereas it was less potent
than IGF-I in R+B22 cells (Fig. 7), in accordance with data
obtained from the anti-phosphotyrosine antibody blot in Fig. 5. These
data confirm that IR-A predominance enhances the cell sensitivity to
IGF-II (which can bind to IGF-IRs, IR-A, and Hybrid-RsA).
Similar results were obtained in HepG2 cells: undifferentiated cells
(mostly expressing Hybrid-RsA) behaved similarly to
R+A25 cells, whereas differentiated cells (mostly
expressing Hybrid-RsB) behaved similarly to
R+B22 cells (data not shown).
|
In cell clones containing only IR-A, both insulin and IGF-II stimulated
Akt and ERK1/2 phosphorylation to a similar extent (Fig.
8). In contrast, in cell clones
containing only IR-B, insulin (but not IGFs) was able to stimulate Akt
and ERK1/2 phosphorylation. In R+ cells (which express only
the IGF-IR), the two IGFs were roughly equally potent in stimulating
Akt and ERK1/2 phosphorylation, whereas insulin was not very effective
(Fig. 8).
|
Biological Effects of Either Insulin or IGFs in Cells Predominantly Expressing Either Hybrid-RsA or Hybrid-RsB
We evaluated whether the presence of Hybrid-RsA or
Hybrid-RsB may affect cell biological responses (such as
cell proliferation and migration) to either insulin or IGFs. To avoid
possible proliferation and migration differences due to the
differentiation state, undifferentiated HepG2 cells were forced to
overexpress either Hybrid-RsA or Hybrid-RsB by
transient IR-A or IR-B cDNA transfection. Control cells were obtained by transfection of an empty vector. Transfection efficiency, evaluated by histone H2B-GFP and -galactosidase, ranged from 15 to 20% (Fig. 9A).
|
Cell proliferation was measured by scoring BrdUrd-labeled nuclei in GFP-positive cells. Both IR-A and IR-B transfection enhanced cell proliferation in response to insulin as compared with empty vector transfection. By contrast, only IR-A transfection significantly enhanced cell proliferation in response to both IGFs. IR-B transfection only slightly enhanced proliferation in response to IGF-I and was totally ineffective for IGF-II-stimulated cell proliferation (Fig. 9B).
We also measured cell migration by scoring -galactosidase-positive
cells that migrated to the lower side of Transwells (Fig. 10A). IR-A transfection
significantly enhanced cell migration in response to all three ligands
as compared with empty vector transfection. In contrast, IR-B
transfection only slightly enhanced cell migration in response to
IGF-I, but not in response to insulin or IGF-II (Fig.
10B).
|
Taken together, these data suggest that the relative abundance of IR
isoforms differentially regulates two major biological effects (such as
cell proliferation and migration) in response to both insulin and IGFs.
IR-A overexpression and subsequent Hybrid-RA formation
markedly enhance cell biological responses to both IGFs, whereas IR-B
overexpression does not. In addition, whereas cell proliferation in
response to insulin is activated via both IR-A and IR-B, only IR-A
increases cell migration in response to insulin, an effect most likely
mediated by the activation of the IGF signaling pathway, via insulin
binding to the Hybrid-RA.
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
The main finding of our study is that the differential expression of the two isoforms of the human IR constitutes a molecular switch for the preferential activation of either the IR or IGF-I pathway. This is determined by both binding and signaling specificities of the two Hybrid-R types that are formed. In particular, predominant IR-A expression in cells coexpressing the IGF-IR leads to increased formation of Hybrid-RsA, which up-regulates the IGF system by two different mechanisms: (a) binding and activation with high affinity by both IGF-I and IGF-II (which do not occur with the Hybrid-RB) and (b) activation of the IGF-IR pathway also after insulin binding.
In contrast, predominant IR-B expression leads to high binding specificity whereby insulin activates only its own receptor and post-receptor signaling. Moreover, IR-B will sequestrate part of the IGF-IR moieties to form Hybrid-RsB, which have a reduced affinity for IGF-I and especially for IGF-II. This combined effect will result in reduced IGF system activity.
Although IR isoforms and insulin/IGF-I hybrid receptors have been extensively studied (18, 19, 22-25, 29), their biological role was unclear. Hybrid-Rs are present in cells and tissues coexpressing both IRs and IGF-IRs and are often the most abundant receptor subtype (14, 16, 17).
