Different Subcellular Localization and Phosphoinositides Binding of Insulin Receptor Substrate Protein Pleckstrin Homology Domains
Giorgia Razzini,
Alessandra Ingrosso,
Anna Brancaccio,
Salvatore Sciacchitano,
Diana L. Esposito and
Marco Falasca
Unit of Physiopathology of Cell Signaling (G.R., A.I., A.B.,
M.F.) Laboratory of Cellular and Molecular Endocrinology
Department of Cell Biology & Oncology Istituto di Ricerche
Farmacologiche "Mario Negri" Consorzio Mario Negri Sud,
66030 Santa Maria Imbaro (CH), Italy
Department of
Experimental Medicine and Pathology (S.S.) Universita "La
Sapienza" di Roma 00161 Roma, Italy
Department of
Oncology and Neuroscience (D.L.E.) Università G. DAnnunzio
Chieti 66100 Chieti, Italy
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ABSTRACT
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Insulin evokes diverse biological effects through
receptor-mediated tyrosine phosphorylation of the insulin receptor
substrate (IRS) proteins. Here, we show that, in vitro, the
IRS-1, -2 and -3 pleckstrin homology (PH) domains bind with different
specificities to the 3-phosphorylated phosphoinositides. In fact, the
IRS-1 PH domain binds preferentially to phosphatidylinositol
3,4,5-trisphosphate (PtdIns-3,4,5-P3), the IRS-2 PH domain
to phosphatidylinositol 3,4-bisphosphate
(PtdIns-3,4-P2), and the IRS-3 PH domain to
phosphatidylinositol 3-phosphate. When expressed in NIH-IR fibroblasts
and L6 myocytes, the IRS-1 and -2 PH domains tagged with green
fluorescent protein (GFP) are localized exclusively in the cytoplasm.
Stimulation with insulin causes a translocation of the GFP-IRS-1 and -2
PH domains to the plasma membrane within 35 min. This translocation
is blocked by the phosphatidylinositol 3-kinase (PI 3-K) inhibitors,
wortmannin and LY294002, suggesting that this event is PI 3-K
dependent. Interestingly, platelet-derived growth factor (PDGF) did not
induce translocation of the IRS-1 and -2 PH domains to the plasma
membrane, indicating the existence of specificity for insulin. In
contrast, the GFP-IRS-3 PH domain is constitutively localized to the
plasma membrane. These results reveal a differential regulation of the
IRS PH domains and a novel positive feedback loop in which PI 3-K
functions as both an upstream regulator and a downstream effector of
IRS-1 and -2 signaling.
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INTRODUCTION
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The insulin receptor (IR) is a tyrosine kinase that phosphorylates
intermediate adapter proteins on multiple tyrosine residues during its
activation. Several insulin receptor substrate (IRS) proteins have been
identified, including IRS-1, IRS-2, IRS-3, and IRS-4 (1). These
multidocking proteins activate downstream signaling pathways by
interacting with SH2-containing proteins, some of which are key players
in insulin signal transduction, such as Grb-2, the regulatory subunit
of phosphoinositide 3-kinase (PI 3-K) p85, and the phosphotyrosine
phosphatase SHP-2 (SH2-containing protein tyrosine phosphatase) (2).
Recently, a new IR-docking protein, called Gab-1 (Grb-2-associated
binding-1) has been identified (3). The activity of PI 3-K is necessary
to elicit many of the effects of insulin on glucose and lipid
metabolism, indicating that it is an essential downstream effector of
insulin signaling (4). The principal mechanisms through which insulin
activates PI 3-K appear to be via IRS proteins. IRS-1 and -2 are widely
distributed, whereas IRS-3 expression has been demonstrated in adipose
tissue, fibroblasts, and liver cells (5, 6). In adipose cells, the
association of the regulatory subunit of PI 3-K p85 is much more rapid
for IRS-3 than for IRS-1 (5). In addition, in the same cells, PI 3-K
activation by IRS-3 is quantitatively higher than that mediated by
IRS-1 or IRS-2. In studies of the specificity of insulin signaling, it
is of great interest to determinate the exact contribution of each
single IRS protein in the promotion of the downstream effects of
insulin. In particular, the formation of complexes catalyzed by IRS
proteins in specific compartments appears crucial. All the IRS proteins
have similar structures, sharing a highly homologous N terminus
pleckstrin homology (PH) domain, and a phosphotyrosine binding (PTB)
domain, both of which are important for mediating interactions with the
insulin receptor (1). PH domains, which have been found in several
signaling proteins, have functional requirements for membrane
association (7). The IRS-1 PH domain has been shown to be essential for
efficient tyrosine phosphorylation of IRS-1 during insulin stimulation
(8, 9). The ligands of IRS PH domains are unknown, although it has been
shown that the IRS-1 and -2 PH domains bind to acidic proteins (10).
