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THE
JOURNAL
OF
BIOLOGICAL
CHEMISTRY
Q
1988
by The
American
Society
for
Biochemistry
and
Molecular
Biology,
Inc.
Vol.
263,
No.
23,
Issue
of
Auguat
15,
pp.
1148€-11492,1988
Printed
in
U.S.A.
Expression and Characterization
of
a
Functional Human Insulin-like
Growth Factor
I
Receptor*
(Received for publication, February
8,
1988)
George Steele-Perkins, Jennifer Turner$, Jeffrey C. Edman$, Joji Hari, Sarah
B.
Pierce,
Cynthia Stover, William
J.
RutterS, and Richard
A.
RothO
From the Department of Pharmacology, Stanford University School
of
Medicine, Stanford, California 94305-5332 and the
$Hormone Research Laboratory, University of California, Sun Francisco, California 94143
Stable transfectants of Chinese hamster ovary (CHO)
cells were developed that expressed the protein en-
coded by
a
human insulin-like growth factor
I
(IGF-I)
receptor
cDNA.
The transfected cells expressed
-25,000
high affinity receptors for IGF-I (apparent
Kd
of
1.5
X
lo-’
M),
whereas the parental CHO cells
expressed only
5,000
receptors per cell (apparent
Kd
of
1.3
X
lo-’
M).
A
monoclonal antibody specific for
the human IGF-I receptor inhibited IGF-I binding to
the expressed receptor and immunoprecipitated poly-
peptides of apparent
M.
values
-135,000
and
95,000
from metabolically labeled lysates of the transfected
cells but not control cells. The expressed receptor was
also capable of binding IGF-I1 with high affinity
(&
-3
nM) and weakly recognized insulin (with about
1%
the potency of IGF-I). The human IGF-I receptor ex-
pressed in these cells was capable of IGF-I-stimulated
autophosphorylation and phosphorylation of endoge-
nous substrates in the intact cell. This receptor also
mediated IGF-I-stimulated glucose uptake, glycogen
synthesis, and
DNA
synthesis. The extent of these re-
sponses was comparable to the stimulation by insulin
of the same biological responses in CHO cells express-
ing the human insulin receptor. These results indicate
that the isolated
cDNA
encodes
a
functional IGF-I
receptor and that there are no inherent differences in
the abilities of the insulin and IGF-I receptors to me-
diate rapid and long term biological responses when
expressed in the same cell type. The high affinity of
this receptor for IGF-I1 also suggests that
it
may be
important in mediating biological responses to IGF-I1
as
well
as
IGF-I.
Insulin-like growth factor(s) (IGF)’ I and I1 are polypeptide
hormones with
-50%
amino acid sequence identity with
proinsulin
(1).
Although the IGFs can bind to the insulin
receptor, each has its own distinct receptor
(2).
The IGF-I
receptor is structurally similar to the insulin receptor, whereas
the IGF-11 receptor is quite distinct
(2,
3). Both the insulin
and IGF-I receptors are synthesized as a single precursor
polypeptide of
M,
-180,000
which is glycosylated and cleaved
*
This work was supported by National Institutes of Health Grant
DK34926
and Research Career Development Award
DK01393
(to
R.
A.
R.).
The costs of publication
of
this article were defrayed in
part by the payment of page charges. This article must therefore be
hereby marked
“advertisement”
in accordance with
18
U.S.C. Section
1734
solely to indicate this fact.
§To whom correspondence should be addressed.
The abbreviations used are: IGF, insulin-like growth factor; CHO,
Chinese hamster ovary cells; Hepes,
4-(2-hydroxyethyl)-l-piperazine-
ethanesulfonic acid;
SDS,
sodium dodecyl sulfate; SSC, saline sodium
citrate; EGTA,
[ethylenebis(oxyethylenenitrilo)]tetraacetic
acid.
to yield two polypeptides of apparent
M,
-135,000 (the
a
chain) and
95,000
(the
@
chain) (4). The
a
chains of both
receptors are predominantly extracellular and are labeled
when the radioactive ligands are cross-linked to their respec-
tive receptors
(5,
6). The B-subunits are transmembrane pro-
teins, and their cytoplasmic domains are tyrosine-specific
kinases (6). Although slight differences in the substrate spec-
ificities
of
the two receptor kinases have been described
(7),
the kinase portions of the two receptors appear to be closely
related by various biochemical and immunological criteria
(7-
9).
The physiological roles for insulin and IGF-I are, however,
quite different. Insulin primarily regulates rapid anabolic
responses, including glucose uptake into muscle and fat cells,
glycogen synthesis in liver and fat synthesis in adipocytes (6,
10).
IGF-I, on the other hand, appears to be one of the primary
regulators of the growth of organisms
(11,
12).
Thus, one
might predict that the IGF-I receptor kinase might be more
potent than the insulin receptor kinase at stimulating long
term biological responses in cells, such as DNA synthesis.
Conversely, the insulin receptor kinase might be thought of
as
being more potent
at
stimulating rapid effects in cells such
as glucose uptake.
Recently, Ullrich
et
al.
(13) have isolated from human
placenta a cDNA clone which they proposed encoded for the
IGF-I receptor. This clone was identified by hybridization to
an oligonucleotide probe whose sequence was based upon the
NH2-terminal amino acid sequence of the a-subunit of the
IGF-I receptor.