Functional studies have consistently shown that Hybrid-Rs behave
similarly to homotypic IGF-IRs rather than to homotypic IRs (14-19,
22, 23). Using immunopurified receptors, Soos et al. (22)
have shown that Hybrid-Rs bind IGF-I with high affinity, similar to
typical IGF-IRs, whereas they bind insulin with much lower affinity
(~20-fold lower compared with IRs). Moreover, insulin does not
effectively displace Hybrid-R-bound IGF-I, possibly because IGF-I
interaction with the -subunit of the IGF-IR allosterically inhibits
insulin binding (23). According to these observations, Hybrid-Rs are
autophosphorylated more efficiently after binding IGF-I compared with
insulin (22).
As Hybrid-Rs are believed to result from random assembly of insulin and IGF-I half-receptors (17), their cell content is directly related to the expression level of the two receptors. Therefore, in cells expressing high IR levels, Hybrid-R content may exceed typical IR and IGF-IR content (18, 19). This will shift the major ligand binding from insulin to IGFs and may have relevant biological consequences in both metabolic disorders and cancer. For instance, increased Hybrid-R formation has been suggested to reduce the availability of typical IRs, thus contributing to insulin resistance in diabetes (44-46); however, these data are controversial. Interestingly, certain human cancers (namely thyroid and breast cancers) (18-21, 28, 47) have been shown to overexpress IRs and, as a consequence, to express very high levels of Hybrid-Rs. In these models, Hybrid-Rs were able to mediate cancer cell growth in response to IGF-I, suggesting that they may provide a selective growth advantage to malignant cells (18, 19).
No previous study has addressed the functional characteristics of the Hybrid-R with relation to the IR isoform involved. Although the precise role of the two IR isoforms is not entirely clear, this issue has become relevant following recent evidence that the relative abundance of IR isoforms is tightly regulated by tissue-specific factors, stage of development, and cell differentiation (24, 25, 29). IR-A is the predominant isoform in fetal tissues; binds IGF-II with high affinity (26); and mediates fetal growth in response to IGF-II, as also suggested by genetic studies carried out in transgenic mice (48, 49). Moreover, when cells transform and become malignant, dedifferentiation is often associated with an increased IR-A relative abundance, providing a selective growth advantage to malignant cells via an autocrine or paracrine loop with locally produced IGF-II (27, 28). IR-B is the predominant IR isoform in normal adult tissues that are major target tissues for the metabolic effects of insulin (adipose tissue, liver, and muscle) (24, 25).
In this study, we have demonstrated that each IR isoform affects
Hybrid-R biology by using three different models: transfected R mouse fibroblasts, undifferentiated and differentiated
HepG2 human hepatoblastoma cells, and a panel of human cell lines with different relative abundance of the two IR isoforms. R
mouse fibroblasts were transfected to coexpress the IGF-IR and either
IR-A or IR-B to obtain cells containing only either the Hybrid-RA or Hybrid-RB. HepG2 cells provide a
natural model expressing up to 80% IR-A of the total IR content under
basal conditions (undifferentiated state), but only ~15% after
differentiation with dexamethasone. In these models, we found that the
two IR isoforms have a similar ability to form hybrids with the IGF-IR
because Hybrid-R content, measured by a specific ELISA, was very close
to the value predicted according to the random assembly model on the
basis of the cell content of IRs and IGF-IRs.
We first studied ligand binding and observed that the two Hybrid-R types bind ligands with different affinity. Immunopurified Hybrid-RsB have a high affinity for IGF-I (ED50 = 2.5 nM IGF-I), bind IGF-II with 6-fold lower affinity, and do not appreciably bind insulin. Accordingly, Hybrid-RsB are activated by IGF-I and to a lesser extent by IGF-II and are not by insulin. In contrast, immunopurified Hybrid-RsA have a higher affinity for IGF-I (ED50 = 0.3 nM IGF-I) compared with Hybrid-RsB and bind IGF-II with a similar affinity (ED50 = 0.6 nM IGF-II) and insulin with a lower affinity (ED50 = 3.7 nM insulin), but still in the physiological range. In agreement with binding data, Hybrid-RsA can be activated by both IGFs and also by insulin.
