Tài liệu Báo cáo khoa học: Compartmentalization and in vivo insulin-induced translocation of the insulin-signaling inhibitor Grb14 in rat liver pptx

Compartmentalization and in vivo insulin-induced
translocation of the insulin-signaling inhibitor Grb14
in rat liver
Bernard Desbuquois
1,2
,Ve
´
ronique Be
´
re
´
ziat
3
, Franc¸ois Authier
4
, Jean Girard
1,2
and Anne-Franc¸oise
Burnol
1,2
1 Institut Cochin, Universite
´
Paris Descartes, CNRS (UMR 8104), France
2 Inserm, U567, Paris, France
3 Centre de Recherche Saint-Antoine, UMR S893, Faculte
´
de Me
´
decine Pierre et Marie Curie, Paris, France
4 Inserm, U756, Faculte
´

de Pharmacie Paris 11, Cha
ˆ
tenay-Malabry, France
Grb14 is a member of the Grb7 ⁄ Grb10 ⁄ Grb14 family
of adaptor proteins, which lack intrinsic enzymatic
activity and share a common multidomain structure.
These adaptors bind to several receptor tyrosine kinases
and signaling proteins, and are involved in the regu-
lation of various processes, including cell growth and
Keywords
endocytosis; insulin receptor; liver;
molecular adaptor; tyrosine kinase activity
Correspondence
B. Desbuquois, De
´
partement
d’Endocrinologie, Me
´
tabolisme, Cancer,
Institut Cochin, 24 rue du Faubourg
Saint-Jacques, 75014 Paris, France
Fax: +33 1 44 41 24 21
Tel: +33 1 53 73 27 08
E-mail:
(Received 14 September 2007, revised 29
April 2008, accepted 2 July 2008)
doi:10.1111/j.1742-4658.2008.06583.x
The molecular adaptor Grb14 binds in vitro to the activated insulin receptor
(IR) and inhibits IR signaling. In this study, we have used rat liver subcel-
lular fractionation to analyze in vivo insulin effects on Grb14 compartmen-

talization and IR phosphorylation and activity. In control rats, Grb14 was
recovered mainly in microsomal and cytosolic fractions, but was also detect-
able at low levels in plasma membrane and Golgi ⁄ endosome fractions. Insu-
lin injection led to a rapid and dose-dependent increase in Grb14 content,
first in the plasma membrane fraction, and then in the Golgi ⁄ endosome
fraction, which paralleled the increase in IR b-subunit tyrosine phosphory-
lation. Upon sustained in vivo IR tyrosine phosphorylation induced by
high-affinity insulin analogs, in vitro IR dephosphorylation by endogenous
phosphatases, and in vivo phosphorylation of the IR induced by injection of
bisperoxo(1,10 phenanthroline)oxovanadate, a phosphotyrosine phospha-
tase inhibitor, we observed a striking correlation between IR phosphoryla-
tion state and Grb14 content in both the plasma membrane and
Golgi ⁄ endosome fractions. In addition, coimmunoprecipitation experiments
provided evidence that Grb14 was associated with phosphorylated IR
b-subunit in these fractions. Altogether, these data support a model whereby
insulin stimulates the recruitment of endogenous Grb14 to the activated IR
at the plasma membrane, and induces internalization of the Grb14–IR com-
plex in endosomes. Removal of Grb14 from fractions of insulin-treated rats
by KCl treatment led to an increase of in vivo insulin-stimulated IR tyrosine
kinase activity, indicating that endogenous Grb14 exerts a negative feedback
control on IR catalytic activity. This study thus demonstrates that Grb14 is
a physiological regulator of liver insulin signaling.
Abbreviations
BPS, between plekstrin homology and SH2; bpV(phen), bisperoxo(1,10-phenanthroline) oxovanadate; EGF, epidermal growth factor; ER,
endoplasmic reticulum; GST, glutathione S-transferase; IR, insulin receptor; IRS-1, insulin receptor substrate-1; IRb, insulin receptor
b-subunit; PDK-1, 3-phosphoinositide-dependent kinase-1; PI3-kinase, phosphoinositide-3-kinase; PIR, phosphorylated insulin receptor-
interacting region; WGA, wheat germ agglutinin.
FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS 4363
metabolism, apoptosis, and cell migration [1–5].
Grb14, which is selectively expressed in insulin target

tissues, interacts with the phosphorylated insulin recep-
tor (IR) through a region called BPS [between plekstrin
homology (PH) and SH2] or phosphorylated insulin
receptor-interacting region (PIR) [6] and inhibits the
tyrosine kinase activity of the IR [7]. On the basis of
the crystal structure of the tyrosine kinase domain of
the IR in complex with the PIR ⁄ BPS domain of Grb14,
Depetris et al. [8] have shown that Grb14 binds as a
pseudosubstrate inhibitor to the peptide-binding groove
of the kinase and thus acts as a selective inhibitor of
insulin signaling. Consistent with this observation,
overexpression of Grb14 in CHO-IR cells impairs Akt
and ERK insulin signaling pathways and inhibits distal
effects of insulin on glycogen and DNA synthesis [6–9],
and microinjection of Grb14 into Xenopus laevis
oocytes inhibits insulin-induced oocyte maturation [10].
Conversely, disruption of the Grb14 gene in mice
ameliorates glucose tolerance in vivo and insulin signal-
ing in both liver and skeletal muscle [11]. However,
although it improves the Akt insulin signaling pathway,
depletion of Grb14 by RNA interference in mouse
primary cultured hepatocytes inhibits the stimulatory
effect of insulin on glycogen synthesis and on glycolytic
and lipogenic gene expression, suggesting that Grb14
action on insulin signaling cannot be restricted to its
inhibitory action on IR catalytic activity [12].
In addition to the IR, three partners of Grb14
involved in insulin signaling have been identified: (a)
protein kinase Cf interacting protein (ZIP), an adaptor
protein that binds to the PIR domain of Grb14