As
expected for the IGF-I receptor, the de-
duced amino acid sequence
of
this protein showed similarities
in overall structure with the insulin receptor, including en-
coding
for
a single polypeptide precursor of
M,
=
151,869 with
a predicted cleavage site which would yield an
a-
and
@-
subunit
(13).
This predicted @-subunit sequence has
a
stretch
of
24
hydrophobic amino acids which is presumably
a
trans-
membrane sequence, and the cytoplasmic domain has a se-
quence which is homologous to other tyrosine kinases. In
agreement with the biochemical and immunochemical data
on the IGF-I receptor
(7-lo),
the deduced sequence
of
the
kinase portion of this receptor exhibited the highest homology
(84%
sequence identity) with the insulin receptor
(13).
In
addition the deduced amino acid sequence contained the
sequences of five other peptides generated from the a-subunit
of the IGF-I receptor. These results supported the hypothesis
that the isolated cDNA encodes for the IGF-I receptor. How-
ever, the isolated cDNA was found to hybridize to a mRNA
which decreased in abundance during the differentiation of
3T3-Ll preadipocytes to adipocytes (13). Since the levels
of
the IGF-I receptor protein have been reported to greatly
increase during the differentiation of the same cells (14),
these results raised the question of whether the isolated cDNA
11486
IGF-I
Receptor cDNA Expressed
in
CHO Cells
11487
does in fact encode for the IGF-I receptor. In addition various
forms
of
the IGF-I receptor have been described in the liter-
ature
(15-21).
These different species of IGF-I receptors vary
in their relative affinities for IGF-I and 11, the molecular
weights of their @-subunits, and their interactions with anti-
bodies.
To characterize further the IGF-I receptor, we have isolated
an IGF-I receptor cDNA, transfected Chinese hamster ovary
(CHO) cells with this cDNA and isolated cell lines which
express the protein encoded by this cDNA. This protein was
analyzed for its ability to bind IGF-I and
I1
and insulin and
to be recognized by a monoclonal antibody to the human IGF-
I
receptor. In addition the biological responses mediated by
this receptor were compared with the responses stimulated
through the human insulin receptor expressed in the same
cell type.
EXPERIMENTAL PROCEDURES
Materials-Recombinant IGF-I (22) and IGF-I1 (23) were the
generous gifts of Drs.
J.
Merryweather (Chiron Corporation) and
M.
Smith (Eli Lilly), respectively. Ascites and hybridoma supernatants
of monoclonal antibody aIR-3 (16) were graciously provided by Dr.
S.
Jacobs (Wellcome Laboratories) and purified on protein A-Seph-
arose. Porcine insulin was purchased from Elanco, gel reagents were
from Bio-Rad, affinity-purified rabbit anti-mouse IgG was from Pel-
Freeze. Other reagents were obtained as previously described (24, 25)
or as stated below.
Isolation and Expression
of
Human Placental IGF-I Receptor cDNA
Clones-A full term human placental cDNA library was prepared in
XgtlO
by published techniques (3). In order to isolate
a
full length
IGF-I receptor cDNA, an oligonucleotide representing bases 136-181
of the published sequence of the human IGF-I receptor cDNA (13)
was synthesized. The oligonucleotide (labeled with [y3'P]ATP and
T4 polynucleotide kinase) was then used to screen 6
X
10' plaques of
the library: hybridization was in 25% formamide, 6
X
SSC, and
5
X
Denhardt's at 42 "C; washing was with 2
X
SSC at
50
"C. Seven
clones that hybridized to this probe were plaque-purified. One of
these clones had a 5.5-kilobase pair EcoRI insert sufficient to encode
the entire IGF-I receptor. The restriction map of this insert agreed
completely with the published sequence, except that this clone had a
substantially longer 5"untranslated region (600 base pairs in our
clone uersus
45
base pairs in the published sequence). Approximately
80%
of our clone has been sequenced, and this sequence agrees
entirely with that of the published sequence (13).
Expression constructs were prepared by ligating the 5.5-kilobase
pair EcoRI insert into the mammalian expression vector, pECE (25).
A plasmid with the insert in the proper orientation for
SV40
early
promoter-dependent transcription was identified by restriction map-
ping and designated by pE-IGFIR. DNA from pE-IGFIR and a
plasmid conferring neomycin-resistance were then cotransfected into
CHO cells by calcium phosphate precipitation (25). Twenty-four
G418-resistant cell lines were isolated by limiting dilution. Cyto-
plasmic RNA was prepared from these lines and analyzed by dot-blot
hybridization for the presence of human IGFIR transcripts. The line
expressing the greatest amount of mRNA was designated CHO-
IGFIR and used for the remainder of this study.
Binding Studies-Confluent 24-well culture plates
of
CHO-IGFIR
or CHO cells were washed and incubated with the indicated labeled
ligand (50-150 pM) and unlabeled competitor for
5
h at 4 'C in buffer
B
(100 mM Hepes, pH 7.8, 120 mM NaC1,
1.2
mM MgS04, 15 mM
sodium acetate, 10 mM glucose, and
1%
bovine serum albumin).
Longer incubations at
4
"C (i.e.
8
or 16 h) resulted in less specific
binding, in part because of the cells becoming detached from the
wells. The cells were solubilized in
0.05%
SDS and counted in a
y
counter. Washings
(2
times) were performed at
4
"C and completed
within 3 min, a time such that less than
5%
of the specifically bound
ligand would dissociate. Labeled ligand was prepared by iodination
of recombinant human IGF-I by the use of the Bolton-Hunter reagent
(Du Pont-New England Nuclear) (105 Ci/g)
or
lactoperoxidase (300
Ci/g). Similar results were obtained with both labeled IGF-I prepa-
rations.