Many PH domains have been found to bind phosphoinositides (11), and
this suggests that the PH domains of the IRS proteins might allow the
interaction of these with membrane phosphoinositides. Here we have
examined the role of the IRS PH domains in membrane targeting and
phosphoinositide interactions by using subcellular localization studies
in vivo and phosphoinositide-binding assays in
vitro. We show that the IRS-1 and IRS-2 PH domains, tagged using
green fluorescent protein (GFP), are localized exclusively in the
cytoplasm in serum-starved NIH-IR and L6 cells. Stimulation with
insulin or insulin-like growth factor I (IGF-I), but not
platelet-derived growth factor (PDGF) or epidermal growth factor (EGF),
induces a rapid translocation of the GFP-IRS-1 and GFP-IRS-2 PH
domains to the plasma membrane. This translocation is blocked by
pretreatment with the PI 3-K inhibitors, wortmannin and LY294002, and
it is stimulated by cotransfection of the constitutively active PI 3-K
(p110F). In addition, we show that the PH domains of IRS-1 and IRS-2
bind the lipid products of PI 3-K, corroborating the idea that PI 3-K
is a determinant for PH domain-mediated anchoring to the plasma
membrane. Finally, the PH domain of IRS-3 binds specifically to
phosphatidylinositol 3-phosphate (PtdIns-3-P), and is present on the
plasma membrane either in serum-starved or insulin-stimulated cells.
These results reveal a novel positive feedback loop in which PI 3-K
functions as both an upstream regulator and a downstream effector of
IRS-1 and IRS-2 signaling in insulin-stimulated cells, and suggest the
differential regulation of IRS-1 and IRS-2 vs. IRS-3.
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RESULTS
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Specific Binding of the IRS Protein PH Domains
To study the lipid binding properties of the PH domains derived
from human (h)IRS-1, murine (m)IRS-2, and mIRS-3, we used a lipid
dot-blot assay to analyze the binding of
[32P]-labeled
glutathione-S-transferase (GST)-PH domain fusions to
different phosphoinositides spotted on nitrocellulose, as previously
described (12). Figure 1
shows the
results of the dot-blot assay for GST alone (Fig. 1A
) and GST-PH domain
fusions from phospholipase C
(Fig. 1B
) and protein kinase B (PKB,
also known as Akt and RAC; Fig. 1C
), used as positive controls, and for
IRS-1, IRS-2, and IRS-3 (Fig. 1
, DF). The control blot using
[32P]-labeled GST alone gave no signals above
background, whereas the phospholipase C
(PLC
) PH domain
bound phosphatidylinositol 4,5-bisphosphate
(PtdIns-4,5-P2) with high specificity, and the
Akt/PKB PH domain bound phosphatidylinositol 3,4,5-trisphosphate
(PtdIns-3,4,5-P3) and phosphatidylinositol
3,4-bisphosphate (PtdIns-3,4-P2) as previously
shown (13, 14, 15). Analysis of these blots indicated that the IRS-1 PH
domain showed a promiscuous pattern with preferential binding to
PtdIns-3,4-P2, but also, although weaker, binding
to PtdIns-3,4,5-P3 and
PtdIns-4,5-P2. Higher specificity was observed in
the case of the IRS-2 PH domain, which showed a preferential
binding to PtdIns-3,4-P2 (Fig. 1E
), whereas the
IRS-3 PH domain showed specific binding for PtdIns-3-P (Fig. 1F
). Only
small quantities of other phosphoinositides were bound by IRS-1, IRS-2,
and -3 PH domains. Other inositol phospholipids tested in the same
assay, such as phosphatidylinositol 4-phosphate, phosphatidylinositol
5-phosphate, and phosphatidylinositol 3,5-bisphosphate, gave much
weaker or no signals (data not shown). Since this assay does not allow
a quantitative comparison, we further investigated the phosphoinositide
binding to IRS protein PH domains by using a different assay. We
incubated a GST-fusion of the IRS PH domains bound to glutathione beads
with a mixture of tritiated inositol phosphate standards (see
Materials and Methods and Ref. 16). The inositol phosphates
present in the mixture (Fig. 2A
) and
those remaining attached to the glutathione beads (Fig. 2
, BE) after
incubation with the mixture and subsequent washing, were analyzed by
HPLC. As shown in Fig. 2B
, the GRP-1 PH domain, our positive control,
bound inositol-1,3,4,5-tetrakisphosphate
(Ins-1,3,4,5-P4) with very high
specificity, as previously shown (17), while the
PLC
1 PH domain bound
inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) (not
shown) (15). The IRS-1 PH domain showed preferential binding to
Ins-1,3,4,5-P4 although substantial binding to
inositol-1,3,4-trisphosphate (Ins-1,3,4-P3) and
Ins-1,4,5-P3 was also evident (Fig. 2C
). The
binding of the IRS-2 and IRS-3 PH domains for
Ins-1,3,4-P3 (Fig. 2D
) and inositol
3-phosphate (Ins-3-P) (Fig. 2E
), respectively, was much more
specific. In the latter case, we also detected a peak around 71 min
that corresponds to inositol-1,3-bisphosphate
(Ins-1,3-P2), which is present as a contaminant
in the standards pool used in the assay. In addition, it is worth
noting that in this pool we used Ins-3-P instead of
Ins-1,3-P2.

