(Received for publication, September 28, 1995)
From the
We examined the signaling function of the IGF-II/mannose
6-phosphate receptor (IGF-IIR) by transfecting IGF-IIR cDNAs into COS
cells, where adenylyl cyclase (AC) was inhibited by transfection of
constitutively activated G cDNA
(G
Q205L). In cells transfected with IGF-IIR cDNA,
IGF-II decreased cAMP accumulation promoted by cholera toxin or
forskolin. This effect of IGF-II was not observed in untransfected
cells or in cells transfected with IGF-IIRs lacking
Arg
-Lys
. Thus, IGF-IIR, through its
cytoplasmic domain, mediates the G
-linked action of IGF-II
in living cells. We also found that IGF-IIR truncated with C-terminal
28 residues after Ser
caused G
-dominant
response of AC in response to IGF-II by activating G
.
Comparison with the G
-dominant response of AC by
intact IGF-IIR suggests that the C-terminal 28-residue region
inactivates G
. This study not only provides further evidence
that IGF-IIR has IGF-II-dependent signaling function to interact with
heteromeric G proteins with distinct roles by different cytoplasmic
domains, it also suggests that IGF-IIR can separate and sequestrate the
G
and G
signals following G
activation.
Insulin-like growth factor II (IGF-II) ()promotes
growth, mainly in fetal development. In cultured cells, it exerts
mitogenic and metabolic stimulation by binding to cell surface
receptors. IGF-IIR is a high-affinity receptor for
IGF-II(1, 2, 3) . It is also a receptor for
M6P(3) . However, these two distinct ligands bind to different
sites in IGF-IIR, which has been indicated by competition experiments
and by the fact that only mammalian IGF-IIR can bind
IGF-II(4) . For several reasons (for review, see (4) ),
it remains unclear whether the IGF-IIR executes signaling functions in
response to IGF-II. Nonetheless, there are multiple lines of
independent evidence that IGF-IIR has signaling function activated by
IGF-II. In multiple cultured-cell systems, IGF-II evokes cellular
responses, most likely through
IGF-IIR(3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) .
We and another group independently showed that IGF-II stimulation of
IGF-IIR promotes calcium influx through G
, a member of the
heteromeric G protein family, in Balb/c3T3 or CHO
cells(12, 13, 14, 15) . In
reconstituted vesicles, purified IGF-IIR directly couples to G
in response to IGF-II(16, 17, 18) , and
human IGF-IIR has a cytoplasmic 14-residue region at
Arg
-Lys
, which can directly
activate G
(18, 19) . In cell-free
systems, this region most likely functions as the effector domain of
IGF-IIR for G
coupling(18, 19, 20) . Although failure
of IGF-IIR coupling to G proteins in cell-free systems was once
reported(21) , a subsequent paper(15) , with two of the
same authors, suggested the G
coupling function of IGF-IIR,
based on the observation that IGF-II stimulates Ca
influx via a pertussis toxin-sensitive G protein in a manner
resistant to tyrosine kinase inhibitors.
Intensive studies of the
molecular signaling function of IGF-IIR have so far been conducted only
on cell-free experimental systems, which have serious limitations. The
present study was conducted to establish a more physiological system,
where one can investigate the signaling functions of IGF-IIR. Here we
report that IGF-II links recombinant IGF-IIR to the G/AC
system in living cells. Furthermore, to the extreme C terminus of
IGF-IIR, we assigned a novel function of inactivating G
,
which is another component of heteromeric G proteins. This study not
only offers further evidence for the interaction of IGF-IIR with
heteromeric G proteins, it provides a novel insight into the
differential regulation of G protein subunit signals by receptors.
G and gip2 (G
Q205L) cDNAs were provided by Dr. H. R.
Bourne. Wild-type G
cDNA, human IGF-IIR cDNA,
2410-2423 cDNA, and the construction method of IGF-IIR
mutants were described previously(20, 22) .
Oligonucleotide-directed mutagenesis was done to construct
CT41
and
CT28 according to the Kunkel method(23) .
Oligonucleotides used were GAGCGTGAGGACGATTGATGAAGGGTGGGGCTGGTC for
CT41, and GCGAGGAAAGGGAAGTGATGATCCAGCTCTGCACAG for
CT28.
COS cells were grown in DMEM plus 10% calf serum and
streptomycin/penicillin. For stable expression of G,
COS cells were transfected by the calcium phosphate method using 10
µg of G
cDNA and 0.3 µg of pBabe/Puro, a
puromycin resistance gene. Cells were then selected with 3 µg/ml
puromycin and tested for immunoblot analysis with AS/7. The COS cell
line used here expresses G
at an approximately half
the level of endogenous G
.