We then studied post-receptor signaling and, more specifically, whether
insulin can induce IGF-IR -subunit phosphorylation in intact cells
expressing Hybrid-Rs. As expected from the binding data, exposure to
insulin caused IGF-IR -subunit phosphorylation in cells expressing
Hybrid-RsA, but not in cells expressing
Hybrid-RsB. Although the -subunits of the IR and IGF-IR
share >80% homology, differences exist in the recruitment of
intracellular mediators and the biological effects elicited by the two
receptors: more pronounced metabolic effects follow activation of the
IR, whereas more pronounced mitogenic, anti-apoptotic, and transforming
effects follow activation of the IGF-IR (1-13). These differences in
biological effects (6-13) are the consequence of the different
activation of intracellular mediators. CrkII is an adapter protein
consisting primarily of SH2 and SH3 domains; is a specific substrate of
the IGF-IR (9, 42, 43); and mediates certain protein-protein interactions involved in signaling pathways that lead to cytoskeletal rearrangement, cell growth, differentiation, apoptosis, and
transformation (41). We found here that CrkII is also a substrate for
IGF-I-stimulated Hybrid-Rs. Moreover, CrkII is also phosphorylated
after insulin stimulation of Hybrid-RsA (but not
Hybrid-RsB), confirming that Hybrid-RsA may
shift typical insulin signaling to IGF-IR signaling.
This phenomenon may have biological relevance in hyperinsulinemic insulin-resistant states and in cancer. In hyperinsulinemic states, elevated insulin levels are suggested to cross-react with the IGF-IR. As insulin binds the Hybrid-RA with an affinity at least 10-fold higher compared with the IGF-IR, it is likely that the most activation of the IGF system by elevated insulin levels (50) occurs via the Hybrid-RA rather than the IGF-IR. Most cancer cells do preferentially express IR-A and consequently Hybrid-RsA. In thyroid cancer, for instance, cell dedifferentiation is associated with both progressive IR-A prevalence and increased autocrine IGF-II production (28). These cancer cells therefore acquire a higher sensitivity not only to IGF-I, but also to IGF-II and insulin.
Finally, we observed that two major biological effects (such as
proliferation and migration) are differentially regulated by the same
factors depending on the prevalence of either Hybrid-RsA or
Hybrid-RsB. In HepG2 cells, proliferation and migration in
response to IGFs were greatly stimulated in cells overexpressing
Hybrid-RsA, but not in cells expressing
Hybrid-RsB. Moreover, insulin stimulated cell migration
only in cells overexpressing Hybrid-RsA, most likely via
activation of IGF-IR -subunit signaling pathways.
This study indicates for the first time that regulation of IR isoform
expression has important implications in both insulin and IGF
signaling. In cells predominantly expressing IR-A (and coexpressing the
IGF-IR), the IGF-IR intracellular cascade may be activated in response
to insulin and IGFs via Hybrid-RA activation. In contrast,
in cells predominantly expressing IR-B (as most differentiated cells
do), insulin will activate only the typical IR signaling pathway,
whereas the response to IGFs will mainly occur via typical IGF-IRs
because Hybrid-RsB have a reduced affinity for IGFs and
because insulin, at physiological concentrations, will not bind. A
better understanding of the molecular mechanisms regulating the
alternative splicing process of the IR gene will therefore provide
important information for the regulation of cell metabolism and
proliferation and other biological functions.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Drs. I. D. Goldfine and K. Siddle for kindly providing anti-IR and anti-IGF-IR antibodies. We warmly thank Dr. R. Baserga for helpful discussion and critical reading of the manuscript.
| |
FOOTNOTES |
|---|
* This work was supported in part by grants from the Associazione Italiana per la Ricerca sul Cancro and Ministero dell'Università e della Ricerca Scientifica e Tecnologica (1999, 2001) (to A. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of a fellowship from the Fondazione Giuseppe Alazio per la Ricerca sul Cancro.
Recipients of fellowships from the Fondazione Italiana per la
Ricerca sul Cancro.
To whom correspondence should be addressed. Tel.:
39-0961-712423; Fax: 39-0957-158072; E-mail: belfiore@unicz.it.
Published, JBC Papers in Press, July 22, 2002, DOI 10.1074/jbc.M202766200
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
IR, insulin
receptor;
IGF, insulin-like growth factor;
IGF-IR, insulin-like growth
factor I receptor;
ERK, extracellular signal-regulated kinase;
Hybrid-R, insulin/insulin-like growth factor I hybrid receptor;
Hybrid-RA, insulin/insulin-like growth factor I hybrid
receptor containing the insulin receptor A isoform;
Hybrid-RB, insulin/insulin-like growth factor I hybrid
receptor containing the insulin receptor B isoform;
BSA, bovine serum
albumin;
PMSF, phenylmethylsulfonyl fluoride;
BrdUrd, bromodeoxyuridine;
ELISA, enzyme-linked immunosorbent assay;
GFP, green
fluorescent protein;
PBS, phosphate-buffered saline;
RT, reverse
transcription;
X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside;
SH, Src
homology.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Avruch, J. (1998) Mol. Cell. Biochem. 182, 31-48[CrossRef] |
