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INTRODUCTION |
Insulin-like growth factor
(IGF)1-I mediates three
distinct regulatory effects on cell growth by activation of the IGF-I
receptor. IGF-I stimulates proliferation of cells and may be required
for optimal growth of these cells (1-4). Transformation and
maintenance of the transformed state also require IGF-I receptor
activation in some cells (4, 5). IGF-I can also protect cells from apoptosis (6, 7).
Three proteins have been identified which are rapidly recruited to the
membrane after IGF-I receptor tyrosine phosphorylation: insulin
receptor substrate (IRS)-1/2, Src-homology/collagen (Shc), and
CT-10-regulated kinase (8-10). Through these substrates IGF-I mediates
activation of two main signaling cascades, the MAP kinase and PI
3-kinase pathways, which can act either in conjunction, in opposition,
or individually to mediate the response to IGF-I whether proliferative,
transforming, or anti-apoptotic (6, 11, 12).
In Rat1 fibroblasts IGF-I activates a pertussis toxin (PTx)-sensitive
heterotrimeric G protein leading to
G
-mediated, Ras-dependent MAP kinase stimulation (13). The
signal is transmitted by G
subunits
dissociated from an IGF-I-activated inhibitory (PTx-sensitive) G
protein. This mechanism of ras-dependent
MAP kinase activation is shared by both the IGF-I receptor tyrosine
kinases and G protein-coupled receptors, such as the lysophosphatidic
acid receptors (13-16). Activation of either receptor tyrosine kinases
or the G protein-coupled receptors induces rapid tyrosine
phosphorylation of docking proteins, e.g. Shc and Grb2,
which function as membrane scaffolds for the recruitment of Ras guanine
nucleotide exchange factors, e.g. mSOS, that regulate Ras activity. The regulation of Ras activity by this
pathway has further been shown to involve the participation of either
Src family nonreceptor tyrosine kinases or focal adhesion kinases depending on the ligand and the cell type examined (15, 16).
IGF-II acting through its cognate IGF-II/mannose 6-phosphate receptor
stimulates growth and metabolic effects. Coupling of this receptor to
the inhibitory heterotrimeric G protein, Gi2, has also been
described in both membranes derived from mouse Balb/3T3 fibroblasts and
COS cells transfected with IGF-II/Man-6-P receptor cDNA (17-19).
Upon stimulation by IGF-II, but not by Man-6-P, the activated
IGF-II/Man-6-P receptor interacts with Gi2 through the Arg2410-Lys2423 sequence in its C-terminal
intracellular domain (19). In contrast to IGF-I-induced IGF-I receptor
activation where MAP kinase stimulation is
G
-dependent, IGF-II-induced
activation of the IGF-II/Man-6-P receptor results in
G
i2-dependent inhibition of
adenylate cyclase activity, an effect only potentiated by the
G
subunits derived from Gi2 activation (19).
Human intestinal smooth muscle cells produce IGF-I, which plays a
autocrine role in the regulation of growth in culture (20). In these
cells IGF-I-stimulated growth is mediated by activation of distinct MAP
kinase-dependent and PI 3-kinase-dependent
signaling cascades (21). Whether IGF-I-stimulated MAP kinase activation and growth in these cells involve the activation of a heterotrimeric G
protein, and the roles of the G protein subunits in mediating this
effect are not known. The specific G protein activated by IGF-I and the
roles of the
and 
subunits derived from G protein activation
on growth have not been examined.
In the present study we show that IGF-I specifically activates the
PTx-sensitive inhibitory G protein, Gi2. Gi2
activation results in concurrent
G
-dependent stimulation of
MAP kinase activity and growth, and
G
i2-dependent inhibition of
adenylyl cyclase activation, cAMP production and results in
disinhibition of cAMP-mediated growth suppression.
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EXPERIMENTAL PROCEDURES |
Isolation and Culture of Muscle Cells from Human
Jejunum--
Muscle cells were isolated from the circular muscle layer
of human jejunum as described previously (20, 21). Segments of normal
jejunum were obtained from patients undergoing surgery according to a
protocol approved by the Institutional Committee on the Conduct of
Human Research. Briefly, muscle cells were isolated by enzymatic
digestion for 60 min at 31 °C in a medium containing 0.2%
collagenase (CLS type II) and 0.1% soybean trypsin inhibitor. The
medium consisted of (in mM): 120 NaCl, 4 KCl, 2.6 KH2PO4, 2 CaCl2, 0.6 MgCl2, 25 HEPES, 14 glucose, and 2.1% Eagle's essential amino acid mixture. Primary cultures of human intestinal muscle cells
were initiated and maintained in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum (DMEM-10) and containing 200 units/ml penicillin, 200 µg/ml streptomycin, 100 µg/ml gentamycin, and 2 µg/ml amphotericin B. The cells were plated at a concentration of 5 × 105 cells/ml and incubated in a 10%
CO2 environment at 37 °C. All subsequent studies were
performed in first passage cultured cells after 7 days, at which time
the cells were confluent.
[3H]Thymidine Proliferation
Assay--
Proliferation of smooth muscle cells in culture was
measured by the incorporation of [3H]thymidine as
described previously (20, 21). Briefly, the cells were washed free of
serum and incubated for 24 h in DMEM-0. After a 24-h incubation in
the absence of serum, the cells were incubated for an additional
24 h with a maximally effective concentration of IGF-I (100 nM) in the presence and absence of various test agents.
During the final 4 h of this incubation period, 1 µCi/ml [3H]thymidine was added to the medium.