Plasmids were
transfected by the lipofection method as described(22) .
Intracellular accumulation of cAMP was measured as
described(24) . A day before transfection, 5 10
cells were seeded on a 12-well plate. Unless otherwise specified,
cDNAs encoding IGF-IIRs or G
/gip2 (0.5 µg/ml
each) were transfected with 1 µl/ml LipofectAMINE (Life
Technologies, Inc.) and incubated for 24 h in a serum-free culture.
After washing cells with fresh media, cells were labeled with 3 µCi
of [
H]adenine for another 23 h 30 min. It should
be emphasized that cells were then washed rigorously with solution
containing M6P, as follows. DMEM-Hepes (DMEM containing 25 mM Hepes/NaOH, pH 7.4) containing 10 mM M6P was added to
cells after discarding media. Cells were then incubated for 15 min at
room temperature and washed four times with DMEM-Hepes. These
procedures, which dissociate M6P and M6P-containing proteins from
IGF-IIR, ensured reproducibility for inhibition of AC by IGF-IIR
stimulation. This was reasonable because M6P binding to IGF-IIR
impaired the action of IGF-II to inhibit AC in cells transfected with
IGF-IIR cDNA (see ``Results''). Cells were then treated with
2.5 µg/ml CTX (Calbiochem) and 1 mM isobutylmethylxanthine
with or without IGF-II (or IGF-I) in DMEM-Hepes at 37 °C for 30
min. Reactions were terminated by aspiration and the immediate addition
of 5% ice-cold trichloroacetic acid (1 ml/well). Acid-soluble
nucleotides were separated on two-step ion-exchange columns as
described(24) , and specific accumulation of cAMP is expressed
as (cAMP/ADP + ATP)
10
.
For binding assay
of IGF-IIR, cells (3 10
/dish) were transfected with
10 µg of IGF-IIR cDNAs and 20 µl of LipofectAMINE in 5 ml of
DMEM plus streptomycin/penicillin. Twenty-four hours after
transfection, the medium was renewed to DMEM plus 10% calf serum and
streptomycin/penicillin. By scraping cells 48 h after transfection,
membranes were prepared and IGF-II binding assay was performed as
described(20) . Specific binding was calculated by subtracting
nonspecific binding, the binding in the presence of 100 nM IGF-II. All other materials were obtained from commercial sources.
Data were analyzed with Student's t test.
We initially examined whether our COS cells were appropriate
to see the effects of G. Indeed, COS cells have not
frequently been used to examine the effects of G
or
G
-coupled receptors, although Bell and co-workers (25) have described the G
-coupled effect of
somatostatin receptors using COS cells. For this reason, we tested the
effect of transfection of wild-type G
or
constitutively activated G
mutant gip2 cDNA
on AC activity. As shown in Fig. 1A, transfection of gip2 resulted in dose-dependent inhibition of CTX-stimulated
cAMP accumulation, whereas that of wild-type G
had no
effect. Therefore, our COS cells seemed to be suitable for examining
the G
-coupling function of receptors with transient
transfection of cDNAs.
Figure 1:
Effects of activated G and intact IGF-IIR on AC activity in COS cells. A, dose
effect of transfection of either gip2 cDNA or wild-type G
cDNA on CTX-stimulated AC activity in COS cells. AC activity is
indicated as a percentage of CTX-stimulated activity in
mock-transfected COS cells, which was 27.2 ± 2.9
(cAMP/(ADP+ATP)
10
). Each value represents the
mean ± S.E. of single determinants done with four independent
transfections. *, p < 0.05;**, p < 0.01 versus no cDNA. B, specific IGF-II binding to the
membranes of transfected COS cells. COS cells were transfected with
each IGF-IIR or mutant cDNA. Forty-eight h after transfection,
membranes were prepared and specific IGF-II binding was measured. The
results are representative of four independent transfections, which
yielded similar results. C, dose effect of IGF-II on
CTX-stimulated AC activity in IGF-IIR-transfected COS cells. COS cells
were transfected with 0.5 µg/ml IGF-IIR cDNA. Cells were stimulated
by 2.5 µg/ml CTX in the presence of various concentrations of
IGF-II. AC activity was assessed by measuring (cAMP/(ADP + ATP))
10
. Each value represents the mean ± S.E. of
single determinants done with four independent transfections. Thus, the
effect of IGF-II was highly reproducible across transfections. *, p < 0.05;**, p < 0.01 versus no IGF-II. D, effect of IGF-II on forskolin-stimulated AC activity in COS
cells transfected with IGF-IIR. After transfection of IGF-IIR cDNA,
cells were treated with increasing concentrations of forskolin with or
without 10 nM IGF-II. AC activity is indicated as n-fold of basal activity in these cells, which was 0.345
± 0.10. The S.E. of AC activity in the presence of 1 µM forskolin stimulation without IGF-II was 2.4. Each value
represents the mean ± S.E. of single determinants done with four
independent transfections.**, p < 0.05 versus no
IGF-II
Intact IGF-IIR cDNA was transfected into
these COS cells, which were treated with IGF-II before cell lysis.