and mediates the assembly of a protein kinase
Cf–ZIP–Grb14 heterotrimer [10]; (b) insulin receptor
substrate-1 (IRS-1), which binds through its phosp-
hotyrosine-binding domain to an NPXY motif of
Grb14 in a phosphorylation-independent manner [13];
and (c) 3-phosphoinositide-dependent kinase-1 (PDK-
1), which binds constitutively to a PDK-1 consensus
binding motif of Grb14 [14].
Although lacking a hydrophobic transmembrane
domain, like many signaling proteins Grb14 is found
in cells in both soluble and membrane-associated
states. In DU 145 human prostate cancer cells, endoge-
nous Grb14 is predominantly associated with a low-
density microsomal fraction, where it colocalizes with
tankyrase 2 [15]. In indirect immunofluorescence stud-
ies, Grb14 is detected as a diffuse but also punctate
cytoplasmic staining that is more concentrated around
the nucleus, suggesting its association with the Golgi; a
pool of Grb14 also localizes at the plasma membrane
[15]. In HEK 293 cells, epitope-tagged Grb14 is mainly
expressed in the cytosol in the resting state, but
redistributes in part to the membrane fraction upon
insulin stimulation [14]. In rat retina, endogenous
Grb14 shows a perinuclear and nuclear localization,
consistent with the identification of a functional
nuclear localization signal in the Grb14 N-terminus
[16]. However, unlike with other insulin signaling
proteins, Grb14 compartmentalization has not been
characterized in physiological insulin target cells.
In the present study, subcellular fractionation and

western blotting procedures have been used to assess
the compartmentalization of Grb14 in rat liver, an
organ that expresses both the IR and Grb14 at a high
level, and where insulin-induced phosphorylation, acti-
vation and internalization of the IR have been previ-
ously documented [17–19]. Our results show that, in
the basal state, Grb14 is localized mainly in high-
density microsomal elements and cytosol. Upon insulin
stimulation, Grb14 is rapidly and dose-dependently
translocated first to plasma membranes and then to
endosomes, in which it associates with phosphorylated
IR. These results suggest that Grb14 is recruited by
the activated IR at the plasma membrane and then
undergoes internalization as a complex with the IR. In
addition, our results provide the first evidence for an
in vivo inhibitory effect of endogenously recruited
Grb14 on IR catalytic activity in both compartments.
Results
Subcellular distribution of Grb14 and the IR in
liver from control and insulin-injected rats
The subcellular distribution of liver immunoreactive
Grb14 was examined using preparative and analytical
fractionation and compared to that of the IR. Upon
differential centrifugation (Fig. 1A), Grb14 was detect-
able as a major protein of 60 kDa in both particulate
and soluble fractions. A minor component of slightly
reduced electrophoretic mobility, which may represent
a phosphorylated form of Grb14 [10], was also
observed. Under basal conditions, Grb14 content was
three-fold to four-fold higher in the light mitochon-

drial–microsomal and cytosolic fractions than in the
nuclear and mitochondrial fractions (P < 0.001).
Analysis of separate light mitochondrial and micro-
somal fractions showed that Grb14 content was about
10-fold higher in the microsomal fraction than in the
light mitochondrial fraction (results not shown). In
recovery studies, the microsomal and cytosolic frac-
tions accounted respectively for about 40% and 49%
of total Grb14 contained in the whole homogenate.
Grb14 was present at relatively low levels in the
plasma membrane and Golgi ⁄ endosome fractions, with
Insulin-induced translocation of Grb14 in rat liver B. Desbuquois et al.
4364 FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS
recoveries of about 1% and 0.2%, respectively. The
subcellular distribution of Grb14 clearly differed from
that of the IR, which was detected only in particulate
fractions with a marked enrichment in the plasma
membrane and Golgi ⁄ endosome fractions, and, to a
lesser extent, in the light mitochondrial–microsomal
fraction, in agreement with a previous report [17].
Two minutes after insulin injection, consistent with
the insulin-induced internalization and phosphoryla-
tion of the IR [17–19], IR content decreased in the
plasma membrane fraction and reciprocally increased
in the light mitochondrial–microsomal and Golgi ⁄
endosome fractions, and IR b-subunit (IRb) phos-
phorylation was detected in each of these fractions.
Concomitantly, a significant increase in Grb14 content
was observed in the plasma membrane and Golgi ⁄
endosome fractions (twofold and 10-fold increase

respectively, P < 0.001) and to a lesser extent in
the light mitochondrial–microsomal fraction (30%
increase, P < 0.05). As the Grb14 content in crude
HN
AB
M LP S
PM
GE
– ins
+ ins
Grb14
+ ins
– ins
+ ins
Grb14
50
150
200
250
100
*
***
***
– ins
+ ins
0
600
800
***
0

200
400
HN
M LP S PM GE
Grb14
–ins
+ ins
–ins
+ ins
number:
Na
+
K
+
ATPase
EEA1
Calnexin
1234567891011121314
40
60
80
100
120
Grb14
control
i2i
***
*
*
100

120
0
20
1 2 3 4 5 6 7 8 9 10 11 12 13 14
ins 2 min
ins 5 min
Fraction number
*
0
20
40
60
80
1234 6 891011121314
*
***
*
*
***
***
1234567891011121314
Fig. 1. Comparative expression of Grb14 and IR proteins in liver subcellular fractions of control and insulin-injected rats. (A) Liver homogen-
ates (H) prepared from five control ()ins) and five insulin-injected (+ins, 2 min postinjection) rats were fractionated into nuclear (N), mito-
chondrial (M), light mitochondrial–microsomal (LP), cytosolic (S), plasma membrane (PM) and Golgi ⁄ endosome (GE) fractions as described in
Experimental procedures. Aliquots (10 lg of protein) were analyzed by western blotting using antibodies against Grb14, IRb and phosphoty-
rosine to detect phosphorylated IRb (p-IRb) as indicated. Upper panel: representative immunoblots. No signal was detected on an anti-phosp-
hotyrosine immunoblot performed in the absence of insulin. Lower panel: quantitation of Grb14 and IRb signals by scanning densitometry,
with results expressed as percentage of signal intensity in the homogenate (mean ± SEM of five determinations on fractions originating
from separate livers). (B) Liver microsomal fractions prepared from three control and six insulin-treated rats (2 and 5 min postinjection, three
rats per time point) were subjected to centrifugation through a continuous sucrose density gradient. Fourteen subfractions with densities