[3H]Thymidine incorporation into the perchloric
acid-extractrable pool was used as a measure of DNA synthesis. DNA
content was measured fluorometrically using Hoescht 33528 with
excitation at 356 nm and emission at 492 nm. Calf thymus DNA was used
as a standard. [3H]Thymidine incorporation was expressed
as cpm/ng DNA.
Identification of IGF-I-activated G Proteins--
G proteins
selectively activated by IGF-I were identified by the method of Okamoto
et al. (22) as described previously by us (23-25).
Confluent human intestinal muscle cells growing in 100-mm dishes were
scraped off the plate and homogenized in 20 mM HEPES medium
(pH 7.4) containing 2 mM MgCl2, 1 mM EDTA, and 2 mM dithiothreitol. After
centrifugation at 27,000 × g for 15 min, the crude
membranes were solubilized for 60 min at 4 °C in 20 mM
HEPES medium (pH 7.4) containing 2 mM EDTA, 240 mM NaCl, and 1% CHAPS. The membranes were incubated for 20 min with 60 nM [35S]GTP
S in a solution
containing 10 mM HEPES (pH 7.4), 100 µM EDTA,
and 10 mM MgCl2. The reaction was stopped with
10 volumes of 100 mM Tris-HCl medium (pH 8.0) containing 10 mM MgCl2, 100 mM NaCl, and 20 µM GTP, and the mixture was placed in wells precoated with specific G protein antibodies. After incubation for 2 h on ice, the wells were washed three times with phosphate-buffered saline
containing 0.05% Tween 20, and radioactivity from each well was
counted. Coating with G protein antibodies (1:1,000) was done after the
wells were coated with anti-rabbit IgG (1:1,000) for 2 h on ice.
The selective IGF-I receptor tyrosine kinase inhibitor, tyrphostin AG
1024 (100 µM) (26), was used to identify to role of IGF-I
receptor tyrosine kinase phosphorylation in G protein activation.
Measurement of IGF-I Receptor Phosphorylation--
Binding of
IGF-I to the IGF-I receptor results in tyrosine phosphorylation of the
IGF-I receptor
subunit (IGF-IR
). Tyrosine phosphorylation of the
IGF-IR
was measured by immunoprecipitation of the IGF-IR
and
subsequent Western blotting of tyrosine-phosphorylated proteins.
Confluent muscle cells growing in 100-mm plates were incubated in
serum-free DMEM for 72 h. The cells were stimulated for 2 min with
increasing concentrations (1-100 nM) of IGF-I, IGF-II, and
del(1-6)IGF-II, a synthetic IGF-II agonist that does not interact with
IGF-binding proteins (33). The reaction was terminated by washing with
ice-cold phosphate-buffered saline. The cells were lysed in
immunoprecipitation buffer consisting of phosphate-buffered saline with
added 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1%
(w/v) SDS, 10 mM phenylmethylsulfonyl fluoride, 1% (w/v)
aprotinin, and 1 mM sodium orthovanadate. Cell lysates
containing equal amounts of protein were incubated with 2 µg/ml
antibody to the IGF-IR
for 2 h at 4 °C. 20 µl of protein A/G-agarose was added, and the incubation was continued overnight. The
resulting immune complexes were collected by centrifugation and washed
four times in immunoprecipitation buffer. The final pellet was
resuspended in 40 µl of electrophoresis sample buffer and boiled for
5 min. Proteins were separated by SDS-polyacrylamide gel
electrophoresis on 10% gels under reducing conditions and then
transferred to nitrocellulose membranes. The membranes were incubated
overnight with a 1:1,000 dilution of anti-phosphotyrosine antibody
(4G10) and then for 90 min with a 1:2,000 dilution of goat anti-mouse
IgG-horseradish peroxidase. The bands were visualized with enhanced
chemiluminescence and quantitated with densitometry.
Measurement of MAP Kinase Activity by in Vitro Kinase
Assay--
MAP kinase activity was measured as described previously
(21). Briefly, confluent muscle cells growing in six-well plates were
incubated in serum-free DMEM for 72 h. The cells were stimulated with 100 nM IGF-I for various time periods, 0-240 min, in
the presence and absence of test agents. The cells were lysed in a buffer containing (in mM): 10 Tris (pH 7.4), 150 NaCl, 2 EGTA, 2 dithiothreitol, 1 orthovanadate, 1 phenylmethylsulfonyl
fluoride, with added 10 µg/ml leupeptin and 10 µg/ml aprotinin.
Cellular debris in the lysates was precipitated by centrifugation at
12,000 × g for 20 min at 4 °C. MAP kinase activity
was measured in duplicate in aliquots of cell lysate containing equal
amounts of protein by the incorporation of phosphate from
[
-32P]ATP (1 µCi/30 µl of reaction volume) into a
synthetic MAP kinase substrate (Amersham Pharmacia Biotech) for
a 30-min incubation at 30 °C. The reaction was terminated, and
phosphorylated peptide substrate was separated using phosphocellulose
microfuge spin tubes (Pierce). The results are expressed in pmol of
phosphate incorporated/min/mg of protein.
Permeabilization of Muscle Cells--
Confluent muscle cells
growing in 100-mm dishes were permeabilized by modification of
techniques described previously (23-25, 27). The effects of specific G
protein subunits were investigated by immunoneutralization using
selective antibodies to specific G protein subunits as described
previously. This technique has been validated previously and used to
identify the signaling mechanisms and functional effects mediated via
specific G protein subunits activated by somatostatin, opioid, and
muscarinic receptors on intestinal smooth muscle cells (23-25).