Parental COS cells expressed 3.6 fmol/µg of endogenous IGF-II
binding sites having the K of 0.90
nM. With cDNA transfection, these cells expressed recombinant
IGF-IIRs with comparable affinities by severalfold of the endogenous
binding level (Fig. 1B, B
and K
were 7.3 fmol/µg and 1.3 nM in intact IGF-IIR transfection, 9.5 fmol/µg and 1.4 nM in
CT41 transfection, 7.1 fmol/µg and 0.85 nM in
CT28 transfection, and 6.0 fmol/µg and 0.88 nM in
2410-2423 transfection, respectively). The endogenous IGF-II
binding site appears to be virtually IGF-IR, because only a
110-kDa protein was cross-linked with radioactive IGF-II in an
IGF-II-inhibitable manner in parental COS cell membranes under the same
condition as in the IGF-II binding assay (data not shown). This
assessment is not only consistent with the report of Steele-Perkins et al.(26) that IGF-IR exhibits considerably high
affinity for IGF-II, but is also strongly supported by the report of
Oshima et al.(27) showing that endogenous IGF-IIR is
scarce in COS cells.
In the cells transfected with either gene, 2.5
µg/ml CTX constantly increased AC activity by 5-fold over the
basal level. In cells transfected with intact IGF-IIR cDNA, IGF-II
significantly impaired the CTX-stimulated AC activity in a
dose-dependent manner (Fig. 1C). IGF-II also inhibited
forskolin-stimulated AC activity (Fig. 1D). It is
underscored that no inhibition of AC was observed by IGF-II without
transfection of IGF-IIR cDNA (Fig. 2). Thus, the effect of
IGF-II was attributable to the recombinant IGF-IIR. In accord with this
idea, 10 nM IGF-I did not reproduce the effect of IGF-II (Fig. 2). Consistent with our previous
study(12, 13, 16, 17, 20) ,
this inhibitory effect of IGF-II was abolished by PTX and by 10 mM M6P (Table 1). These data indicate that IGF-II triggers the
signaling function of IGF-IIR and couples it to the
G
/AC system in living cells.
Figure 2: Effect of IGF-IIR and cytoplasmic mutants on AC activity in COS cells. COS cells were transfected with recombinant IGF-IIR cDNAs or pECE vector (each 0.5 µg/ml). Cells were then stimulated by 2.5 µg/ml CTX in the presence or absence of 10 nM IGF-II, and AC activity was measured. As a control, the effect of 10 nM IGF-I was examined in the IGF-IIR-transfected COS cells. Each value represents the mean ± S.E. of single determinants done with three independent transfections. AC activity is indicated as a percentage of CTX-stimulated activity in mock-transfected COS cells, which was similar to that in the left panel C.***, p < 0.005. n. s., not significant. Inset, illustration of IGF-IIR mutants.
In cell-free systems,
the Arg-Lys
region of IGF-IIR has
been implicated in its G
coupling
function(18, 19, 20) . To examine whether
this is the case in living cells, we constructed mutant IGF-IIRs
lacking the C-terminal 41 residues after Arg
(
CT41)
or the 28 residues after Ser
(
CT28) and lacking
Arg
-Lys
(
2410-2423).
Despite remarkable expression of
CT41 (Fig. 1B),
IGF-II failed to inhibit CTX-stimulated AC activity in cells
transfected with this mutant (Fig. 2), indicating an essential
role of the C-terminal 41 residues for AC suppression. We unexpectedly
found a novel AC-linked function of
CT28. In
CT28-transfected
COS cells, CTX augmented AC activity to the same level as in COS cells
transfected with other IGF-IIRs; however, only in the
CT28-transfected cells, did IGF-II further potentiate
CTX-stimulated AC activity (Fig. 2). This effect of IGF-II
depended on the amount of
CT28 cDNA used for transfection (Fig. 3A). In the same
CT28-transfected cells,
IGF-II did not affect AC activity without CTX (not shown). These
suggest that AC potentiation by
CT28 is mediated by the
G
subunit of heteromeric G
proteins(28, 29) .