increasing linearly from 1.065 (fraction 1) to 1.195 gÆmL
)1
(fraction 14) were collected, and aliquots (10 lg of protein) were analyzed by wes-
tern blotting using antibodies directed against Grb14, IRb, EEA1 (endosomal marker), Na
+
⁄ K
+
-ATPase (plasma membrane marker) and caln-
exin (ER marker) as indicated. Top: representative immunoblots in control ()ins) and insulin-injected (+ins, 5 min postinjection) rats. Bottom:
quantitation of Grb14 and IRb signals, with results expressed as percentage of maximum (mean ± SEM of three determinations on micro-
somal fractions originating from separate livers). Significant differences between control and insulin-treated liver fractions [2 min in (A) and
5 min in (B)] using the LSD test are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001.
B. Desbuquois et al. Insulin-induced translocation of Grb14 in rat liver
FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS 4365
homogenates was unchanged, these results suggest a
translocation of cytosolic Grb14 to IR-enriched com-
partments. However, the increased recovery of Grb14
in IR-enriched fractions did not exceed 5% of the total
Grb14 liver pool, explaining why no decrease in cyto-
solic Grb14 could be detected.
To further characterize the distribution of membrane-
associated Grb14 and IR under basal and insulin-
stimulated conditions, the microsomal fraction, which
contained the majority of both proteins, was subjected
to analytical density gradient centrifugation. The distri-
bution of Grb14 and IRb was analyzed and compared
to that of three organelle markers: EEA1 (endosomes);
Na
+
⁄ K

+
-ATPase (plasma membrane); and calnexin
[endoplasmic reticulum (ER)] (Fig. 1B). In control rats,
Grb14 was expressed predominantly in the high-density
region of the gradient (subfractions 8–14; density range,
1.13–1.20 gÆmL
)1
), as was calnexin. In contrast, IRb
was expressed mainly at intermediate densities (subfrac-
tions 6–10; density range, 1.11–1.16 gÆmL
)1
), similarly
to Na
+
⁄ K
+
-ATPase, and to a lesser extent at
low densities (subfractions 2–5; density range, 1.07–
1.11 gÆmL
)1
), coinciding with EEA1. Insulin treatment
caused a shift in the distribution of both Grb14 and
IRb towards lower densities, which was more
pronounced at 5 min than at 2 min. The shift in Grb14
distribution resulted from an increased Grb14 content
at low and intermediate densities (subfractions 3–8;
density range, 1.08–1.14 gÆmL
)1
), whereas the shift in
IRb distribution involved both a decreased content at

intermediate and high densities (subfractions 8–12;
density range, 1.13–1.18 gÆmL
)1
) and an increased con-
tent at low densities (subfractions 2–5; density range,
1.07–1.11 gÆmL
)1
). Insulin treatment also resulted in an
increased content of phosphorylated IRb, the distribu-
tion of which was superimposable on that of IRb (data
not shown). Taken together, these results extend those
obtained with preparative procedures and confirm that,
upon insulin-induced IR internalization and activation,
Grb14 associates in part with plasma membranes and
endosomes.
Time course and dose-dependence of the insulin
effect on Grb14 content in plasma membrane
and endosomal liver fractions
In time-course studies (Fig. 2A), Grb14 content in the
plasma membrane fraction reached a maximum (three
PM
A B
GE
00 .5 2 5 15 30 60
Grb14
00 .5 2 5 15 30 60 ins (min)
0
2
4
** *

** *
*
0
4
8
** *
** *
** *
**
p-
0
10
20
** *
** *
**
**
**
15
0
30
*
** *
** *
** *
** *
0.5
1
** *
** *

** *
**
4
2
*
** *
** *
** *
** *
** *
0
0 0.5 2 5 15 30 60
0
00 .5 2 5 15 30 60
00 .3 3 3 00 0.3 3 3 0
ins ( µg) :
PM GE
Grb14 Grb14
0
5
10
15
*
*
0
1
2
3
*
p-

0
10
20
**
***
0
10
20
30
*
*
Fig. 2. Time course and dose-dependence of in vivo insulin effects on Grb14, phosphorylated IRb (p-IRb) and IRb content in liver plasma
membrane (PM) and Golgi ⁄ endosome (GE) fractions. PM and GE fractions were prepared from livers of rats studied at the indicated time
points after injection of 30 lg of insulin (four to six rats per time point) (A), or studied 2 min after injection of the indicated dose of insulin
(two rats per dose) (B). Aliquots (10 lg of protein) were analyzed by western blotting using antibodies against Grb14, phosphotyrosine and
IRb, as indicated. The figure shows representative immunoblots and quantitation of protein signals by scanning densitometry, with results
expressed as fold change relative to basal value [mean ± SEM of four to six determinations for (A) or two determinations for (B), on frac-
tions originating from separate livers]. Significant changes relative to control (no insulin) using the LSD test are indicated as follows:
*P < 0.05; **P < 0.01; ***P < 0.001.
Insulin-induced translocation of Grb14 in rat liver B. Desbuquois et al.
4366 FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS
times basal level) as early as 30 s after insulin injection,
and subsequently declined to almost basal values
within 30 min, whereas Grb14 content in the Golgi ⁄
endosome fraction was maximal at 2 min (about six
times basal level) and declined more slowly, remaining
elevated at 60 min. The insulin-induced increase in
Grb14 content in the plasma membrane and Golgi ⁄
endosome fractions was significantly correlated with the
increase in phosphorylated IRb content in the same