For measurement of the binding of lZ5I-IGF-I to the isolated recep-
tor, a plate binding assay was utilized (26). In brief, microtiter wells
were sequentially coated with
50
pl of affinity-purified rabbit anti-
mouse IgG (40 pg/ml) (Pel Freeze) and 10 nM monoclonal antibody
17A3 and then incubated with 25 pl of a 1:2 dilution of lysate of
CHO-IGFIR cells
(lo7
cells lysed with
0.5
ml of 2% Triton X-100 in
50
mM Hepes, pH 7.6,
5
mM EDTA,
5
mM EGTA, 150 mM NaCl,
1
mM phenylmethylsulfonyl fluoride,
1
mg/ml bacitracin). After
12
h
at 4 "C, wells were washed 3 times, and 25 pl of the mix of labeled
(600
PM) and unlabeled ligand were added. After
5
h at 4 "C, wells
were washed
4
times and cut out and counted.
Metabolic Labeling-Confluent 100-mm plates of CHO-IGFIR or
CHO cells were incubated in methionine- and cysteine-free Dulbec-
co's modified Eagle's medium containing 10% dialyzed fetal calf
serum and 250 pCi of [%]methionine and cysteine (Tran 3'S-label,
ICN). After 12 h at 37 "C, complete medium was added to the cells
and the incubation continued for an additional 7 h. The cells were
then lysed with
2
ml of 2% Triton X-100 in
50
mM Hepes, pH 7.6,
100 mM NaC1,5 mM EDTA,
5
mM EGTA,
1
mM phenylmethylsulfonyl
fluoride, and
1
mg/ml bacitracin. After
1
h at
0
'C, the lysates were
centrifuged for 20 min
at
3000 rpm and the supernatant immunopre-
cipitated with 20 pl of protein A-Sepharose (Pharmacia LKB Bio-
technologies Inc.) coated with
50
pg of affinity-purified rabbit anti-
mouse IgG and either
15
pg of monoclonal antibody or control mouse
IgG. After 19 h at 4 "C, the protein A-Sepharose was washed 4 times,
and the bound proteins released by heating for 2 min at
100
"C in
1%
SDS and 4% P-mercaptoethanol. The released proteins were analyzed
on 7.5% polyacrylamide SDS gels.
Biological Assays-For measurements of thymidine incorporation,
monolayers of cells in 24-well plates were incubated for 36 h at 37 'C
in serum-free Ham's F-12 medium containing 20 mM Hepes, pH 7.3,
100 units/ml of penicillin and 100 pg/ml of streptomycin. Hormones
and fresh medium were added, and the cells incubated for an addi-
tional
8
h. Cells were then pulsed with 0.75 pCi of [methyL3H]
thymidine (ICN) (20 Ci/mmol) for 45 min, washed 2 times, and lysed
with 0.075% SDS. Trichloroacetic acid (final concentration,
10%)
was added to the lysate, and the resulting precipitate was collected
by filtration on Whatman glass fiber filters, washed with
5%
trichlo-
roacetic acid, and counted for radioactivity.
For measurements of ligand-stimulated glycogen synthesis, con-
fluent monolayers of cells in 24-well plates were preincubated for 30
min at 37 'C in DB buffer (pH 7.4) containing 140 mM NaCI, 2.7 mM
KC1,
1
mM CaC12, 1.5 mM KHzPO,,
8
mM Na2HP04,
0.5
mM
MgC12,
and
1%
bovine serum albumin. Hormone and 4 pCi of ~[6-~H]glucose
(0.5
mM, final concentration) (ICN, 25 Ci/mmol) were added and the
assay continued for
2
h at 37 "C. After washing the cells 2 times, the
cells were lysed by the addition of 750 pl of 30% KOH containing 3
mg/ml carrier glycogen. The mixture was boiled for 30 min, and the
glycogen was precipitated by the addition of ethanol (final concentra-
tion, 70%). After 16 h at 4 "C, the precipitate was pelleted, washed
with 70% ethanol, dissolved in water, and counted for radioactivity.
For measurements of 2-deoxyglucose uptake, confluent monolayers
of cells in 24-well plates were washed with DB buffer and then
preincubated for 15 min. The assay was initiated by the addition of
hormone and 30 min later, 0.25 pCi of deoxy-~-[I-"C]glucose (Path-
finder) in 0.1 mM 2-deoxy-~-glucose. After 10 min at 37 "c, cells were
washed with ice-cold buffer containing 200
p~
phloretin, solubilized
with 0.03% SDS, and the lysates were counted for radioactivity.