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Figure 1. Comparison of the Phosphoinositide-Binding
Specificities of the GST-IRS PH Domains Using a Dot-Blot Assay
[32P]-labeled GST-PH domain fusion proteins were used to
probe nitrocellulose filters onto which specific phosphoinositides had
been spotted. The amount of lipid spotted in each row was 0.2, 0.4,
0.6, 0.8, and 1.0 µg.
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Figure 2. Inositol Phosphate Binding Specificities of the
GST-IRS PH Domains
GST fusion proteins attached to beads were incubated with a mixture of
standard [3H]-inositol phosphates. The beads were washed,
and then the bound inositol phosphates were eluted with 0.1
N HCl, separated by HPLC, and quantified by an on-line
radioactivity flow detector. A, HPLC profile of the mixture of standard
[3H]-inositol phosphates (1/10 of mixture incubated with
the beads); B, [3H]-inositol phosphates bound to the GST
Grp-1 PH domain; C, to the GST IRS-1 PH domain, D, to the GST IRS-2 PH
domain, and E, to the GST IRS-3 PH domain. No binding was observed with
GST alone.
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Quantitative analyses of the HPLC patterns, expressing the amount of
radioactivity of each compound as a percentage of the total
radioactivity present in the mixture, and then, in the case of the
IRS-1 PH domain, as a percentage of
Ins-1,3,4,5-P4 bound, demonstrate that the IRS-1
PH domain binds most strongly to Ins-1,3,4,5-P4
(100.0 ± 12.0) and, to a lesser extent, to
Ins-1,3,4-P3 (38.5 ± 2.5), Inositol
1,4,5-P3 (20.0 ± 5.4), and Ins-3-P
(8.5 ± 5.1). The same analyses for the IRS-2 and IRS-3 PH domains
show strong preferential binding to Ins-1,3,4-P3
and Ins-3-P, respectively, and weak (<10% of preferential inositol
phosphates) or no binding to the other inositol phosphates. Similar
data were obtained when the GST fusion of the IRS-1, IRS-2, and IRS-3
PH domains was incubated with deacylated cell-lipid extracts obtained
from [3H]-inositol-labeled L6 cells stimulated
with insulin. Furthermore, the same relative affinities for binding to
standard inositol headgroups were observed when a quantitative analysis
was performed using deacylated phosphoinositides (data not shown).
Subcellular Localization of the IRS Protein PH Domains
To explore the possible roles of the PH domains in
signal-dependent localization of the IRS proteins, we analyzed the
subcellular localization of the IRS PH domains fused to a GFP. We
transfected the GFP-IRS PH domains into NIH-IR and L6 cells. In
serum-starved NIH-IR cells transfected with the GFP-IRS-1 and GFP-IRS-2
PH domains, all the fluorescence was dispersed in the cytoplasm (Fig. 3
, A and D). Stimulation of cells with
100 nM insulin induced a translocation of the GFP-IRS-1 and
IRS-2 PH domains to the plasma membrane, which was visible after 23
min of stimulation, and more evident at longer times (510 min; Fig. 3
, B, C, E, and F). A different behavior was observed when the IRS-3 PH
domain was transfected. In fact, as shown in Fig. 3G
, in serum-starved
cells there was a clear plasma membrane localization, and after insulin
stimulation there were no qualitative differences, although the
localization appeared to be more pronounced (Fig. 3
, H and I). To
obtain a quantitative analysis of these observations, a blind scoring
of the intracellular localization was performed and has been shown in
Fig. 3J
; clear quantitative differences exist in the plasma membrane
localization of the GFP-IRS-1 and IRS-2 PH domains in unstimulated and
insulin-stimulated NIH-IR cells, whereas no difference was found in the
case of the GFP-IRS-3 PH domain. The nuclear staining present in both
unstimulated and stimulated cells was also found with the empty vector
and therefore represents nonspecific staining due to the small size of
the fusion protein that permits it to diffuse through the nuclear pores
(16).