Briefly, muscle cells were released from the culture plate by treatment
with 0.5% (w/v) trypsin containing 0.53 mM EDTA. The cells
were washed free of enzymes by centrifugation at 150 × g and resuspended in a cytosol-like buffer containing (in
mM): 20 NaCl, 100 KCl, 1 MgSO4, 25 NaHCO3, 1 EGTA, 0.18 Ca2+, and 1% IGF-I-free
bovine serum albumin. Cells were permeabilized by incubation with 35 µg/ml saponin at 31 °C for 10 min. The cells were washed free of
saponin by centrifugation at 150 × g and resuspended in the same medium with 1.5 mM ATP and ATP-regenerating
system (5 mM creatine phosphate and 10 units/ml creatine
phosphokinase). The cells were incubated for 1 h with neutralizing
antibody to G
i2 (10 µg/ml) or
G
(10 µg/ml). The reaction was initiated by addition
of 100 nM IGF-I and terminated after 10 min by placing the
cells on ice. The cells were rapidly centrifuged at 150 × g and 4 °C and the supernatant removed. The cells were
resuspended in MAP kinase lysis buffer (see above), and MAP kinase
activity was measured by in vitro kinase assay as described above.
Measurement of PI 3-Kinase Activity by in Vitro Kinase
Assay--
PI 3-kinase activity was measured by a modification of the
method of Higaki et al. (28) as described previously (21). Briefly, muscle cells grown to confluence in 100-mm dishes were incubated in serum-free DMEM for 72 h. Cells were stimulated for 10 min with 100 nM IGF-I in the presence and absence of
various test agents. The cells were lysed in a buffer consisting of (in mM): 50 Tris-HCl (pH 7.4), 150 NaCl, 1 Na2VO3, 2 EDTA, 1 MgCl2, 1 CaCl2, and 30 nM leupeptin, with added 1%
(w/v) trasylol and 1% (v/v) Nonidet P-40. Aliquots of cell lysate
containing equal amounts of protein were incubated with 25 µl of
anti-phosphotyrosine antibody (PY20) coupled to agarose beads with
gentle mixing for 2 h at 4 °C. The beads were collected by
centrifugation at 12,000 × g for 5 min at 4 °C and
washed three times with lysis buffer and two times with kinase assay
buffer. Kinase assay buffer consisted of (in mM): 50 Tris-HCl (pH 7.8), 50 NaCl, 2 MgCl2, and 0.5 EDTA. After
the final washing the beads were resuspended in 30 µl of kinase assay
buffer to which 10 µl of sonicated 1 mg/ml phosphatidylinositol was
added. The reaction was initiated by the addition of 5 µl of 50 mM ATP containing 0.5 µCi of [
-32P]ATP
and continued for 10 min at 30 °C. The reaction was terminated by
the addition of 0.5 ml of 1 N HCl and 2 ml of
chloroform-methanol (2:1, v/v). Phospholipids were recovered from the
lower organic phase and dried under N2 gas. The dried
phospholipids were dissolved in chloroform and spotted on Silica H Gel
TLC plates impregnated with 1% potassium oxalate. Chromatograms were
developed in chloroform, methanol, 28% NH3, water
(70:100:15:25, v/v). The plates were air dried and phospholipids
visualized with autoradiography. The spots corresponding to authentic
phosphatidylinositol 3,4,5-trisphosphate were scraped off the plates
and incorporated 32P quantified by
-scintillation
counting. Results are expressed as the increase in 32P
incorporation into PI-3-P in cpm above basal values.
Measurement of Adenylyl Cyclase Activity--
Adenylyl cyclase
activity was measured by the method of Salomon et al. (29).
Briefly, a 0.1-mg sample of membrane protein was incubated for 15 min
at 37 °C in 50 mM Tris-HCl (pH 7.4), 1 nM
ATP, 2 mM cAMP, 0.1 mM GTP, 0.1 mM
isobutylmethylxanthine, 5 mM MgCl2, 100 mM NaCl, 5 mM creatine phosphate, 50 units/ml creatine phosphokinase, and 4 × 106 cpm of
[3H]ATP. The reaction was terminated by the addition of
2% SDS, 45 mM ATP, and 1.5 mM cAMP.
[3H]cAMP was separated from [3H]ATP by
sequential chromatography on Dowex AG50W-4X and alumina columns. The
results were expressed as pmol of cAMP/mg of protein/min.
Measurement of cAMP by Radioimmunoassay--
cAMP production was
measured by modification of methods described previously (23-25).
Briefly, confluent muscle cells growing in six-well plates were
incubated in serum-free DMEM for 72 h. Cells were incubated for 10 min in 1 µM isobutylmethylxanthine. cAMP in the cells was
stimulated by activation with 10 µM forskolin, and 100 nM IGF-I was added for an additional 5 min. The reaction was terminated using ice-cold 10% trichloroacetic acid in which the
cells were incubated for 15 min at 4 °C. The supernatants were
extracted three times with water-saturated diethyl ether. The resulting
aqueous phase was frozen and lyophilized. The samples were
reconstituted for radioimmunoassay in 500 µl of 50 mM
sodium acetate (pH 6.2) and acetylated with triethylamine/acetic
anhydride (3:1 v/v) for 30 min. cAMP was measured in duplicate using
100-µl aliquots and expressed as pmol/mg of protein.
Statistical Analysis--
Values given represent the mean ± S.E. of n experiments where n represents the
number of experiments on cells derived from separate primary cultures.
Statistical significance was tested by Student's t test for
either paired or unpaired data as was appropriate.