fractions (plasma membrane, r = 0.72, P < 0.001;
Golgi ⁄ endosome, r = 0.60, P < 0.01). When normal-
ized to IRb content, insulin-induced changes in Grb14
content were similar in the two fractions (twofold to
threefold maximal increase), as were the changes in
phosphorylated IRb content (10–20-fold maximal
increase). These findings suggest that the ability of
phosphorylated IR b to recruit Grb14 is the same in the
plasma membrane and Golgi ⁄ endosome fractions.
In dose–response studies (Fig. 2B), the insulin-
induced increase in Grb14 content was detectable for
3 lg of insulin in the plasma membrane fraction and
0.3 lg in the Golgi⁄ endosome fraction, and was maxi-
mal for 30 lg in both fractions, again in good agree-
ment with the increase in phosphorylated IRb content.
Functional relationships between membrane
association of Grb14 and IR tyrosine
phosphorylation
The parallel increase in Grb14 content and phosphory-
lated IRb content in the plasma membrane and
Golgi ⁄ endosome fractions of insulin-treated rats sug-
gested that IRb phosphorylation state was involved in
the association of Grb14 with these compartments. To
gain further insight into the functional relationship
between these two events, we used three complemen-
tary approaches. First, we assessed the response of
endosomal Grb14 to [GluA13,GluB10]insulin and
[HisA8,HisB4,GluB10,HisB27]insulin, two high-affinity
insulin analogs that were previously reported to induce
prolonged tyrosine phosphorylation of the endosomal

IR [20], administered in vivo. As shown in Fig. 3, these
analogs also induced a more sustained association of
Grb14 with the Golgi⁄ endosome fraction, which paral-
leled their effects on phosphorylated IRb content.
Second, we examined the ability of Grb14 associated
with the Golgi ⁄ endosome fraction to dissociate upon
incubation at 37 °C, conditions under which the phos-
phorylated IR has been shown to be rapidly dephos-
phorylated by endogenous phosphatases [21]. As
shown in Fig. 4, incubation of Golgi ⁄ endosome frac-
tions from insulin-treated rats indeed resulted in a
progressive, time-dependent dephosphorylation of
phosphorylated IRb. Concurrently, Grb14 content
decreased in sedimented membranes, while remaining
unchanged in total incubation mixtures (data not
shown), indicating a dissociation of membrane-bound
Grb14. Membrane-bound Grb14 and phosphorylated
IRb were significantly correlated (r = 0.56; P < 0.05).
Similar results were obtained using the plasma mem-
brane fraction (data not shown). Importantly, both
processes were almost fully inhibited by bisper-
oxo(1,10-phenanthroline) oxovanadate [bpV(phen)], a
potent inhibitor of endosomal phosphotyrosine phos-
phatases.
Finally, we examined the response of endosomal
Grb14 to bpV(phen) administered in vivo; this was pre-
viously shown to increase IRb phosphorylation [22]
and to prevent the dephosphorylation of activated IRb
that occurs shortly after insulin injection [23]. Our
results confirm these observations, and further show

that the changes in phosphorylated IR b content
induced by bpV(phen) in the Golgi⁄ endosome fraction
are accompanied by somewhat parallel changes in
Grb14 content (Fig. 5). When injected alone,
bpV(phen) led, at 15 and 45 min postinjection, to a
12–16-fold increase in phosphorylated IRb content and
to a nearly two-fold increase in Grb14 content in
the Golgi ⁄ endosome fraction. When injected 15 min
prior to an inframaximal dose of insulin, bpV(phen)
A3
Grb14
Grb14
H2
WT
Grb14
0 2 5
15 30 60
min :
Fig. 3. Time course of in vivo effects of human insulin analogs on
Grb14 and phosphorylated IRb (p-IRb) content in liver Golgi ⁄ endo-
some fractions. Golgi ⁄ endosome fractions were isolated from livers
of rats studied at the indicated time points (one rat per time point)
after injection of 30 lg of [GluA13,GluB10]insulin (A3),
[HisA8,HisB4,GluB10,HisB27]insulin (H2) or wild-type human insulin
(WT). Aliquots (30 lg protein) were analyzed by western blotting
using antibodies against Grb14 and phosphotyrosine.
B. Desbuquois et al. Insulin-induced translocation of Grb14 in rat liver
FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS 4367
prevented the decrease in phosphorylated IRb and
Grb14 content that occurred between 2 and 15 min

after insulin injection. A significant correlation was
observed between phosphorylated IRb and Grb14
(r = 0.81, P < 0.01). Comparable effects of
bpV(phen) treatment on IRb phosphorylation and
Grb14 content, albeit less marked, were also observed
in the plasma membrane fraction of untreated and
insulin-treated rats (data not shown).
Taken together, these data strongly suggest that the
phosphorylation status of IRb is implicated in the
in vivo association of Grb14 with IR-containing subcel-
lular compartments.
Nature of the association of Grb14 with liver
membrane fractions from control and
insulin-treated rats
As expected for a peripheral protein lacking a trans-
membrane domain, Grb14 associated with the micro-
somal, plasma membrane and Golgi ⁄ endosome
fractions was partially extracted by treatment with
KCl at concentrations above 1 m. On the basis of the
comparative quantitation of sedimentable Grb14 in
untreated and KCl-treated fractions, the proportion of
Grb14 removed by 2 m KCl was about 60–75% in
–
+
bpV
Grb14
0 5 10 20 30 40
min
+
–

+
100
50
0
Fig. 4. In vitro dissociation of membrane-bound Grb14 upon
dephosphorylation of in vivo activated IRs in liver Golgi ⁄ endosome
fractions. Golgi ⁄ endosome fractions from five insulin-injected rats
(2 min postinjection) were incubated at 37 °C for the indicated
times in the absence or presence of bpV(phen) (0.2 m
M), and then
subjected to high-speed centrifugation as described in Experimental
procedures. Total incubation mixtures and resuspended pellets
were analyzed by western blotting using antibodies against phosp-
hotyrosine and Grb14 as indicated. Incubation did not affect the
intensity of Grb14 signals in total incubation mixtures (data not
shown). Top: blots representative of five experiments carried out
on Golgi ⁄ endosome fractions originating from separate livers. Bot-
tom: quantitation of Grb14 (gray bars) and phosphorylated IRb
(p-IRb) signals (white bars) in the absence of bpV(phen), with results
expressed as percentage of the 0 time value (mean ± SEM of five
determinations). Both membrane-bound Grb14 and p-IR show a sig-
nificant, time-dependent decrease according to ANOVA (P < 0.0001).
Grb14
05 15 45
bpV (min)
A