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Figure 3. Insulin-Induced GFP-IRS PH Domain Membrane
Translocation in NIH-IR Cells
Fluorescence micrographs, obtained as described previously (9 ), of
NIH-IR cells expressing GFP-IRS-1 (AC), GFP-IRS-2 (DF), and
GFP-IRS-3 (GI) PH domains at the indicated times after insulin (100
nM) stimulation. Bar, 10 µm. J,
Statistical analysis of the plasma membrane localization in control and
insulin-stimulated NIH-IR cells transfected with GFP IRS-1, IRS-2, and
IRS-3 PH domains. Means ± SD from three separate
experiments, each performed in duplicate, are shown.
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Effects of Other Growth Factors on the Subcellular Localization of
the IRS Protein PH Domains
To determine whether a similar translocation of IRS PH domains
takes place in response to stimulation of other receptors that activate
PI 3-K, NIH-IR 3T3 and L6 cells were also stimulated with IGF-I, PDGF,
and EGF. IGF-I evoked a translocation response of the IRS-1 and -2 PH
domains very similar to that observed when insulin was used as the
agonist (Fig. 4
, C and D). Interestingly in this case, under the same
conditions, PDGF (Fig. 4
, E and F) and
EGF (Table 1
) were not able to induce any
translocation of the GFP-IRS-1 and GFP-IRS-2 PH domains in L6 and
NIH-IR cells after up to 10 min of stimulation, remaining instead like
the untreated cells (Fig. 4
, A and B). In parallel, as a positive
control, PDGF was able to induce plasma membrane translocation of
GFP-Akt-PH (data not shown). To further analyze this selectivity in
IRS-1 PH domain translocation induced by different growth factors, we
microinjected the GFP-IRS-1 PH domain DNA into differentiated 3T3 L1
adipocytes, where differences in insulin and PDGF signaling have been
well studied. As shown in Fig. 5A
, in
serum-starved adipocytes, no specific localization of the IRS-1 PH
domain was observed, while after 10 min of insulin stimulation there is
a clear plasma membrane localization (Fig. 5B
); this was not seen with
PDGF stimulation (Fig. 5C
), confirming the specificity of insulin in
inducing plasma membrane localization of the IRS-1 PH domain.

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Figure 4. IGF-I, but Not PDGF, Induces IRS-1 and IRS-2 PH
Domain Membrane Translocation in NIH-IR Cells
Cells were transiently transfected with GFP-IRS-1 and GFP-IRS-2 PH.
After 24 h of serum starvation, the cells were stimulated with
IGF-I (10 ng/ml, panel B), or PDGF (25 ng/ml; panel C).
Bar, 10 µm.
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Figure 5. Insulin, but Not PDGF, Induces IRS-1 PH Domain
Membrane Translocation in 3T3 L1 Adipocytes
3T3 L1 adipocytes were serum-starved for 12 h and then
microinjected with GFP-IRS-1. After 4 h, cells remained
unstimulated (A), were stimulated with insulin (100 nM,
panel B), or PDGF (25 ng/ml, panel C).
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Effect of PI 3-K Inhibitors on the IRS Protein PH Domain
Localization
To test whether this event was PI 3-kinase-dependent, as our
binding data suggested, we preincubated NIH-IR and L6 cells with the PI
3-K inhibitors wortmannin (Fig. 6
, B and D) and LY294002 (Table 1
)
before insulin stimulation. This pretreatment completely abolished
insulin-dependent translocation to the plasma membrane of the GFP-IRS-1
and 2 PH domains in NIH-IR (Fig. 6
, B
and D) and L6 cells (Table 1
). In addition, similar data were obtained
by coexpression with a dominant negative p85 (the regulatory subunit of
PI 3-K) mutant (Table 1
). When the effect of the inhibitors of PI 3-K
on IRS-3 PH domain localization was examined, we found that wortmannin
inhibited GFP IRS-3 PH domain plasma membrane targeting in both
insulin-stimulated (Fig. 6
, E and F) and serum-starved NIH-IR (Fig. 6
, G and H) and L6 cells (Table 1
), whereas coexpression with a dominant
negative p85 had no effect (Table 1
). Next, we examined the effect of
expression of a constitutively activated PI 3-K (p110F) on the cellular
localization of the GFP-IRS-1 and -2 PH domains. In L6 cells, membrane
localization of the IRS-1 and -2 PH domains was not seen in cells that
express a membrane-targeted kinase-inactive form of PI 3-K (p110KD)
(Fig. 6
, I and J). In contrast, in L6 cells that overexpress the
membrane-targeted PI 3-K, the PH domains of IRS-1 and IRS-2 were
targeted to the plasma membrane even in serum-starved cells (Fig. 6
, K
and L). These data suggest that the IRS-1 and -2 PH domain binding to
the PI 3-K products plays a role in their membrane translocation. In
addition, the membrane targeting of the IRS-3 PH domain seems not to be
dependent on p110 activity, but should be dependent on the level of
PtdIns-3-P itself.