Phosphorylation Experiments-Confluent 100-mm plates of CHO-
IGFIR cells were washed twice with phosphate-free Krebs-Ringer
bicarbonate buffer containing 0.1% bovine serum albumin, 10 mM
glucose, and 20 mM Hepes, pH 7.6, and then incubated in 4 ml of the
same buffer containing
0.5
mCi carrier-free [3ZP]orthophosphate
(Amersham Corp.). After 2.5 h at 37 "C, the cells were treated with
either IGF-I, aIR-3, or buffer. The reaction was stopped after 5 min
by rapidly removing the media, washing the cells, and adding
1
ml of
lysis buffer
(1%
Triton X-100,
100
mM NaC1,50 mM Hepes, pH 7.6,
1
mg/ml bacitracin,
1
mM phenylmethylsulfonyl fluoride,
1
mM
sodium vanadate, 10 mM NaF, and 10 mM EDTA). The lysate was
cleared by centrifugation (11,000
X
g for 10 min) and the IGF-I
receptor was immunoprecipitated with aIR-3 bound to protein A-
Sepharose (Pharmacia LKB Biotechnology Inc.) coated with rabbit
anti-mouse IgG. For lysates of cells treated with aIR-3, additional
rabbit anti-mouse
IgG
coated protein A-Sepharose was added to
ensure the precipitation of IGF-I receptor complexed with aIR-3. The
immunoprecipitates were analyzed by SDS gel electrophoresis and
autoradiography. The P-subunit band of the IGF-I receptor was also
excised from the dried gel by homogenization in
0.05
M
NaHC03
containing
0.5%
SDS and 5% P-mercaptoethanol. The extracted
protein was precipitated with 20% trichloroacetic acid and hydrolyzed
in 6
N
HCl for
2
h at 100 "C. The samples were then lyophilized in a
11488
IGF-I
Receptor
cDNA
Speed
Vac
and the phosphoamino acids were separated by
high
voltage electrophoresis
on
thin
layer
silica
gels
(0.2-mm thickness,
E.
Merck)
(27).
In vivo
tyrosine phosphorylation
of
the
receptor
and
other proteins
were
assessed
by
immunoblotting with anti-phosphotyrosine antibod-
ies.
Confluent
100-mm
plates
of
CHO-IGFIR
cells
were
washed
and
treated with either
10
nM
IGF-I
or
10
nM
aIR-3
in
serum-free
Ham’s
F-12
media
for
5-15
min.
For
the experiments
shown
in
this
paper,
cells
were
washed
and lysed as described above
in
the phosphorylation
studies. In other experiments (not
shown),
the
cells were
directly
lysed with
2%
SDS containing
5%
8-mercaptoethanol and loaded
onto the SDS-polyacrylamide
gels.
Lysates (the equivalent
of
2
X
10‘
cells/lane)
were
electrophoresed
on
10%
polyacrylamide-SDS
gels,
transferred
to
nitrocellulose filters, and probed with antiphosphoty-
rosine antibodies
(28).
The bound rabbit
immunoglobulin
was
de-
tected
by
use
of
alkaline
phosphatase-conjugated anti-rabbit
immu-
noglobulin
(Promega Biotech, as detailed
in
their
handbook).
In
some
experiments
cells
were treated with IGF-I
and
aIR-3
in
the presence
of
the phosphatase inhibitor
0.5
mM
vanadate
or
35
p~
phenylarsene
oxide.
RESULTS
Binding Studies-Chinese hamster ovary cells (called
CHO-IGFIR) stably expressing the presumptive IGF-I recep-
tor cDNA (see “Experimental Procedures” for details on this
cDNA) were found to specifically bind
-10
times more “‘I-
IGF-I as the parental CHO cells (Table I). These transfected
cells were found to specifically bind approximately the same
amount of ‘“I-IGF-11 and
Y5
the amount of “‘I-insulin as “‘I-
IGF-I (Table I). Scatchard analyses of the binding of
“‘1-
IGF-I to CHO-IGFIR indicated the presence of -25,000 recep-
tors/cell with an apparent
Kd
of 1.5
X
lo-’
M
for the hormone
(Fig.
hi).
In comparison the parental CHO cells were found
to have
-5,000
IGF-1 receptors/cell with an apparent
Kd
of
1.3
X
lo-’
M
(Fig.
L4).
To verify that the “‘I-IGF-I was binding to the human
IGF-I receptor, various monoclonal antibodies were tested for
their ability to inhibit the binding of ’“I-IGF-I to the CHO-
IGFIR cells. One monoclonal antibody utilized, aIR-3, rec-
ognizes the human IGF-I receptor but does not recognize
either the rodent IGF-I receptor or the human insulin and
IGF-I1 receptors (16).
At
10
nM this antibody was found to
maximally inhibit
70%
of the IGF-I binding to CHO-IGFIR
cells (Fig. 1B). The remaining binding could be due to the
endogenous hamster IGF-I receptor. In contrast two mono-
clonal antibodies to the human insulin receptor (5D9 and
MC51) (19) and control IgG at the same concentrations had
no effect on the binding of IGF-I to these cells (Fig. 1B).
To define further the specificity of the receptor expressed
in CHO-IGFIR cells, “‘I-IGF-I was incubated with these cells
TABLE
I
Binding
of
labeled
insulin, IGF-I,
and
IGF-11
to
CHO-IGFIR
and
CHO
cells
Confluent monolayers
of
cells
were
incubated
for
5
h
at
4
“C
with
100
p~
of
labeled
ligand
in
the
presence
or
absence
of
the
indicated
competing
unlabeled
ligand.
The
cells
were
washed,
lysed,
and
counted
as
described
under
“Experimental Procedures.” The
specific
activities
of
the ligands
were
105, 130,
and
102
pCi/pg
for
IGF-I, -11,
and
insulin,
respectively. Results
shown
are
averages
of
duplicate
wells
and the duplicates
differed
from
each
other
by
less
than
10%.