Materials--
Recombinant human IGF-I and IGF-II were from
Austral Biologicals (San Ramon, CA). Collagenase and soybean trypsin
inhibitor were from Worthington. HEPES was from Research
Organics (Cleveland, OH). DMEM was from Mediatech Inc. (Herndon, VA).
Fetal bovine serum was from Summit Biotechnologies, Inc. (Fort Collins,
CO). The MAP kinase assay kit, [
-32P]ATP (specific
activity 3,000 Ci/mmol), [3H]thymidine (specific activity
6 Ci/mmol), [3H]ATP (specific activity 26.5 Ci/mmol), and
[125I]cAMP (specific activity 2,000 Ci/mmol) were from
Amersham Pharmacia Biotech. [35S]GTP
S (specific
activity 1,250 Ci/mmol) was from PerkinElmer Life Sciences.
Western blotting materials and the protein assay kit were from Bio-Rad.
Anti-phosphotyrosine PY20- agarose beads were from Transduction
Laboratories (Lexington, KY). Phosphocellulose spin columns were from
Pierce. Thin layer chromatography plates were from Analtech (Newark,
DE). Plastic cultureware was from Corning (Corning, NY). Antibodies to
G
i1, G
i3,
G
q/11 and G
were from Santa
Cruz Biotechnology (Santa Cruz, CA). Antibody to
G
i2 was from Chemicon (Temecula, CA). Anti-phosphotyrosine antibody 4G10 was from Upstate Biotechnology (Lake
Placid, NY). Tyrphostin AG 1024, forskolin, and PTx were from
Calbiochem. Phosphatidylinositol and all other chemicals were obtained
from Sigma.
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RESULTS |
Effect of PTx on IGF-I-induced Growth--
IGF-I increased
[3H]thymidine incorporation by 289 ± 9% above
basal (basal, 34 ± 2 cpm/ng DNA) (Fig.
1). The growth elicited by IGF-I was
partially inhibited, 35 ± 5%, in the presence of 200 ng/ml PTx.
We have shown previously that IGF-I-induced growth is mediated jointly
by activation of distinct MAP kinase-dependent, PI
3-kinase-independent and a MAP kinase-independent, PI
3-kinase-dependent pathways (21). To determine whether
PTx-sensitive growth elicited by IGF-I was mediated by activation of
the MAP kinase-dependent or the PI
3-kinase-dependent pathways, cells were incubated with the
MAP kinase kinase inhibitor PD98059 (10 µM) or the
PI 3-kinase inhibitor LY294002 (10 µM). We have shown
previously that at the concentrations used these inhibitors selectively
block activation of MAP kinase and PI 3-kinase, respectively, in these
cells (21). Thymidine incorporation in response to 100 nM
IGF-I was partly inhibited (42 ± 4%) by the MAP kinase kinase
inhibitor, partly inhibited (52 ± 3%) by the PI 3-kinase
inhibitor, and partly inhibited (35 ± 5%) by PTx (Fig. 1) (21,
30, 31). The MAP kinase kinase inhibitor had only a minor additive
effect to that of PTx (MAP kinase kinase inhibitor + PTx: 42 ± 10% inhibition versus PTx alone: 35 ± 5% inhibition,
p = not significant) on growth stimulated by IGF-I
(Fig. 1). In contrast, the addition of the PI 3-kinase inhibitor and
PTx was fully additive, strongly inhibiting growth stimulated by IGF-I
(PI 3-K inhibitor + PTx: 90 ± 2% versus PTx alone:
35 ± 5%, p < 0.01) (Fig. 1). These results
suggest that the portion of IGF-I-induced growth mediated by activation
of the MAP kinase pathway was sensitive to PTx, whereas the portion mediated by activation of the PI 3-kinase pathway was insensitive to
PTx.

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Fig. 1.
IGF-I stimulates growth by activation of
distinct PTx-sensitive, MAP kinase-dependent, and PI
3-kinase-dependent pathways. IGF-I-stimulated growth
of confluent human intestinal muscle cells was partly inhibited by the
MAP kinase kinase inhibitor PD98059 (10 µM), partly
inhibited by the PI 3-kinase inhibitor LY294002 (10 µM),
and partly inhibited by PTx (200 ng/ml). The effect of PTx was not
augmented by 50 µM PD98059. The effects of PTx and 50 µM LY294002 were additive and strongly inhibited
IGF-I-induced growth. Growth was measured by the incorporation of
[3H]thymidine as described under "Experimental
Procedures." Results were expressed as a percentage of the response
to IGF-I, 289 ± 9% above basal (basal, 34 ± 2 cpm/ng DNA).
Values represent the mean ± S.E. of three separate experiments. *
denotes p < 0.05 versus IGF-I alone; **
denotes p < 0.01 versus IGF-I + PTx.
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IGF-II and insulin were also examined for their ability to stimulate
growth of these cells. 100 nM IGF-II increased
[3H]thymidine incorporation by 40 ± 25% above
basal, and 100 nM insulin increased
[3H]thymidine incorporation by 35 ± 15% above
basal values. The increase in [3H]thymidine incorporation
elicited by IGF-II or insulin, however, was not inhibited in the
presence of PTx (38 ± 8 and 36 ± 20% above basal,
respectively). The rank order of potency of these three peptides to
stimulate growth, IGF-I > IGF-II > insulin, implied that
the growth stimulated by IGF-I reflected predominantly activation of
its cognate IGF-I receptor.