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Figure 6. Effect of PI 3-Kinase Inhibition or Activation on
Membrane Localization of the GFP-IRS PH Domains
AD, Fluorescence micrographs of serum-starved NIH-IR cells expressing
the GFP-IRS-1 and IRS-2 PH fusion proteins, stimulated with 100
nM insulin for 10 min, with 100 nM wortmannin
pretreatment for 10 min. EH, Fluorescence micrographs of
serum-starved NIH-IR cells expressing the GFP-IRS-3 PH fusion protein,
stimulated (E and F) or unstimulated (G and H) with 100 nM
insulin for 10 min, with 100 nM wortmannin pretreatment for
10 min (F and H). IL, Fluorescence micrographs of serum-starved L6
cells that had been cotransfected with a vector expressing the
GFP-IRS-1 and GFP-IRS-2 PH fusion proteins and inactivated (p110KD)
p110ß (PI 3-K) and membrane-targeted activated (p110F). All p110
variants have been described and characterized previously (37 ). In all
transfection experiments the total amount of DNA in each transfection
was held constant by the addition of empty vector. Bar,
10 µm.
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Phosphoinositide Specificity and Subcellular Localization of the
IRS-1 PH Domain Mutated in the Variable Loop
Our localization studies indicated that the IRS-1 PH
domain translocation is PI 3-K dependent, while our binding data
indicated that the IRS-1 PH domain binds weakly to PI 3-K lipid
products. To further investigate whether the IRS-1 PH domain is able to
bind to membrane PtdIns-3,4,5-P3 in intact cells,
we examined the binding of an IRS-1 PH domain mutant. In PH domains of
proteins known to bind products of PI 3-K, such as Bruton tyrosine
kinase, Grp-1, and Akt, it has been shown that an arginine located in
the ß2-strand (Fig. 7a
) is critical for
lipid binding as well as plasma membrane localization. Therefore, we
mutated this residue (R28C) and tested whether the PH domain was still
able to localize to the plasma membrane, and whether the lipid binding
was affected. As shown in Fig. 7
, B and C, the IRS-1 mutant neither
localized to the plasma membrane upon insulin stimulation, nor bound
the phosphoinositides in our binding assays (Table 1
), indicating that
phosphoinositide binding of the IRS-1 PH domain is essential for its
recruitment to the plasma membrane.

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Figure 7. Insulin-Induced Changes in the Localization of the
Wild-Type and R28C Mutant GFP IRS-1 PH Domain in Transfected NIH-IR
Cells
a, Schematic representation of the IRS-1 structure and alignment of the
ß2-strand of the IRS-1 PH domain with other PH domains of proteins
known to bind the products of PI 3-K. b, Fluorescence micrographs of
NIH-IR cells expressing GFP-IRS-1 (A and B) and GFP-IRS-1 R28C (C).
Bar, 10 µm.
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DISCUSSION
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In this study, we have examined the subcellular localization of
the PH domains derived from IRS-1, IRS-2, and IRS-3, and their binding
to inositol phospholipids and phosphates. In summary, our results
clearly show that the IRS-1 PH domain has a weaker preferential
affinity for PtdIns-3,4-P2 and
PtdIns-3,4,5-P3, the IRS-2 PH domain binds to
PtdIns-3,4-P2, and the IRS-3 PH domain binds to
PtdIns-3-P. In addition, insulin and IGF-I stimulate the translocation
of the IRS-1 and IRS-2 PH domains from the cytosol to the plasma
membrane in NIH-IR and L6 cells, and this translocation is dependent
upon insulin-stimulated PI 3-K activity; the IRS-3 PH domain did not
show this insulin-dependent translocation.
These observations enlarge on the concept that several PH domains play
a determinant role in the translocation of cytosolic proteins to the
plasma membrane, where the lipid products of PI 3-K are formed. The
observed PI 3-K-dependent localization of the IRS-1 and IRS-2 PH
domains to the plasma membrane supports the intriguing idea that PI 3-K
is both required for, and a consequence of, IRS-1 signaling. PI 3-K is
an essential enzyme in the insulin-signaling cascades that mediate both
metabolic and mitogenic cellular responses (4). Insulin can activate
both IRS-associated and non-IRS-associated PI 3-K activities (4, 18).