Figure 3:
IGF-II-dependent augmentation of AC
stimulation by IGF-IIRCT28. A, effect of
CT28 cDNA
on CTX-stimulated AC activity in COS cells. COS cells were transfected
with increasing doses of
CT28 cDNA and stimulated by 2.5 µg/ml
CTX with or without 10 nM IGF-II. Basal AC activity in
mock-transfected COS cells was 2.0 ± 0.1. Each value represents
the mean ± S.E. of single determinants done with four
independent transfections. *, p < 0.02;**, p <
0.05 versus no IGF-II. B, effect of
CT28 on
CTX-induced AC stimulation in COS cells overexpressing G
(COS/G
). COS/G
cells were
transfected with
CT28 cDNA (or vector), and stimulated by 2.5
µg/ml CTX with or without 10 nM IGF-II. AC activity is
indicated as a percentage of CTX-stimulated activity in
mock-transfected COS/G
cells, which was 22.7 ±
2.6. Each value represents the mean ± S.E. of four independent
experiments. Note that IGF-II augmented CTX-stimulated AC activity by
200% in parental COS cells that received
CT28 transfection
under the same condition. C, effect of PTX on
IGF-II/
CT28-induced augmentation of CTX-stimulated AC activity.
COS cells were transfected with
CT28 for 24 h and treated with 10
ng/ml PTX for another 24 h. During the last 30 min, cells were
stimulated by 2.5 µg/ml CTX with or without 10 nM IGF-II.
AC activity is indicated as a percentage of CTX-stimulated activity in
CT28-transfected COS cells. Each value represents the mean
± S.E. of four independent experiments. D, effect of
IGF-IIR
2410-2423 on AC activity in COS cells. COS cells were
transfected with
2410-2423 cDNA and then stimulated by 2.5
µg/ml CTX in the presence or absence of 10 nM IGF-II, and
AC activity was measured. Each value represents the mean ± S.E.
of four independent experiments. AC activity is indicated as percentage
of the CTX-stimulated activity in
2410-2423-transfected COS
cells, which was 20.7 ± 2.8.
To confirm the G
mediation, we examined the effect of G
on the function
of
CT28 (Fig. 3B). In COS cells overexpressing
G
(COS/G
), CTX stimulated AC activity
with a similar fold of the basal activity to that observed in parental
COS cells and
CT28-transfected COS cells. In COS/G
cells transfected with
CT28, IGF-II hardly potentiated the
CTX-stimulated AC activity, indicating that G
impaired
this function of
CT28. The reason why
CT28 could not
significantly inhibit AC activity in COS/G
cells was
likely that G
expression was not sufficient to totally
absorb the G
released by
CT28 stimulation. Since
G
acts as a specific inhibitor of G
without
affecting AC(29) , these data confirm that G
mediates
the IGF-II-triggered potentiation of AC by
CT28.
As high
concentrations of G are required to enhance AC activity, the
source of G
should be G
in non-neuronal
cells(30) . It was thus likely in our COS cells, that
CT28
releases G
by activating G
. To confirm the source
of G
, we examined the effect of PTX on this function of
CT28. As in Fig. 3C, 24-h treatment of 10 ng/ml
PTX blocked the stimulatory effect of 10 nM IGF-II in COS
cells transfected with
CT28, while the CTX response was not
changed by PTX. These data indicate that the action of
CT28 on
G
is through G
, again suggesting that the
Arg
-Lys
region is interactive with
G
.
We confirmed the inability of 2410-2423 to
affect AC (Fig. 3D). In COS cells transfected with
2410-2423, IGF-II could neither inhibit nor augment AC
activity, despite the expression of this mutant comparable to that of
intact IGF-IIR (Fig. 1B).
2410-2423 is
mutant IGF-IIR that lacks Arg
-Lys
but retains the extreme C-terminal 28 residues that
CT28
lacks. Therefore, this finally demonstrates that the domain that is
essential for the interaction with G
is not the extreme C
terminus but the Arg
-Lys
region.
We have herein established a whole-cell system in which
IGF-II-triggered signaling function of IGF-IIR can be examined. Using
this system, multiple lines of evidence show that recombinant human
IGF-IIR activates G and suppresses AC in response to
IGF-II. IGF-IIR transfection was required to observe the effect of
IGF-II on AC, consistent with the scarceness of endogenous IGF-IIR in
COS cells. IGF-I could not reproduce the effect of IGF-II. In addition,
M6P treatment of transfected COS cells blocked the effect of IGF-II,
reproducing our in vitro data(16) . It is thus
emphasized that insufficient removal of lysosomal enzymes from IGF-IIR
precludes this receptor from responding to IGF-II. Furthermore, the
Arg
-Lys
region is shown here to be
essential for AC suppression by IGF-IIR, as predicted by our in
vitro study(18, 19, 20) .