B
1
2

*
*
1 6.2 12.5 16.5
p-
0
bpV (min) 5 0 15 45
5 2 0 15
Grb14
–
bp V
6
4
2
***
***
–
+
0
5
10
***
**
+
0
2
–
+
0 2 5 15 ins
(
min

)

ins (min)
0
2
4
3
1
Fig. 5. In vivo effects of bpV(phen), alone or in association with
insulin, on Grb14, phosphorylated IRb (p-IRb) and IRb content in
liver Golgi ⁄ endosome fractions. Golgi ⁄ endosome fractions were
prepared from livers of rats studied at the indicated time points
after injection of 1.2 lmol of bpV(phen) (A) (three to four rats per
time point) or 3 lg of insulin (B). In (B), rats were pretreated or not
with 1.2 lmol of bpV(phen) 15 min prior to insulin injection (two to
three rats per time point and per condition). Fractions (10 lg of pro-
tein) were analyzed by western blotting using antibodies against
Grb14, phosphotyrosine and IRb. The blots (left part) are represen-
tative of experiments carried out on Golgi ⁄ endosome fractions orig-
inating from separate livers, and densitometric measurements of
Grb14, p-IRb and IRb signals are shown on the right. In (A), results
are expressed as fold increase in Grb14 (white bars) and p-IRb
(numbers under the blot) above the 0 bpV(phen) control (mean ±
SEM of the three or four determinations). In (B), results are
expressed as fold increase above the 0 insulin control, with (gray
bars) or without (white bars) bpV(phen) pretreatment (mean ± SEM
of the two or three determinations). A significant effect of
bpV(phen) treatment using the LSD test is indicated as follows:
*P < 0.05; **P < 0.01; ***P < 0.001.
Insulin-induced translocation of Grb14 in rat liver B. Desbuquois et al.

4368 FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS
fractions of control rats and 40–60% in fractions iso-
lated shortly after (30 s to 5 min) insulin injection
(data not shown). Under these conditions, at least
90% of the IR remained membrane-associated as
assessed by both western blotting and insulin binding.
NaCl, at 1 m, was less effective than KCl at the same
concentration, and so was urea at 4 m. On the other
hand, treatment with 0.1 m Na
2
CO
3
(pH 11.5)
removed at least 60–70% of Grb14, again leaving the
IR membrane-associated (data not shown). These find-
ings indicate that Grb14 recruited under insulin stimu-
lation is tightly associated with membranes.
To determine whether Grb14 translocated to the
plasma membrane and Golgi ⁄ endosome fractions in
insulin-treated rats interacts with the phosphorylated
IR, fractions were solubilized with Triton X-100 and
soluble extracts were subjected to immunoprecipitation
using antibodies against IRb and Grb14, followed by
western blotting using antibodies against Grb14 and
phosphotyrosine, respectively. As shown in Fig. 6,
insulin treatment increased Grb14 content in IR immu-
noprecipitates, and phosphorylated IRb content in
Grb14 immunoprecipitates, in a time-dependent
manner. As in crude lysates, these changes were maxi-
mal at 30 s in the plasma membrane fraction and at

2 min in the Golgi ⁄ endosome fraction, and subse-
quently declined. These findings strongly favor a direct
interaction between Grb14 and the phosphorylated
IRb induced by in vivo insulin stimulation. Further-
more, they suggest that Grb14 recruited by the phos-
phorylated IR at the plasma membrane may undergo
cointernalization along with the IR.
Specificity of insulin-induced changes in Grb14
content in liver subcellular fractions
The IR also binds to Grb7 and Grb10, two adaptor
proteins that are structurally related to Grb14, and
besides the IR, the epidermal growth factor (EGF),
fibroblast growth factor and platelet-derived growth
factor receptors also can bind Grb14 [5]. As liver
expresses both Grb7 and the EGF receptor at high
levels, we performed a comparative analysis, in time
studies, of the in vivo effects of insulin and EGF on
the contents of Grb7 and Grb14 in the plasma mem-
brane and Golgi ⁄ endosome fractions. As shown in
PM
00 .5 25 15 30 60 ins. (min)
GE
00 .5 2 5 15 30 60
Grb14
IP
100
100
0
40
20

60
80
0
40
20
60
80
IP Grb14
100
40
60
80
100
40
60
80
0

20
0
20
Fig. 6. Time course of in vivo insulin-induced coimmunoprecipitation of Grb14 with phosphorylated IRb (p-IRb). Plasma membrane (PM) and
Golgi ⁄ endosome (GE) fractions were prepared from livers of rats killed at the indicated time points after injection of 30 lg of insulin (two
rats per time point). Following solubilization by Triton X-100, fractions were immunoprecipitated with monoclonal antibody against IRb or
polyclonal antibodies against Grb14 as indicated. Immunoprecipitates (IP) were analyzed by western blotting using antibodies against Grb14
and phosphotyrosine, and polyclonal antibodies against IRb. Immunoblots are representative of two experiments, carried out on fractions
originating from separate livers. Densitometric measurements of Grb14 (white bars) and p-IRb (gray bars) signals are expressed as percent-
age of maximum (mean ± SEM of these duplicate determinations).
B. Desbuquois et al. Insulin-induced translocation of Grb14 in rat liver
FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS 4369