It is of interest to determine the exact way through which insulin
activates PI 3-K-mediated recruitment of the IRS-1 and IRS-2 PH
domains. It is thus possible that the direct activation of PI 3-K by
insulin or IGF-I receptors (19, 20), or the PI 3-K activity recruited
by the other IR docking proteins, could be involved in IRS-1 and IRS-2
membrane targeting. One possible explanation for these results is that
IRS-3 is responsible for PI 3-K activation after insulin stimulation,
and thereby promotes IRS-1 and IRS-2 translocation. Interestingly,
PDGF, a potent stimulator of PI 3-K, whose activity in terms of lipid
production was confirmed in the cell types used in the present study
(data not shown), does not induce membrane translocation of the IRS-1
and IRS-2 PH domains. This result suggests that the PI 3-K-induced
translocation of certain PH domains is specific for certain growth
factors. This specificity could be explained as previously proposed
(21, 22, 23), as a consequence of different quantitative or spatio-temporal
accumulation of PI 3-K products, possibly due to differential
localization and regulation of PI 3-K by insulin vs. PDGF.
It has been shown that in 3T3-L1 adipocytes, insulin and PDGF activate
PI 3-K rapidly, although the time courses of activation differ, since
insulin activation of PI 3-K is more persistent compared with the
transient activity induced by PDGF (24). Alternatively, and as recent
results suggest (10), it could be that PI 3-K is necessary, but not
sufficient, to induce this translocation, and that other factors, such
as specific proteins, are involved. Our binding data establish clearly
that the PH domains of IRS-2 and IRS-3 preferentially bind
PtdIns-3,4-P2 and PtdIns-3-P, respectively,
whereas the binding specificity of the IRS-1 PH domain appears less
clear. In fact, the dot-blot analysis seems to indicate a higher
affinity of the IRS-1 PH domain for
PtdIns-3,4-P2, whereas the binding on glutathione
beads suggests a preferential binding to
PtdIns-3,4,5-P3. This discrepancy can be
explained by the fact that PtdIns-3,4,5-P3 is
more water soluble and hence can be washed out of the filter, thus
giving an underestimation of its binding to the IRS-1 PH domain, as
already discussed (16). At the same time, and taking into consideration
that there is preferential binding to
PtdIns-3,4,5-P3, we also observed substantial
binding to PtdIns-4,5-P2; the latter is much more
abundant than the 3-phosphorylated phosphoinositides (25), even in
activated cells, and may suggest that in the cell the PH domain would
interact with PtdIns-4,5-P2. In addition, as
shown in Fig. 2
and according to previous data (14), Grp-1 PH domain
binding to PtdIns-3,4,5-P3 is much stronger
compared with that observed with the IRS-1 PH domain. This evidence may
indicate the absence of any physiological role for this interaction.
However, our experiments demonstrating the importance of PI 3-K in
IRS-1 PH domain translocation imply that this event is most likely
regulated by PI 3-K products. In addition, the evidence that the IRS-1
PH domain does not localize in serum-starved cells indicates that the
potential interaction with PtdIns-4,5-P2 is not
relevant. In other words, we can hypothesize that binding of IRS-1 to
PtdIns-3,4,5-P3 occurs only where there is a
substantial and localized accumulation of
PtdIns-3,4,5-P3. Consistent with this hypothesis
is the evidence that constitutively active PI 3-K alone was sufficient
to cause the translocation of the IRS-1 PH domain. A possible
explanation for these data, other than cooperation between the
phosphoinositides and further proteins, as already proposed for the
ß-adrenergic receptor kinase PH domain (26), could be that the IRS-1
PH domain requires oligomerization for high-affinity phosphoinositide
binding, as already shown for PH domains of dynamin isoforms (27). Our
binding data cannot rule out a role for protein binding to IRS PH
domains, as recent data have suggested for the IRS-1 and IRS-2 PH
domains (10). In fact, this interaction could give further specificity,
together with lipid binding, or may be involved in a different step of
IRS activation. Previous studies using cell fractionation have shown
that IRS-1 and -2 are mainly located in the low density microsome
fraction, showing a different pattern of localization compared with
IRS-3, which is located mainly in the plasma membrane fraction. IRS-1
and -2 localization to the intracellular membranes seems not to involve
the PH domains. The mechanism by which the binding of PI 3-K lipid
products to the isolated PH domains of the IRS proteins play a role in
the regulation of the intact proteins in the cells remains unknown, but
is at present under investigation. In the case of IRS-1 and IRS-2, this
interaction could play a role in the correct orientation of the docking
protein for efficient tyrosine phosphorylation by the receptor. A
similar model for IRS-1 has been recently proposed on the basis of
structural and functional data (28). Therefore, in the case of IRS-1
and IRS-2, although the PH domain localization to the plasma membrane
is physiologically relevant, it is not sufficient to localize the
entire protein. This explains why its localization has not been found
previously on the plasma membrane, although it is fundamental for its
correct orientation and targeting to the plasma membrane. In fact, as
depicted in our model (Fig. 8
), we
hypothesize that the IRS-1 and IRS-2 proteins are targeted to
intracellular membranes, and upon insulin stimulation the protein
could, through its PH domain, interact with the plasma membrane; this
could potentially occur in specific areas, such as with the
cytoskeleton. This could thus be an intriguing explanation of the
specificity of insulin vs. PDGF signaling and also
underlines the importance of the localization of IRS-1 and IRS-2 to
specific intracellular compartments.