IGF-I Receptor Tyrosine Phosphorylation in Response to IGF-I and
IGF-II--
The ability of IGF-I and IGF-II to stimulate tyrosine
phosphorylation of the IGF-IR
was examined in anti-phosphotyrosine Western blots of IGF-IR
immunoprecipitates. IGF-I caused
concentration-dependent tyrosine phosphorylation of the
IGF-IR
(maximal stimulation: 329 ± 5% above basal with 100 nM IGF-I) (Fig. 2). In
contrast, IGF-II did not cause tyrosine phosphorylation of the
IGF-IR
in concentrations up to 100 nM (Fig. 2).

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Fig. 2.
IGF-I causes
concentration-dependent tyrosine phosphorylation of the
IGF-I receptor. Panel A, representative
anti-phosphotyrosine Western blot of IGF-IR immunoprecipitates
stimulated with IGF-I, IGF-II, or del(1-6)IGF-II. Panel B,
IGF-I (closed circles) caused
concentration-dependent tyrosine phosphorylation of the
IGF-IR subunit. IGF-II (closed squares) and
del(1-6)IGF-II (open circles) did not cause tyrosine
phosphorylation of the IGF-IR subunit. Quiescent muscle cells were
incubated with 1-100 nM IGF-I, IGF-II, or del(1-6)IGF-II
for 2 min. IGF-IR tyrosine phosphorylation was measured in
anti-phosphotyrosine Western blots of IGF-IR immunoprecipitates as
described under "Experimental Procedures." Results are expressed as
a percent above basal phosphorylation in relative densitometric units.
Values represent the mean ± S.E. of three or four separate
experiments. ** denotes p < 0.01 versus
basal.
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The interaction of IGF-II with IGF receptors like that of IGF-I, can be
modulated by the presence of up to six IGF-binding proteins (IGFBPs).
We have shown previously that human intestinal muscle cells express
IGFBP-3, IGFBP-4, and IGFBP-5 exclusively (32). To determine whether
the inability of IGF-II to activate the IGF-IR in these cells reflected
binding of IGF-II to one of these IGFBPs, we used del(1-6)IGF-II, an
IGF-II agonist with reduced affinity for all IGFBPs (33).
del(1-6)IGF-I did not cause tyrosine phosphorylation of the IGF-IR
in concentrations up to 100 nM (Fig. 2). The results imply
that the effects of IGF-I in these cells were mediated by activation of
the IGF-IR, whereas those mediated by IGF-II were not.
Effect of PTx on IGF-I-mediated Activation of MAP Kinase--
We
have shown previously that activation of MAP kinase by IGF-I is
concentration-dependent and time-dependent
(21). MAP kinase activation in response to a maximally effective
concentration of IGF-I (100 nM) is rapid, occurring within
2 min, attains a maximum at 10 min, and declines to basal levels within
240 min (21). In the presence of 200 ng/ml PTx, activation of MAP
kinase by 100 nM IGF-I is inhibited at 2 and 10 min
(64 ± 9% and 48 ± 16% inhibition from untreated control,
respectively, p < 0.05) (Fig.
3). MAP kinase activation by IGF-I at
longer time points (30-240 min) was abolished, i.e.
returned to basal unstimulated levels. The results implied that the
early activation of MAP kinase by IGF-I is partially dependent on
activation of an inhibitory G protein. Sustained MAP kinase activation
is fully dependent on activation of an inhibitory G protein.

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Fig. 3.
PTx inhibits IGF-I-stimulated MAP kinase
activation. IGF-I ( ) elicited time-dependent MAP
kinase activation. In the presence of 200 ng/ml PTx ( ),
IGF-I-induced MAP kinase stimulation was strongly inhibited at 2 and 10 min and was abolished (returned to basal unstimulated levels) at longer
time periods. MAP kinase activity was measured by an in
vitro kinase assay as the incorporation of
[ -32P]ATP into synthetic MAP kinase substrate as
described under "Experimental Procedures." Results are expressed as
the increase in MAP kinase activity above basal (basal, 1.77 ± 0.35 pmol of Pi/min/mg of protein). Values represent the
mean ± S.E. of three separate experiments. * denotes
p < 0.05 versus IGF-I alone.
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Identification of the Inhibitory G Protein Activated by
IGF-I--
The ability of PTx to inhibit both IGF-I-induced MAP kinase
activation and growth implied that an inhibitory G protein is activated
by IGF-I in human intestinal muscle cells. The identity of the G
protein that accounts for the sensitivity to PTx was measured by the
binding of [35S]GTP
S to G protein subunits in
membranes prepared from these cells. 100 nM IGF-I
selectively increased the activity of G
i2, 252 ± 17% above basal (basal, 7,020 ± 118 cpm/mg of
protein) (Fig. 4). IGF-I did not affect
the activity of G
i1, G
i3, or G
q/11. In the presence of a selective inhibitor of the IGF-I receptor tyrosine kinase, tyrphostin AG 1024 (100 µM), IGF-I-induced
G
i2 activation was inhibited 78 ± 4%
(p < 0.01). The results implied that the activation of
Gi2 by the IGF-I receptor occurred as a result of IGF-I
receptor tyrosine kinase autophosphorylation.

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Fig. 4.
IGF-I selectively activates
G i2. Panel A, 100 nM IGF-I caused a significant increase in the binding of
[35S]GTP S·G complexes to wells
precoated with G i2 antibody but not to wells
precoated with antibody to G i1,
G i3, or G q/11.
Panel B, 100 nM IGF-II did not increase the
binding of [35S]GTP S·G complexes.