‘%I-Ligand Competitor
Total ‘Z611-ligand bound
CHO-IGFIR cells CHO cells
cpm
cpm
IGF-I
0
3421 547
IGF-I
100
nM
IGF-I
231 216
IGF-I1
0
3259 1656
IGF-I1
100
nM
IGF-I1
440 467
Insulin
0
828 332
Insulin
10
PM
insulin
247 236
Expressed
in
CHO
Cells
in the presence of increasing concentrations of unlabeled IGF-
I, IGF-11, and insulin (Fig. IC). In agreement with the binding
studies, IGF-I1 was found to be only 2-3 times less potent
than IGF-I at inhibiting binding, whereas insulin was -100
times less potent. These results could have been affected by
the high levels of endogenous IGF-I1 receptors in CHO cells
(Table I). The IGF-I receptors from the CHO-IGFIR cells
were therefore isolated and tested for their relative affinities
for IGF-I and
11.
As
with the intact cells, IGF-I1 was found to
be only slightly less potent (<%fold) than IGF-I at inhibiting
the binding of ”‘I-IGF-I to the purified receptor, whereas
insulin was 100 times less potent (Fig. 1D).
These results indicate that the CHO-IGFIR cells express a
receptor with almost equal affinity for IGF-I and
11.
Similar
results were obtained with other preparations of IGF-I and
11.
For comparison we therefore examined the binding of IGF-
I to its receptor in the rat hepatoma cells BRL-3A2
(7)
and
in fetal human brain. As with the expressed receptor, IGF-I1
was found to be only 2-3 times less potent than IGF-I at
displacing “‘I-IGF-I from its receptor in these other tissues.
Metabolic Labeling Studies-To examine the structure of
the expressed protein, lysates of metabolically labeled CHO-
IGFIR and CHO cells were immunoprecipitated by either
control IgG or monoclonal antibodies to the human insulin
(19) or IGF-I receptor (16) (aIR-3). Polypeptides of M,
-135,000 and 95,000 (corresponding to the
a-
and @-subunits,
respectively) were immunoprecipitated from the CHO-IGFIR
cells by the monoclonal antibody to the human IGF-I receptor
but not by control IgG (Fig.
2).
The expressed protein was
also not precipitated by the monoclonal antibody to the
insulin receptor, which was previously shown to be capable of
precipitating the human insulin receptor expressed in CHO
cells (29). The antibody to the human IGF-I receptor did not
precipitate similar polypeptides from the parental CHO cells,
even in greatly overexposed autoradiographs.
Biological Studies-To investigate whether the expressed
receptor was functional, the CHO-IGFIR cells were compared
to the parental CHO cells for their ability to respond to IGF-
I.
At
0.3 and 10 nM, IGF-I stimulated thymidine incorporation
in the CHO-IGFIR cells -2- and 4-fold, respectively (Fig.
3A). In contrast the parental CHO cells were stimulated
0.5-
and 2.5-fold at these concentrations of IGF-I. The increased
responsiveness of the transfected cells to IGF-I was consistent
with the functioning of the expressed receptor. To further
verify that the expressed human IGF-I receptor was mediating
this response, we utilized monoclonal antibody aIR-3 which
recognizes the human but not the hamster IGF-I receptor
(Fig. 2). This antibody was found to stimulate thymidine
incorporation in the CHO-IGFIR cells but not in the parental
CHO cells (Fig. 3A). The maximal response to this antibody
was approximately 2-fold and a response was observed at 100
PM.
Similar results were obtained with preparations of the
antibody isolated from mouse ascites and hybridoma super-
natants.
IGF-I was also found to stimulate glycogen synthesis to a
greater extent in the CHO-IGFIR cells than in the CHO cells,
with responses of
800
and
50%
of control at 10 nM IGF-I,
respectively (Fig. 3B). In addition the monoclonal antibody
to the human IGF-I receptor could stimulate this response in
the transfected cells but not the parental CHO cells (Fig. 3B).
As
with thymidine incorporation, the maximal response elic-
ited by the antibody was approximately
Yz
of the response
observed with IGF-I. Finally, the two cell types were compared
for the ability of IGF-I to stimulate glucose uptake (Fig. 3C).
Again, IGF-I was more potent in the CHO-IGFIR cells than
IGF-I
Receptor
cDNA
Expressed
in
CHO Cells
11489
N
X
0
c
.
E
D
a
m
0
20
40
60
80
100
120
Bound
(pMI
3c
1
I
I
z
Fi
-2
v
U
Ti
':I\
Insulin
-1
P
IGF-It
LL
IGF-I
0
0
10""
1
o-n
1
c6
lb
Competing ligand
(MI
Competing ligand
(MI
FIG.
1.
IGF-I binding
to
the expressed IGF-I receptor.
A,
Scatchard plot of IGF-I binding to either CHO-
IGFIR or CHO cells. Duplicate confluent wells of cells were incubated for
5
h at
4
"C with lactoperoxidase-labeled
IGF-I, washed, lysed, and counted. Nonspecific binding was determined in the presence of 100 nM IGF-I and was
subtracted from all the values. Least squares analysis was used to determine the slope and intercept of the
Scatchard plot.
B,
inhibition of IGF-I binding to CHO-IGFIR cells by various monoclonal antibodies and normal
IgG
(NZgG).
Binding was performed as in
A
except the '"I-IGF-I was labeled with the Bolton-Hunter reagent.