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Figure 8. Model Outlining the Role of Phosphoinositide-IRS-1
and IRS-2 PH Domain Interactions in Insulin Signaling
LDM, Low-density microsome.
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Alternatively, a different mechanism could be hypothesized. It has
recently been shown that a truncated IRS-1 protein that retains only
the PH and the PTB domains inhibits apoptosis and replication of
32DIR cells during insulin stimulation, and that
although PI 3-K was not activated, its lipid products were required
since LY294002 inhibited these responses (29). These results indicate
that novel phosphotyrosine-independent signaling pathways may be
regulated by the PH and PTB domains of IRS proteins, suggesting a
possible functional role of 3-phosphoinositide interactions with IRS-PH
domains.
Recent results confirm that the IRS-1 and -2 PH domains are
functionally similar and are required for interaction with
3-phosphorylated phosphoinositides to facilitate interactions between
IRS proteins and the insulin receptor (30). In fact, the IRS-2 domain
can substitute for the IRS-1 domain of a chimeric IRS protein, whereas
heterologous PH domains from the ß-adrenergic receptor kinase,
phospholipase C
(the putative split PH domain not the N-terminal) or
spectrin, which did not show binding preference for 3-phosphorylated
phosphoinositides in our assays (data not shown and Ref. 16), fail to
mediate tyrosine phosphorylation of chimeric IRS-1 proteins (30). The
preferential binding of the IRS-2 PH domain to
PtdIns-3,4-P2 suggests an important role for this
lipid in insulin signaling by the addition of a downstream target
together with Akt/PKB. In addition, these data underscore the potential
implication of a PtdIns-3,4,5-P3 5'-phosphatase,
the enzyme that has PtdIns-3,4,5-P3 as substrate,
and produces PtdIns-3,4-P2. In fact, it has been
previously shown that insulin activates the formation of complexes
containing PtdIns-3,4,5-P3 5'-phosphatase (31).
Thus, it is possible that the insulin-sensitive
PtdIns-3,4,5-P3 5'-phosphatase has a positive
signaling function.
The finding that the IRS-3 PH domain binds to PtdIns-3-P
introduces the novelty that a PH domain binds specifically to
PtdIns-3-P, and that this interaction occurs at the plasma membrane.
Previous data have shown that a protein domain called FYVE, from the
initials of the proteins where it was first characterized, binds
specifically to PtdIns-3-P and localizes the protein to the Golgi
compartment (32, 33, 34). Interestingly, the FYVE domain of EEA1 also binds
to Rab5 (35), suggesting that the binding specificity is given not only
by PtdIns-3-P, but that it also requires a protein interaction. The
localization of the IRS-3 PH domain to the plasma membrane, even in
serum-starved cells, and its displacement by pretreatment with
wortmannin (known to reduce the level of PtdIns-3-P) suggest not only
that a PH domain may bind specifically to this lipid, but also that a
portion of the PtdIns-3-P is constitutively present on the plasma
membrane.
When our results are combined with previous reports (36), it appears
that the regulation of IRS-1 and -2 is different from that of IRS-3.
Further studies will be required to clarify which IRS protein
transduces the signal inducing the individual insulin-induced cell
activities; perhaps the use of single PH domains as dominant negatives
will help in the comprehension of signals transduced by each of the IRS
proteins.
 |
MATERIALS AND METHODS
|
---|
PDGF-BB and IGF-I were purchased from PeproTech Ltd. (London,
UK). DMEM, calf serum (CS), and FCS were obtained from Life Technologies, Inc. (Gaithersburg, MD). All other biochemicals
were of the highest possible purities and were obtained from
Sigma (St. Louis, MO).
Expression of GST-PH Fusion Proteins
The cDNA fragments corresponding to the PH domains from human
IRS-1 (residues 1134), mouse IRS-2 (residues 1144), and mouse IRS-3
(20145) were amplified using PCR. The PCR products were digested with
BamHI plus EcoRI and were ligated into the
appropriately digested pGEX-2TK (Amersham Pharmacia Biotech, Arlington Heights, IL) bacterial expression vector to
direct expression of the GST-PH domain fusion proteins. The DNA
sequence of each PH domain insert was verified by dideoxynucleotide
sequencing. The GSTPH domain fusions were expressed and purified
using glutathione-agarose, as described previously (16). The point
mutation in IRS-1 was generated by PCR mutagenesis using the Quick
Change Mutagenesis System (Stratagene, La Jolla,
CA).
Dot-Blot Assay
Phosphoinositides (Matreya, Inc., Pleasant Gap, PA; and Echelon,
Salt Lake City, UT) in 1:1 chloroform-methanol containing 0.1% HCl (12
N) were spotted onto nitrocellulose filters, as shown.