Fig. 7A, insulin induced an increase in Grb7 content
that paralleled the increase in Grb14 content, with a
maximal effect at 30 s in the plasma membrane frac-
tion and at 2 min in the Golgi ⁄ endosome fraction. The
response of Grb7 to insulin was similar to that of
Grb14 in the plasma membrane fraction but somewhat
greater in the Golgi ⁄ endosome fraction. EGF did not
significantly affect Grb14 content in the plasma mem-
brane and Golgi ⁄ endosome fractions, except for for a
three-fold increase at 15 min (P < 0.001) in the
Golgi ⁄ endosome fraction. It did, however, significantly
increase Grb7 content in these two fractions, with a
maximal effect at 0.5 min in the plasma membrane
fraction (3.5-fold increase, P < 0.001) and at 5–
15 min in the Golgi ⁄ endosome fraction (sixfold
increase, P < 0.001) (Fig. 7B). These changes were
temporally correlated with the increase in phosphory-
lated EGF receptor content in both the plasma mem-
brane and Golgi ⁄ endosome fractions and in EGF
receptor content in the Golgi ⁄ endosome fraction
(Fig. 7C).
Effect of insulin-induced association of Grb14 on
IR tyrosine kinase activity
We have previously shown that glutathione S-transfer-
ase (GST)–Grb14 inhibits the in vitro tyrosine kinase
activity of the recombinant IR as measured using
poly(Glu,Tyr) as substrate [7]. Consistent with this
observation, GST–Grb14 also inhibited the ability of
the endogenous liver IR partially purified from a
microsomal fraction to phosphorylate RCAM-lyso-

zyme, a high-affinity substrate of the receptor (Fig. 8).
The inhibitory effect of GST–Grb14 on insulin-stimu-
lated RCAM-lysozyme phosphorylation by the IR was
dose-dependent, being detectable at 0.15 lgÆmL
)1
(1.5 nm) and almost complete at 5 lgÆmL
)1
(50 nm).
Inhibition by exogenous Grb14 of the IR activated
in vitro suggested that removal of endogenous Grb14
from the IR activated in vivo would elicit an opposite
effect. To address this question, we first assessed the
ability of insulin administered in vivo to increase the
level of RCAM-lysozyme in cell fractions, and then
assayed the fractions of insulin-injected rats for Grb14,
IR and phosphorylated IR content and RCAM-lyso-
zyme phosphorylation following treatment or no treat-
ment with 2 m KCl. As shown in Fig. 9A, intact
Golgi ⁄ endosome fractions isolated 2 min after insulin
injection displayed a four-fold increase (4.03 ± 0.32,
mean ± SEM, n = 4) in the extent of RCAM-lyso-
zyme phosphorylation relative to control fractions. KCl
treatment of the Golgi ⁄ endosome fractions from insu-
lin-injected rats affected neither the content nor the
tyrosine phosphorylation state of the IR, but led to a
60% decrease in Grb14 content (Fig. 9B). Removal of
Grb14 from these fractions led to a nearly two-fold con-
comitant increase in the extent of RCAM-lysozyme
phosphorylation (Fig. 9C). KCl treatment of the
plasma membrane and microsomal fractions isolated 2

and 5 min after insulin injection induced a similar
increase in the extent of RCAM-lysozyme phosphoryla-
GE
0 0.5 2 5 15 30 60
PM
A
B
C
0 0.5 2 5 15 30 60ins (min):
Grb14
Grb7
6
4
2
8
3
2
0
1
0
0 0.5 15 305
GE
0 0.5 15 305
PM
Grb14
EGF (min) :
Grb7
3
4
4

6
0 0.5 155EGF (min) :
PM
00.5 155
GE
0
2
1
0
2
()
p-EGFR
EGFR
Fig. 7. Comparative in vivo effects of insulin and EGF on Grb7 and
Grb14 content in liver subcellular fractions. Plasma membrane (PM)
and Golgi ⁄ endosome (GE) fractions were isolated from livers of
rats studied at the indicated time points after injection of 30 lgof
insulin (A) (four rats per time point) or 100 lg of EGF (B, C) (three
rats per time point). Aliquots (10 lg of protein) were analyzed by
western blotting using antibodies against Grb14 and Grb7 as indi-
cated (A, B). Following EGF treatment, aliquots were also immuno-
blotted with antibodies against phosphotyrosine and EGF receptor
(EGFR) (C). The blots are representative of experiments carried out
on fractions originating from separate livers. Densitometric
measurements of Grb14 (white bars) and Grb7 (gray bars) signals
are expressed as fold increase above control (mean ±
SEM of four
determinations for insulin and three determinations for EGF). See
text for statistical analysis of insulin and EGF effects on Grb7 and
Grb14 content in the PM and GE fractions. p-EGFR, phosphorylated

EGFR.
Insulin-induced translocation of Grb14 in rat liver B. Desbuquois et al.
4370 FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS
tion by subsequently prepared wheat germ agglutinin
(WGA)-purified insulin receptors (data not shown).
Altogether, these results suggest that Grb14 endoge-
nously recruited by the phosphorylated IR after in vivo
insulin stimulation exerts an inhibitory action on IR
catalytic activity.
Discussion
Insulin signaling proteins in adipocytes [24–27] and
liver [28,29] have been shown to be compartmentalized
and to undergo activation and ⁄ or redistribution to
specific subcellular compartments in response to insu-
lin. In liver, plasma membranes and endosomes are
major sites to which IRS-1, phosphoinositide-3-kinase
(PI3-kinase) and Akt1 redistribute upon in vivo insulin
stimulation, and where IRS-1 and PI3-kinase interact
with phosphorylated IRs [28,29]. Our results extend
these observations to the molecular adaptor Grb14,
thus reinforcing its role as an insulin signaling protein.
Specifically, we show that following insulin treat-
ment, endogenous Grb14 undergoes a time-dependent
and reversible translocation to plasma membranes and
endosomes, in which it is recruited by phosphorylated
IRb. Furthermore, we present evidence that Grb14
exerts a physiological negative feedback control on IR
catalytic activity in these compartments.
Under basal conditions, liver Grb14 was recovered
mainly in the cytosolic and microsomal fractions, and

about 80% of microsomal Grb14 was recovered in
high-density subfractions, as was calnexin, a marker of
the ER. Although final evidence that a pool of Grb14 is
localized in the ER awaits morphological confirmation,
this localization is not unprecedented. Many peripheral
membrane proteins, including signaling proteins such
as Shc [30], mammalian target of rapamycin [31] and
tyrosine phosphatase PTP-1B [32], have been shown to
be localized on the cytosolic surface of the ER mem-
brane [33]. Localization of Grb14 to the ER may
involve the interaction of its PH domain with mem-
brane phosphoinositides, possibly phosphatidylinositol
4,5-bisphosphate, which was ultrastructurally identified
in intracellular membranes [34], and shown to bind to
Grb14 in an insulin-independent manner [16].
The changes in the subcellular distribution of Grb14
induced by insulin are consistent with a model
whereby cytosolic Grb14 is recruited to the phosphory-
lated IR at the plasma membrane and is then translo-
cated to endosomes as a protein complex with the
receptor. First, as expected from insulin-induced inter-
nalization of the IR, insulin led to an increase in
Grb14 level earlier in plasma membrane fractions than
in endosomal fractions. Second, in kinetics and dose–
response studies with insulin and superactive insulin
analogs, a striking correlation between the IR phos-
phorylation state and the content of Grb14 in the
plasma membrane and ⁄ or endosomal fractions was
observed. Third, a similar correlation was found upon
in vitro dephosphorylation of the activated IR by