C,
inhibition
of
IGF-I binding to CHO-IGFIR cells by insulin, IGF-I, and IGF-11. Binding was performed as in
A.
0,
inhibition of IGF-I binding to the isolated IGF-I receptor by insulin, IGF-I, and IGF-11. Binding was performed
on the IGF-I receptor expressed in CHO-IGFIR cells after adsorption of this receptor onto microtiter wells coated
with a monoclonal antibody (17A3) which recognizes the @-subunit of the IGF-I receptor. This antibody does not
bind to the IGF-I1 receptor
(24).
Comparable lysates of the parental CHO cells had less than 5% of the IGF-I
binding
of
transfected cells.
abcdef
-200
.
116
-
92
-
66
45
FIG.
2.
Identification
of
the polypeptide subunits
of
the ex-
pressed human IGF-I receptor.
Metabolically labeled lysates of
either CHO
(a-c)
or CHO-IGFIR
(d-f)
cells were immunoprecipitated
with either control IgG
(a-d),
monoclonal antibody
to
the human
insulin receptor, MC51
(b
and
e)
or a monoclonal antibody to the
human IGF-I receptor, aIR-3 (c andf). The immunoprecipitates were
analyzed on SDS-polyacrylamide gels and an autoradiograph of the
gel is shown. The positions of molecular weight markers (in thou-
sands) are indicated.
in the CHO cells and aIR-3 could only stimulate a response
in the transfected cells.
To determine whether the IGF-I receptor had a different
potency than the insulin receptor at stimulating responses,
the same biological responses were studied in CHO cells
expressing the human insulin receptor. These transfected cells
have previously been described and shown to have -15,000
human insulin receptors/cell(29,30). Insulin maximally stim-
ulates glucose uptake 60-110% over controls in these cells
(29,30), a value close to that found for IGF-I in CHO-IGFIR
cells (average stimulation over control cells was
90%
in three
experiments).
At
10 nM, insulin was found to stimulate thy-
midine incorporation and glycogen synthesis in the
CHO-
HIR cells 4.5- and 6.5-fold, respectively (Fig.
4),
values that
were not significantly different from that observed in CHO-
IGFIR cells (4- and &fold, respectively).
Receptor Phosphorylation-To investigate in
vivo
receptor
phosphorylation, CHO-IGFIR cells were labeled with
["PI
orthophosphate, treated with IGF-I
or
aIR-3, lysed, and the
lysates were immunoprecipitated and analyzed. IGF-I and
aIR-3 were both found to stimulate the in
vivo
phosphoryla-
tion of the IGF-I receptor in CHO-IGFIR cells (Fig.
5A).
As
in the biological responses, IGF-I was about twice as potent
11490
IGF-I
Receptor
cDNA
Expressed
in
CHO
Cells
A'
I
I
I
I
I
100
c
I
I
1
'C
4
-
2
Z?
8
x
3-
c=
2e
E2
'p
E
2-
$-E
c
:E
3
.;;$---Jj
&;
h
m
gg
60-
.-
a);
I3m
E.:
40-
U€
$?
$-
2%
Im
/
.-
5%
4
aQ
20-
-
I
v
k
-
-a
OL
I
::
-,
,!
,
I
I
1-
0
I
I I
0
IO'"
10'
lo8
10'
0
10'"
IO'
lo8
0
10"
1
0'"
10'
1
O8
Concentration
(M)
Concentration
(M)
Concentration
(M)
FIG.
3.
Stimulation
of
biological responses in CHO-IGFIR
(closed symbols)
and CHO
(open
symbols)
cells
by
IGF-I
(circles)
and aIR-3
(boxes).
A,
thymidine incorporation. Cells were incubated with the indicated
concentrations of ligands and with [rnethyl-[3H]thymidine as described under "Experimental Procedures," Results
shown are averages of triplicate wells and are expressed as percent controls without hormone. The average basal
thymidine incorporations in nine experiments were 102 and
60
fmol for the CHO and CHO-IGFIR cells,
respectively.
B,
glycogen synthesis. Cells were incubated with the indicated concentrations of ligands and with
D-
[3H]glucose as described under "Experimental Procedures." Results shown are averages
of
triplicate wells and are
expressed as percent controls without hormone. The average basal incorporations for the CHO-IGFIR and CHO
cells were 52 and
60
pmol, respectively, of glucose/4
X
10'
cells in 2 h.
C,
glucose uptake. Cells were incubated with
the indicated concentrations of ligands and with ["C]2-deoxyglucose as described under "Experimental Proce-
dures." Results shown are the averages of three independent experiments and are expressed as the percent maximal
stimulation in each experiment. The average maximal stimulations for the three experiments were 92 and 96% for
the CHO-IGFIR and CHO cells, respectively, and the average basal 2-deoxyglucose uptake for the two cell types
was 1.4 and
1.1
nmol/4
x
lo6
cell in
10
min.
[A
'
0
10'
lon
10'
o
IO'"
IO'
I$
Insulin
(M)
Insulin
(MI
FIG.
4.
Insulin stimulation
of
biological responses in either
CHO cells expressing the human insulin receptor
(closed sym-
bols)
or the parental CHO cells
(open symbols).
A,
thymidine
incorporation. Cells were incubated with either the indicated concen-
tration of insulin
(circles)
or
30
nM monoclonal antibody to the insulin
receptor (5D9) plus the indicated concentration
of
insulin
(boxes).