Membranes isolated from human intestinal muscle cells were solubilized
in CHAPS and incubated with [35S]GTP S in the presence
and absence of IGF-I or IGF-II for 20 min as described under
"Experimental Procedures." Results are expressed as the increase in
bound radioactivity in cpm/mg of protein. Values represent the
mean ± S.E. of four separate experiments. ** denotes
p < 0.01 versus GTP S alone.
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Coupling of the IGF-II/Man-6-P receptor to a PTx-sensitive inhibitory G
protein has been described in Balb/3T3 fibroblasts (18). In human
intestinal muscle cells, IGF-II did not increase the activity of
G
i1, G
i2, G
i3 , or G
q/11
(Fig. 4).
Functional Role of G
in IGF-I-mediated MAP Kinase
Activation--
Permeabilized muscle cells were used to determine
which of the G protein subunits activated by the IGF-I receptor
mediated IGF-I-induced MAP kinase activation. Activation of
G
i2 by IGF-I implied the concomitant
activation of G
. Therefore, the
individual roles of the G
i2 and the
G
subunits in IGF-I-mediated MAP kinase
activation were examined by incubation of permeabilized muscle cells
with either a selective G
i2-neutralizing antibody or a common G
-neutralizing antibody (23-25).
IGF-I increased peak MAP kinase activity (measured at 10 min) by
0.39 ± 0.10 pmol of Pi/min/mg of protein above basal
(basal, 0.94 ± 0.09 pmol of Pi/min/mg of protein,
p < 0.05). The increase was inhibited by 97 ± 9% (p < 0.05) in the presence of the common G
antibody but was not affected by the
G
i2 antibody (3 ± 8% inhibition)
(Fig. 5). The pattern of response elicited by treatment of permeabilized muscle cells with the G protein
antibodies implied that the IGF-I-induced increase in MAP kinase
activity was mediated via the G
subunits
derived from Gi2.

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Fig. 5.
G subunits
mediate the IGF-I-induced activation of MAP kinase. The 100 nM IGF-I-stimulated increase in MAP kinase activity was
inhibited by immunoneutralization of G subunits but was
not affected by immunoneutralization of G i2
subunits. Saponin-permeabilized human intestinal muscle cells were
preincubated with immunoneutralizing antibody to G or
G i2 for 1 h (23-25). Cells were then
stimulated with 100 nM IGF-I for 10 min, and lysates were
prepared from the cells. MAP kinase activity was measured by in
vitro kinase assay as the incorporation of 32P into a
synthetic MAP kinase substrate as described under "Experimental
Procedures." Results are expressed as the increase in MAP kinase
activity above basal levels in pmol of Pi/min/mg of protein
(basal, 0.94 ± 0.09 pmol of Pi/min/mg of protein).
Values represent the mean ± S.E. of five separate experiments. *
denotes p < 0.05 versus IGF-I alone.
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Inhibition of cAMP Production by IGF-I--
The activation of
G
i2 by IGF-I suggested that IGF-I may
inhibit the accumulation of cAMP. This notion was tested in two
complementary ways. In the first, the role of the IGF-I-activated
G
i2 subunit in regulating adenylyl cyclase
activity was determined. In the second, the effect of IGF-I on cAMP
levels was measured.
The activity of adenylyl cyclase was measured in membranes prepared
from human intestinal muscle cells as the conversion of [3H]ATP into cAMP. Incubation of membranes with 10 µM forskolin increased adenylyl cyclase activity by
362 ± 44% above basal (basal, 6.8 ± 0.5 pmol of cAMP/mg of
protein/min) (Fig. 6).
Forskolin-stimulated adenylyl cyclase activity was inhibited 68 ± 2% in the presence of 100 nM IGF-I (Fig. 6). Inhibition of
forskolin-stimulated adenylyl cyclase activity was attenuated in the
presence of the selective G
i2-neutralizing
antibody but not affected by the common G
-neutralizing
antibody (fig. 6).

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Fig. 6.
G i2
subunits mediate IGF-I-induced inhibition of adenylyl cyclase
activity. Forskolin-stimulated adenylyl cyclase activity was
inhibited 68 ± 2% by IGF-I. IGF-I-induced inhibition of adenylyl
cyclase activity was reversed in the presence of the selective
G i2-neutralizing antibody but not affected
by the presence of the common G -neutralizing antibody.
Membranes prepared from human intestinal muscle cells were incubated
for 15 min with forskolin alone or forskolin and 100 nM
IGF-I in the presence and absence of 10 µg/ml
G i2- or G -neutralizing
antibody. Adenylyl cyclase activity was measured from the conversion of
[ 32P]ATP into cAMP as described under "Experimental
Procedures." Results are expressed as pmol of cAMP/mg of protein/min
above basal (basal, 6.5 ± 0.5 pmol of cAMP/mg of proteinmin).
Values represent the mean ± S.E. of six separate experiments. **
denotes p < 0.01 versus forskolin alone; + denotes p < 0.01 versus forskolin + IGF-I.
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Intact human intestinal smooth muscle cells were incubated with IGF-I
alone, to determine its effects on basal cAMP levels, and with IGF-I in
the presence of forskolin, to determine the effect of IGF-I on
forskolin-stimulated cAMP levels. 100 nM IGF-I by itself
was capable of decreasing basal cAMP levels by 30 ± 4% (basal,
23.5 ± 1.4 pmol/mg of protein) (Fig.
7A). The increase in cAMP
stimulated by 10 µM forskolin (144 ± 4 pmol/mg of
protein above basal) was strongly inhibited, 89 ± 1%, in the
presence of 100 µM IGF-I (Fig. 7B). The
inhibition of forskolin-stimulated cAMP production by IGF-I was
attenuated, 58 ± 2%, by pretreatment with PTx (Fig.