endogenous phosphatases and, reciprocally, in vivo
phosphorylation of the IR by bpV(phen), an inhibitor
of tyrosine phosphatases. Finally, coimmunoprecipita-
tion experiments showed that, following insulin treat-
ment, Grb14 was associated with the phosphorylated
IR in both the plasma membrane and endosomal frac-
tions. On the other hand, although at least part of the
Grb14–IR complex present in endosomes may derive
from internalization of a complex formed at the
plasma membrane, direct recruitment of cytosolic
Grb14 to activated IR internalized in the endosomes
– ins.
p-RCAM-L
A

B
0 10 20 40
10 20 40
+
+ ins.
–
GST-Grb14 :
Time (min) :
p-RCAM-L
0 0.15 0.5 1.5 5 15 15
µg GST-Grb14
GS T
p-RCAM-L
100
0

20
40
60
80
Fig. 8. In vitro effects of GST–Grb14 on the tyrosine kinase activity
of liver IRs. IRs were partially purified from a liver microsomal frac-
tion by adsorption on WGA–Sepharose, and examined for their abil-
ity to phosphorylate RCAM-lysozyme as described in Experimental
procedures. (A) Time course of RCAM-lysozyme phosphorylation in
the presence (+ins) or absence ()ins) of 0.5 l
M insulin, and in the
presence (+) or absence ())of5lgÆmL
)1
GST–Grb14. (B) Dose-
dependent effect of GST–Grb14 on insulin-stimulated RCAM-lyso-
zyme phosphorylation (20 min incubation of the receptor with
RCAM-lysozyme). The blots in (A) and (B) are representative of two
experiments on separate microsomal fractions, and the densitomet-
ric measurements in (B) are expressed as percentage of the value
in the absence of GST–Grb14 (mean ± SEM of two determina-
tions). p-RCAM-L, phosphorylated RCAM-lysozyme.
B. Desbuquois et al. Insulin-induced translocation of Grb14 in rat liver
FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS 4371
cannot be excluded. It is noteworthy that the recovery
of phosphorylated IRb was about two to four times
lower in Grb14 immunoprecipitates than in IR immu-
noprecipitates (data not shown). As Grb14 is less effec-
tively immunoprecipitated by antibodies against Grb14
than is the IR by antibodies to IR, these data would

be compatible with a high proportion of phosphory-
lated receptors having Grb14 bound to them.
Structural studies of the kinase domain of the IR in
complex with the PIR–BPS of Grb14 and of the
Grb14 SH2 domain have allowed us to propose a
model for the Grb14–IR interaction. The PIR–BPS
binds as a pseudosubstrate inhibitor in the substrate-
binding groove of the kinase, whereas the SH2 domain
interacts with phosphorylated tyrosine residues of the
IR kinase loop, which help position the PIR–BPS and
increase binding affinity [8]. Although the interaction
of Grb14 with the IR is probably the major determi-
nant of the insulin-induced membrane translocation of
Grb14, several lines of evidence suggest that the associ-
ation of Grb14 via its PH domain with locally pro-
duced phosphatidylinositol 3,4,5-trisphosphate may
also contribute to this process. First, insulin stimulates
the association of the regulatory p85 subunit of PI3-
kinase with both plasma membranes and endosomes
[28]. Second, full-length Grb14, as well as its PH
domain, bind D3 phosphoinositides in vitro in a pro-
tein–lipid overlay assay, and in retina lysates Grb14 is
coimmunoprecipitated by antibodies to phosphatidyl-
inositol 3,4,5-trisphosphate in an insulin-dependent
manner [16]. Finally, insulin-induced membrane trans-
location of epitope-tagged Grb14 in HEK 293 cells is
inhibited by wortmanin, a PI3-kinase inhibitor [14].
Previous studies have shown that the adaptor pro-
tein Grb7 interacts with the IR in two-hybrid, GST
pull-down and coimmunoprecipitation assays [35]. On

the other hand, although the EGF receptor binds both
Grb14 and Grb7 in cloner of receptor targets (CORT)
and GST pull-down assays, neither endogenous Grb14
in DU145 cells nor epitope-tagged Grb14 in transfect-
ed HEK 293 cells is recruited by the EGF receptor
[36]. Consistent with the association of Grb7 with the
activated IR in crude liver lysates [35], insulin treat-
ment led to an increase in Grb7 content in liver plasma
membrane and endosomal fractions, the kinetics and
extent of which were comparable to those observed
with Grb14. These findings suggest that the relative
affinities of Grb7 and Grb14 for the IR are similarly
increased by insulin, and that, like Grb14, Grb7 is
recruited by the activated IR at the plasma membrane
– ins-RCAM-L
A

B
C
150
A
U
– ins
+ ins
** *
** *
+ ins
81 62 43 2
min:
p

p-RCAM-L
0
50
100
81 62 4
min:
32
** *
** *
– KCl
50
100
** *
+ KCl
0
Grb14
p-
AU
– KCl
**
p-RCAM-L
–KCl
+ KCl
p-RCAM-L
10 30 20
min:
0
1
2
3