Results shown are averages of triplicate wells and are expressed as
the percent controls without hormone. The average basal thymidine
incorporations were 215 and 201 fmol for the CHO-HIR and CHO
cells, respectively.
B,
glycogen synthesis. Cells were incubated with
the indicated concentrations of insulin and assayed as described under
"Experimental Procedures." Results shown are averages of triplicate
wells and are expressed as percent controls without hormone. The
average basal glucose incorporations for the CHO-HIR and CHO
cells were 54 and 42 pmol, respectively, per 4
x
10' cells in
2
h.
as aIR-3. (In three experiments, aIR-3 stimulated
'13
to
'12
as
much
32P
incorporation into the receptor as IGF-I). Phospho-
amino acid analyses, however, indicated that aIR-3 stimulated
the phosphorylation of the receptor only on serine residues,
whereas IGF-I stimulated the phosphorylation of both tyro-
sine and serine residues (Fig.
5B).
To
assess
in
vivo
substrates of the IGF-I receptor and to
verify the phosphoamino acid analyses, intact cells were
treated with either IGF-I or aIR-3, lysed, and the lysates were
examined by immunoblotting for the presence of phosphoty-
rosine-containing proteins.
As
in the
in vivo
phosphorylation
studies, IGF-I but not aIR-3 stimulated the phosphorylation
of the receptor on tyrosine residues (Fig.
6).
In addition IGF-
I but not aIR-3 stimulated the phosphorylation on tyrosine
residues of several additional proteins, including proteins of
apparent
M,
of 150,000 and
40,000
(Fig.
6).
As
previously
reported by Chou
et
ul.
(31), insulin stimulated the phospho-
rylation in the CHO-HIR cells of proteins with similar mo-
lecular weights (data not shown). The inability to detect
proteins phosphorylated on tyrosine residues in CHO-IGFIR
cells in response to aIR-3 could have been due to these
proteins being insoluble in the Triton X-100 used to lyse the
cells. However, the same results were obtained even when the
treated cells were directly solubilized with
1%
SDS (data not
shown). Since a low stoichiometry of tyrosine phosphorylation
could be missed in such an experiment, we also treated cells
with 35
PM
phenylarsine oxide which has been reported to
potentiate the ability of insulin to stimulate the phosphoryl-
ation of proteins in the intact cell (32). Although IGF-I caused
a marked increase in tyrosine phosphorylation of a number
of proteins in these cells, we still could not detect any effect
of aIR-3 on the tyrosine phosphorylation of any of these
proteins (Fig.
6,
lunes
d-f).
Similarly, vanadate treatment of
the intact cells (33) potentiated the effect of IGF-I on the
tyrosine phosphorylation of various endogenous proteins but
again no effect of aIR-3 could be detected in these cells (data
not shown).
DISCUSSION
The present studies demonstrate that CHO cells transfected
with a presumptive IGF-I receptor cDNA with a sequence
identical to that reported (13) express a protein which binds
lZ5I-IGF-I with a high affinity (apparent
Kd
=
1.5 nM) (Fig.
1).
Moreover, the expressed protein was recognized by a
monoclonal antibody which specifically binds the human IGF-
I receptor
(16)
and has the appropriate structure (Figs.
1
and
2).
These results further establish that this cDNA encodes for
an IGF-I receptor. Surprisingly, this receptor also recognized
IGF-I1 with high affinity
(Kd
-3 nM) (Fig.
1).
Although several
reports have described IGF-I receptors with almost equal
affinities for IGF-I and I1 (15,
17),
most studies have found
IGF-I
Receptor
cDNA
Expressed
in
CHO
Cells
11491
A
a
b
C
-
-200
-
116
I)-
-
92
-
66
Tyr
-D
Thr
-b
Ser
-b
FIG.
5.
In
vivo
receptor phosphorylation.
A,
stimulation of
receptor phosphorylation.
CHO-IGFIR
cells were labeled with
["PI
orthophosphate, treated for
5
min at
37
"C
with either buffer
(a),
10
nM
IGF-I
(b)
or
10
nM
aIR-3
(c),
lysed, and the lysates were immu-
noprecipitated with
aIR-3.
The immunoprecipitates were analyzed
by SDS-polyacrylamide gel electrophoresis and autoradiography. The
positions of molecular weight markers (in thousands) are indicated.
The phosphorylated band at 200,000 was observed in a second exper-
iment with both
IGF-1
and
aIR-3
but it was not present in a third
experiment.
B,
phosphoamino acid determinations. The phosphoryl-
ated 0-subunits from
(A)
were hydrolyzed and analyzed by high
voltage electrophoresis on thin layer silica gels. The positions
of
standards (located by ninhydrin staining) are indicated.
that the IGF-I receptor binds IGF-I with 10-30 times higher
affinity than IGF-I1 (15,
18,
20, 34-38). One explanation for
these different results could be the use of partially impure
preparations of IGF-I1 in these latter studies. The present
work utilized highly purified recombinant IGF-I1 (23) which
also was almost equipotent with IGF-I in displacing lZ5I-IGF-
I
from its receptor in BRL rat hepatomas and in fetal human
brain.
It is possible, however, that another IGF-I receptor exists
which binds IGF-I more selectively. In several studies the
IGF-I receptor P-subunit was found to give two species on
SDS-gel electrophoresis
(8,
16). In contrast the expressed
receptor in CHO-IGFIR cells exhibited only a single @-subunit
band on SDS-gel electrophoresis (Fig. 2). In addition a mono-
clonal antibody (5D9) which recognizes the IGF-I receptor in
some tissues (19) did not bind to the expressed receptor (Fig.