In this
study, an IGF-IIR mutant has pointed to a novel function of the
receptor C terminus. CT28 enhanced cAMP production in response to
IGF-II. Multiple lines of evidence indicate that this response was
mediated by G
, the source of which was the activated
G
. In contrast, intact IGF-IIR activated G
and
mainly generated the signal of G
(inhibition of AC) in
response to the same stimulation. These indicate that the C-terminal
Ser
-Ile
region of IGF-IIR can inactivate
G
. This inactivation suggests direct or indirect interaction
of this C-terminal region with G
. In further support of this
idea, the Ser
-Ile
region is homologous to
a part of the PH domains (31) of multiple proteins including
-adrenergic receptor kinase (Fig. 4), which are proven to
bind G
(32) . It has also been shown that the isolated
PH domain of
-adrenergic receptor kinase inactivates the action of
G
(33) . These findings suggest that this region of
IGF-IIR inactivates G
through interaction. Because of
technical difficulty, we were not able to examine the effects of the
isolated regional peptides on AC augmentation by
CT28 observed in
this whole-cell system.
Figure 4:
The C terminus of human IGF-IIR is
homologous to multiple PH domains. Ile is the C-terminal
end of human IGF-IIR. Identical residues are in black, and
similar residues are shaded. The sequences of the PH domains
all correspond to their sixth subdomains.
Among known AC subtypes, no single AC that
responds to both G (inhibitory) and G
(stimulatory) has been specified. However, among AC types I-VI,
our COS cells express type VI, which is inhibited by
G
, and type IV, which is stimulated by G
(data not shown). It is thus reasonable to assume that the whole
response of AC to G
in intact COS cells is the sum of the
respective effects of G
and G
on these AC
subtypes, thus allowing the total AC activity to respond to both
G-protein subunits.
In summary, this study shows that IGF-IIR, in
living cells, activates G and affects AC through
differential actions of multiple cytoplasmic domains of its own. In our
cells, it activates G
through
Arg
-Lys
and inactivates G
through Ser
-Ile
, resulting in the
predominant action of G
. Therefore, the distinct roles
played by multiple domains of IGF-IIR separate and sequestrate the
G
and G
signals following G
activation. This
is potentially a very interesting mechanism that allows a receptor to
differentially activate G
and G
and selectively turn on
each subunit-specific pathway. With this novel mechanism, there is no
longer necessity that a receptor must always turn on both subunit
pathways by activating one heteromeric G protein complex. It is thus
important to investigate whether a similar mechanism is possessed by
other receptors.
It is also conceivable that this novel property of
IGF-IIR may contribute to its unique signaling function in
vivo. Multiple effects of G depend on
G
-released G
(34) . Thus, IGF-IIR-induced
G
activation may lack some of the G
outputs
induced by conventional receptors. It is also conceivable that the
G
inactivating effect of the C terminus of this receptor may
be affected by the amount of free G
inside the cell. There
may be an intracellular free G
pool with different sizes in
different cells(35) . Excess G
may thus occupy the C
terminus and attenuate its inhibitory effect. In accord with this idea,
IGF-II binding to IGF-IIR can potentiate AC stimulation in human
fibroblasts (36) but not in COS cells (this report) and can
stimulate PI turnover in renal cells (5) but not in Balb/c3T3 (12) or CHO cells (15) . Alternatively, the
G
-linked function of IGF-IIR might be involved in its
trafficking function as an M6P receptor, as one of the established
functions of G
is translocation of target proteins (37) . This is, however, less likely, because the residues
essential for IGF-IIR trafficking have been mainly localized near the N
terminus of the cytoplasmic domain, particularly before
Arg
(38) . This possibility is further lowered by
the fact that the cation-dependent M6P receptor, another trafficking
receptor for M6P, has no cytoplasmic regions homologous to the PH-like
domain in the extreme C terminus of IGF-IIR. In conclusion, this study
demonstrates the coupling of IGF-IIR with heteromeric G proteins in
native cell environments. While calcium influx is one of its most
likely outputs(12, 13, 14, 15) , it
is important to determine which cellular function is executed by the
demonstrated IGF-IIR interaction with the G proteins.