7B).

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Fig. 7.
IGF-I inhibits cAMP production.
Panel A, 100 nM IGF-I inhibits basal cAMP levels
by 30 ± 4%. Panel B, 10 µM
forskolin-stimulated cAMP production is inhibited 89 ± 1% by 100 nM IGF-I. In the presence of 200 ng/ml PTx, the ability of
IGF-I to inhibit forskolin-stimulated cAMP production is decreased by
58 ± 2%. Human intestinal muscle cells were incubated with IGF-I
for 10 min, and cAMP levels were measured in trichloroacetic acid
extracts by radioimmunoassay as described under "Experimental
Procedures." Results are expressed as pmol of cAMP/mg of protein
(basal, 23.5 ± 1.4 pmol of cAMP/mg of protein). Values represent
the mean ± S.E. of three separate experiments. ** denotes
p < 0.01 versus basal levels; + denotes
p < 0.01 versus forskolin alone; ++ denotes
p < 0.01 versus forskolin + IGF-I.
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Functional Role of cAMP in IGF-I-mediated Inhibition
Growth--
Activation of adenylyl cyclase and the resulting increase
in cAMP levels are generally accompanied by inhibition of growth in
smooth muscle cells (34, 35). The effect of adenylyl cyclase activation
on growth of human intestinal smooth muscle has not been examined
previously. Accordingly, it was investigated by the addition of
forskolin to activate adenylyl cyclase directly. 10 µM
forskolin alone inhibited basal [3H]thymidine
incorporation by 60 ± 5% (p < 0.05) (Fig.
8). In the presence of forskolin,
[3H]thymidine incorporation elicited by 100 nM IGF-I was inhibited by 65 ± 6% (p < 0.05) (Fig. 8). The results imply that the ability of IGF-I to
activate Gi2 and inhibit adenylate cyclase activity decreases the levels of growth-inhibitory cAMP and thereby reinforces the growth-stimulatory effects of IGF-I mediated by activation of MAP
kinase.

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Fig. 8.
The cAMP-dependent pathway
inhibits growth of confluent human intestinal muscle cells.
Confluent muscle cells were incubated with 100 nM IGF-I
alone, 10 µM forskolin alone, or a combination of IGF-I
and forskolin. Activation of the cAMP cascade with forskolin inhibited
basal growth. IGF-I-stimulated growth was also inhibited in the
presence of forskolin. Test agents were incubated with muscle cells for
24 h and growth measured as the incorporation of 1 µCi/ml
[3H]thymidine during the final 4 h into perchloric
acid-extractable pools. Results are expressed as a percentage of basal
[3H]thymidine incorporation (34 ± 4 cpm/ng of DNA).
Values represent the mean ± S.E. of four experiments. * denotes
p < 0.01 versus basal; ** denotes
p < 0.01 versus IGF-I alone.
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IGF-I-mediated Activation of PI 3-Kinase Occurs Independently of a
PTx-sensitive G Protein--
The independence of IGF-I-induced PI
3-kinase activation from the PTx-sensitive G protein was examined
directly by measurement of PI 3-kinase activation in the presence of
PTx. Preincubation of muscle cells with 200 ng/ml PTx did not alter
maximal IGF-I-induced increase in PI 3-kinase activation (IGF-I,
112 ± 8% above basal; IGF-I + PTx, 132 ± 9% above basal)
(Fig. 9). The results confirmed the Gi2-independent nature of IGF-I-mediated PI 3-kinase
activation and PI 3-kinase-dependent growth in these
cells.

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Fig. 9.
IGF-I-induced PI 3-kinase activation occurs
independently of Gi2 activation. The role of the
inhibitory G protein, Gi2, activated by IGF-I on
IGF-I-induced PI 3-kinase activation, was measured in muscle cells
stimulated with 100 nM IGF-I for 10 min (maximal stimulus)
in the presence and absence of 200 ng/ml PTx. The increase in PI
3-kinase activity induced by IGF-I was not inhibited by PTx. PI
3-kinase activity was measured by an immune complex kinase assay as the
incorporation [32P]ATP into phosphatidylinositol
3,4,5-trisphosphate using phosphatidylinositol as substrate as
described under "Experimental Procedures." Results are expressed as
the percentage of basal PI 3-kinase activity (basal 76 ± 12 cpm).
Values represent the mean ± S.E. of four separate
experiments.
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DISCUSSION |
In human intestinal muscle cells IGF-I activates two distinct
signaling pathways coupled to stimulation of growth: a MAP
kinase-dependent, PI 3-kinase-independent and a MAP
kinase-independent, PI 3-kinase dependent pathway (21). These two
pathways jointly mediate growth in response to IGF-I. The present paper
shows that activation of MAP kinase by IGF-I occurs through the
activation of Gi2 by the tyrosine-phosphorylated IGF-I
receptor and is mediated by the G
subunits. The G
i2 subunits derived from
Gi2 activation may also participate in the regulation of
growth by IGF-I by attenuating the levels of growth inhibitory cAMP.