1B). These results support the hypothesis that another IGF-
I receptor type exists, and this species could bind IGF-I with
higher selectivity. These two IGF-I receptors could arise from
the same gene by differential processing of the mRNA or by
a post-translational modification of the protein. The high
affinity of the present IGF-I receptor for IGF-I1 could also be
interpreted to mean that this receptor is responsible for
mediating the biological responses of cells to IGF-11.
In the present studies the expressed IGF-I receptor was
also found to be functionally active. It was capable of media-
a
bcd
e
f
-
68
-
43
-
25
FIG.
6.
Effect of
aIR-3
and
IGF-I
on
the phosphorylation of
endogenous substrates.
CHO-IGFIR
cells were treated
for
10
min
at
37 "C
with either buffer
(a),
10
nM
IGF-I
(b),
or
10
nM
aIR-3
(c),
lysed, and the lysates were analyzed by immunoblotting with anti-
phosphotyrosine antibodies. To enhance the detection
of
endogenous
substrates,
CHO-IGFIR
cells were also treated with
35
p~
phenylar-
sine oxide
(31)
for
10
min at
37 "C,
then either buffer
(d),
10
nM
IGF-
I
(e),
or
10
nM
aIR-3
(f)
was added, and after an additional
10
min
at
37 "C
the cells were processed as above. The positions of prestained
molecular weight markers (in thousands) are indicated.
ting long-term (DNA synthesis), intermediate (glycogen syn-
thesis), and rapid effects (glucose uptake) of IGF-I. Evidence
that the human receptor was mediating these responses was
obtained by the finding that the CHO-IGFIR cells were more
responsive to IGF-I than the CHO cells, and that a mono-
clonal antibody (aIR-3), which is specific for the human IGF-
I receptor (16), could stimulate a response in the transfected
cells but not the parental cells (Fig. 3). Also of interest was
the finding that the IGF-I receptor had about the same
potency at stimulating these three responses as the human
insulin receptor expressed in the same parental CHO cells
(Fig.
4
and Refs. 29-31). These results are consistent with the
reports that the major endogenous substrates for the insulin
receptor tjrl.osine kinase are the same as those for the IGF-I
receptor (39-41). These results indicate that there are no
inherent differences in the abilities of these two receptor
kinases to mediate various responses and suggest that the
different physiological roles of these two hormones (6,lO-12)
are determined by the distribution of the two receptors on
different cells and/or the pharmacodynamics of the two hor-
mones.
Quite unexpected was the finding that the monoclonal
antibody aIR-3 was capable of mimicking the ability of IGF-
I to stimulate three different biological responses in the CHO-
IGFIR cells. The antibody was approximately
l/z
as potent as
IGF-I in the maximal responses it could stimulate. This
antibody has previously been found to be an antagonist of
IGF-I in other cell types (42-46). The mechanism whereby
this antibody stimulates a response is unclear. Unlike IGF-I
the antibody did not stimulate either the tyrosine phospho-
rylation of the IGF-I receptor or the tyrosine phosphorylation
of the major
in
vivo
substrates (Figs.
5
and 6). The antibody
did, however, stimulate the serine phosphorylation of the
receptor (Fig. 5). This is further evidence that the antibody
is capable of stimulating the same responses as IGF-I. These
data could be interpreted to mean that the antibody stimulates
responses independently of the intrinsic tyrosine kinase ac-
tivity of the receptor. However, this is unlikely since other
antibodies (47, 48) which had been thought to act in this
fashion were incapable of stimulating a response in cells
11492
IGF-I
Receptor
cDNA
Expressed
in
CHO
CelLs
expressing mutated receptors lacking kinase activity
(30,49).
Alternately, it is possible that this antibody, like other anti-
receptor antibodies
(50-53),
can efficiently induce the clus-
tering and internalization of the receptor without stimulating
its intrinsic tyrosine kinase activity. If the internalized recep-
tor is responsible for mediating the biological responses to
hormone, this internalized basally active kinase may be suf-
ficient to induce
a
partial response. It
is
also possible that the
antibody stimulates a low stoichiometry of receptor tyrosine
phosphorylation which
is
not detected in our assays. However,
even when inhibitors of phosphatases or potentiators of phos-
phorylation were added, we could not detect antibody-stimu-
lated tyrosine phosphorylation of the receptor
or
other pro-
teins. Finally, it is possible that the antibody induces a con-
formational change in the receptor which mimics the effect
of tyrosine autophosphorylation of the receptor. According to
this hypothesis, the autophosphorylation of the P-subunit of
the receptor induces a conformational change in the receptor
which can activate associated proteins without their being
phosphorylated on tyrosine residues. Additional studies on
the mechanism whereby this antibody stimulates
a
response
may aid in elucidating how tyrosine kinases transduce their
signals.
Acknowledgments-We
are grateful to Dr. James Merryweather
(Chiron Corp.) for IGF-I, Dr. Michele Smith (Eli Lilly) for IGF-11,
Dr. Jean Wang (University of California, San Diego) for the anti-
phosphotyrosine antibodies, Dr. Steve Jacobs (Wellcome Labs) for
aIR-3, Dr. Anita Payne for a critical reading of the manuscript, and
Karen Bird for preparation of the manuscript.
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