After drying, the nitrocellulose filters were blocked for 68 h at 4 C
in Tris-buffered saline (TBS) containing 1% BSA, without detergent.
The [32P]-labeled GST-PH domain fusion
proteins, labeled as previously described (12), were then added to
TBS/1% BSA to a final concentration of 0.5 µg/ml, and this solution
was used to probe the phosphoinositide-containing nitrocellulose for 30
min at room temperature. Filters were washed five times with TBS and
dried, and the bound radioactivity was visualized and quantified using
a PhosphorImager (Molecular Dynamics, Inc.).
Analysis of [3H]-Inositol Phosphate and
[3H]-Glycerophosphoinositide Binding to PH
Domains
Mixtures of standard [3H]-inositol
phosphates [NEN Life Science Products (Boston, MA), 21
Ci/mmol; ARC, Inc. (St. Louis, MO)], used at the same concentrations,
were added to the GST-PH fusion proteins (50 µg) on
glutathione-agarose beads. After incubation at 25 C for 10 min, the
beads were washed three times with 10 mM Tris, pH 7.4, and
the bound counts eluted with 0.1 N HCl. The eluted material
was analyzed by strong anion exchange (SAX) HPLC as described
previously (16), using a Partisil 10 SAX analytical column, eluting
with a shallow gradient from 0.0 to 1.0 M
(NH4)2HPO4
(pH 3.80). Radioactivity in the eluate was monitored with an on-line
radioactivity flow detector (FLO ONE A-525, Packard Instruments,
Meriden, CT). Quantitative analysis was obtained by calculating the
amount of radioactivity under the peaks and using the same protein
concentrations and specific activities of the different
[3H]-inositol phosphates. This assay was
initially tested by using several PH domains, and other putative
lipid-binding domains, derived from different proteins, and gave very
reproducible results, consistent with those obtained with other binding
assays. In addition, with several PH domains, such as those from
pleckstrin and dynamin, we did not find any binding at all (as for GST
alone), indicating that only specific binding was detected.
Subcellular Localization Studies
cDNA encoding the different PH domains was subcloned (in frame
with GFP) into the GFP fusion protein expression vector pEGFP-C1
(CLONTECH Laboratories, Inc., Palo Alto, CA), using the
BglII and EcoRI sites for expression of an
EGFP-PH domain fusion protein in mammalian cells. NIH-IR cells, kindly
provided by Professor E. Skolnik (New York University, New York, NY)
and L6 cells were maintained in DMEM supplemented with 10% CS and FCS,
respectively. Cells were seeded onto 12-mm circular glass coverslips in
wells of a six-well plate and transfected with 1 µg of EGFP
fusion protein. LipofectAMINE (Life Technologies, Inc.)
was used for the transfections according to the manufacturers
suggestions. Twenty-four hours after transfection, the NIH-IR and L6
cells were serum-starved overnight in 0.5% CS and 0% FCS,
respectively. After stimulation with growth factors, cells were washed
in PBS and fixed in 4% paraformaldehyde/PBS and mounted for
fluorescence microscopy. Microscopy was performed using a LSM 510 laser
confocal microscope system connected to an Axiovert 100M (Carl Zeiss, Oberkochen, Germany) and using a 63x objective.
Statistical analysis of the plasma membrane localization (no score,
025% membrane localization; score 1, >25% membrane localization)
was performed by blind scoring of at least 100 cells from three
separate experiments, each performed in duplicate.
Microinjection
3T3 L1 adipocytes, differentiated as described previously (24),
were plated at a density of 3.5 x 104 onto
13-mm glass coverslips. The following day, the cDNA of PH-IRS1 GFP in
PBS was microinjected in cells in HEPES-buffered culture medium, using
an Axiovert IM 35 microscope (Carl Zeiss), a
micromanipulator 5171 (Eppendorf, Hamburg, Germany), and
microinjector 5246. At 4 h after microinjection, the cells were
serum starved in 0.5% for 12 h in DMEM. Cells were then
stimulated, as described above.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Dr. G. Rodrigues and Dr. C. P. Berrie
for constructive comments on the manuscript, Dr. M. A. Lemmon for
many valuable discussions, and A. Marchegiani and C. Iurisci for
excellent technical assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: M. Falasca, Unit of Physiopathology of Cell Signaling, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (CH) Italy.
This work was supported by Telethon Italy (Grant 328/bi to M.F. and
Grant E.606 to D.L.E.) and in part by the Italian National Research
Council (Convenzione C.N.R., Consorzio Mario Negri Sud).
Received for publication November 15, 1999.
Revision received March 15, 2000.
Accepted for publication March 22, 2000.
 |
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