The evidence supporting the activation of Gi2 by IGF-I and
distinct roles for G
subunits in the stimulation of MAP kinase leading to growth, and for
G
i2-subunits in the inhibition of adenylyl cyclase and
cAMP leading to growth can be summarized as follows. 1) IGF-I induced
specific activation of Gi2 which was sensitive to the IGF-I
tyrosine kinase inhibitor, tyrphostin AG 1024. 2) IGF-I-induced MAP
kinase activation was inhibited by PTx and by immunoneutralization of
G
subunits but not by immunoneutralization of
G
i2 subunits. 3) IGF-I-induced inhibition of
forskolin-stimulated adenylyl cyclase activity was sensitive to
immunoneutralization of G
i2 but not
G
subunits. 4) IGF-I inhibited both basal cAMP
production and forskolin-stimulated cAMP production, and the inhibition
was attenuated in the presence of PTx. 5) IGF-I-induced growth was
partially inhibited by PTx, by a MAP kinase kinase inhibitor, or the PI
3-kinase inhibitor (21); the effects of PTx and the PI 3-kinase
inhibitor were additive, whereas those of PTx and the MAP kinase kinase
inhibitor were not additive. 6) IGF-I-induced growth was inhibited by forskolin.
IGF-I receptor tyrosine phosphorylation on residues Tyr-1158, Tyr-1162,
and Tyr-1163 is required for the mitogenic response mediated by IGF-I
receptor activation (8). Several studies have provided evidence that
following IGF-I receptor autophosphorylation both IRS-1 and Shc
are activated (8, 9). IRS-1 associates with PI 3-kinase (36, 37), Nck
(38), Grb2 (38, 39), and PTP1D (40) through an SH2 interaction, whereas
Shc interacts with Grb2 (39). The Grb2-docking protein provides a
scaffold for recruitment of the Ras-guanine nucleotide exchange
factor, mSOS, to the plasma membrane and results in
p21ras activation. Activation of Ras
initiates the raf/MAP kinase cascade, a key signaling pathway in
IGF-I-induced growth.
Although evidence exists linking IGF-I receptor phosphorylation to
IRS-1 and Shc activation, in contrast to IRS-1, Shc does not associate
directly with either the IGF-I receptor or with IRS-1 (9). Recent
evidence demonstrates that not only do receptor tyrosine kinases like
the IGF-I receptor but also some G protein-coupled receptors activate
Shc (14, 15). In the case of G protein-coupled pathways, Shc activation
is mediated by G
subunits derived from
Gq-coupled receptors and by G
subunits derived from Gi-coupled receptors (15). Both the
receptor tyrosine kinase and G protein-coupled receptor signaling
cascades involve the participation of Src family nonreceptor tyrosine
kinases and lead to the activation of p21ras
through the Shc-Grb2-mSOS pathway (15, 16, 41). Thus,
Ras-dependent MAP kinase activation is a signaling pathway
shared by both receptor tyrosine kinases and G protein-coupled
receptors. The participation of
G
-dependent MAP kinase
activation is growth factor-specific: the IGF-I receptor is coupled to
this pathway in Rat1 fibroblasts, the epidermal growth factor receptor is not (13).
G
-dependent mechanisms of PI
3-kinase activation in the signaling pathways coupling G
protein-coupled receptor and receptor tyrosine kinase activation to MAP
kinase stimulation have also been elucidated. Participation of the p85 and associated p110
/
PI 3-kinase subunits (42, 43), as well as a
recently described PI 3-kinase, p110
(44), have been implicated in
this signaling pathway. We have shown previously that in human intestinal muscle cells MAP kinase activation and PI 3-kinase activation by IGF-I were fully distinct events based on the selective inhibitors, LY294002 and PD98059 (21). In the present study, we further
show that activation of MAP kinase but not PI 3-kinase is PTx-sensitive
and involves G
subunits. The distinct
nature of these two pathways in mediating IGF-I-induced growth is
supported by the finding that the effect of the PI 3-kinase inhibitor
and PTx to inhibit growth is additive, whereas the effects of the MAP
kinase inhibitor and PTx are not additive.
In Balb/3T3 fibroblasts and in COS cells transfected with an
IGF-II/Man-6-P receptor cDNA, IGF-II has been shown to activate the
inhibitory G protein, Gi2 (17, 19). In human intestinal muscle cells, IGF-II does not activate an inhibitory G protein, and the
effects of exogenous IGF-II are not inhibited by PTx. We have shown
previously that although IGF-II is produced by these cells, the levels
of IGF-II produced are beneath the threshold that can stimulate growth
(45).
In Rat1 fibroblasts, the IGF-I receptor is coupled to
G
activation and leads to PTx-sensitive, Ras-dependent MAP kinase activation, implying the
involvement of Gi (13). Recently, the association of
Gi and role of dissociated G
subunits from the IGF-I-activated IGF-I receptor in MAP kinase
activation have been characterized in rat cerebellar granule neurons
and in the NG-108 neuroblastoma and Balb/c3T3 cell lines (45). However,
the specific inhibitory G protein activated and the roles of the
G
and G
subunits in
IGF-I-induced growth were not determined. Our data demonstrate that in
human intestinal smooth muscle cells IGF-I selectively activates
Gi2. This interaction depends on the intrinsic tyrosine
kinase activity of the IGF-I receptor. Gi2 activation mediates the MAP kinase-dependent, PI 3-kinase-independent
portion of IGF-I-induced growth through the
G
subunits derived from Gi2.
In these cells net growth is determined not only by pathways that
stimulate growth, i.e. the MAP kinase-dependent and PI 3-kinase-dependent pathways, but also by pathways
that suppress growth, i.e. the cAMP pathway. Thus,
IGF-I-induced growth mediated by activation of the
G
-dependent MAP kinase, and
the PI 3-kinase pathways can be augmented by inhibition of the cAMP
pathway by the G
i2 subunits derived from
Gi2 activation by IGF-I.