+ KCl
*
10min: 20 30min:
Fig. 9. Effect of Grb14 recruited in vivo on
IR kinase activity in the Golgi ⁄ endosome
fraction. (A) Golgi ⁄ endosome fractions iso-
lated from three control rats and four rats
studied 2 min after insulin injection were
assayed for RCAM-lysozyme (RCAM-L)
phosphorylation over an 8–32 min incuba-
tion. (B, C) Golgi ⁄ endosome fractions from
four insulin-injected rats (2 min postinjec-
tion) were divided into two identical aliqu-
ots, one of which was treated with 2
M KCl
as described in Experimental procedures.
Untreated and KCl-treated fractions were
assayed for Grb14, IRb and phosphorylated
IRb (p-IRb) content (B) and for ability to
phosphorylate RCAM-L (C). The figure
shows representative immunoblots and
results of densitometric measurements
expressed as arbitrary units (AU) for phos-
phorylated RCAM-L (p-RCAM-L) and per-
centage of control (no KCl treatment) for
Grb14, IRb and p-IRb [mean ± SEM of three
or four determinations in (A), four to six
determinations in (B), four determinations in
(C)]. Significant effects of insulin in (A) and
KCl in (B) and (C) are indicated as follows:

*P < 0.05; **P < 0.01; ***P < 0.001.
Insulin-induced translocation of Grb14 in rat liver B. Desbuquois et al.
4372 FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS
and undergoes internalization as a complex with the
receptor. Like insulin, EGF induced an increased con-
tent of Grb7 in liver membrane fractions that was tem-
porally correlated with the increased tyrosine
phosphorylation of the EGF receptor. However, such
treatment affected Grb14 only marginally, suggesting a
preferential association of the EGF receptor with
Grb7, as previously documented [36]. Whether the
membrane translocation of Grb14 induced by EGF
involves the direct interaction of this protein with the
activated EGF receptor or the association of the
Grb14 PH domain with membrane phosphoinositides
remains to be established.
Inhibition by Grb14 of IR catalytic activity was pre-
viously found using GST–Grb14 and IR purified from
CHO-IR cells, and poly(Glu,Tyr) as a substrate [7].
The inhibitory effect of the GST–Grb14 fusion protein
was confirmed in the present study using IR prepared
from rat liver and RCAM-lysozyme as a substrate.
Importantly, partial removal of endogenous Grb14 by
KCl treatment from the plasma membrane and
endosomal fractions of insulin-treated rats led to a sub-
stantial increase in IR tyrosine kinase activity. This
observation argues for a role of endogenous Grb14,
physiologically recruited to the IR after in vivo insulin
stimulation. Although KCl treatment removed Grb7
from cell fractions to the same extent as Grb14 (data

not shown), this is unlikely to contribute significantly
to the increased kinase activity of the IR, as Grb7 is
both less potent and less efficient than Grb14 in its abil-
ity to inhibit in vitro activation of IR tyrosine kinase
[7]. At the present time, we cannot assess whether the
kinase activity of the IRs before KCl treatment repre-
sents residual activity of receptors with Grb14 bound to
them, the activity of a fraction of receptors lacking
bound Grb14, or a combination of both.
The finding that, following insulin treatment, the
majority of liver Grb14 remains associated with the
microsomal and cytosolic fractions suggests that,
besides its ability to interact with the activated IR and
to inhibit IR catalytic activity, Grb14 may play addi-
tional roles in the liver. As a molecular adaptor,
Grb14 interacts constitutively in cells with several part-
ners, including ZIP [10], PDK-1 [14], IRS-1 [13], and
tankyrase 2 [15]. The binding of ZIP to Grb14, by
recruiting protein kinase Cf, enhances the serine phos-
phorylation of Grb14 and its inhibitory effect on IR
kinase and insulin signaling [10]. The interaction of
Grb14 with PDK-1, an upstream kinase that activates
Akt in response to insulin, appears to be required for
insulin-induced membrane translocation of PDK-1 and
Akt activation in transfected HEK cells [14]. On the
other hand, the functional significance of the inter-
action of Grb14 with tankyrase 2, a poly(ADP-
ribose)polymerase closely related to tankyrase, is still
unclear [15]. Conceivably, it could explain the colocal-
ization of these two proteins in a low-density micro-

somal fraction in DU 145 prostate cancer cells and in
transfected HEK 293 cells. Future identification of
other Grb14 partners should lead to interesting new
information on the role of this adaptor protein.
The endosomal colocalization and ⁄ or cointernaliza-
tion of the activated IR and Grb14 raises the question
as to whether Grb14 could, directly or via its partners,
regulate IR traffic and ⁄ or degradation of the IR. How-
ever, despite a recent report showing the involvement
of Grb10 in the ubiquitination and proteasomal degra-
dation of the IR in transfected cells [37], there is so far
no evidence to support a physiological role of Grb10
and Grb14 in the regulation of IR degradation [5].
Additional studies are thus needed to determine
whether Grb14 is involved in insulin-induced endocy-
tosis and ⁄ or degradation of the IR.
In summary, our results show that, whereas Grb14
is located in the basal state mainly in high-density
microsomal elements and the cytosol, upon insulin
stimulation endogenous Grb14 is in part translocated
to plasma membranes and endosomes, where it is asso-
ciated with phosphorylated IRb. Our data also suggest
that Grb14 recruited by the IR at the plasma mem-
brane undergoes cointernalization with the receptor.
Finally, evidence is presented that Grb14 exerts nega-
tive feedback control on IR catalytic activity in these
compartments, indicating that it is a physiological reg-
ulator of liver insulin signaling, and that it may repre-
sent an interesting molecule with which to regulate
liver insulin action.

Experimental procedures
Materials
Porcine insulin, recombinant human insulin, [GluA13,
GluB10]insulin and [HisA8,HisB4,GluB10,HisB27]insulin
were gifts from Novo Nordisk A ⁄ S (Bagsvaerd, Denmark).
Mouse EGF (receptor grade) was from Collaborative Bio-
medical (Bedford, MA, USA). Monoclonal and polyclonal
antibodies against IRb, polyclonal antibodies against EGF
and polyclonal antibodies against Grb7 were from Santa
Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal
antibody against phosphotyrosine (clone 4G10) was from
Upstate Biotechnology Incorporated (Lake Placid, NY,
USA). Monoclonal antibody against EEA1 (clone 14) was
from BD Transduction Laboratories (San Jose, CA, USA).
Monoclonal antibody against Na
+
⁄ K
+
-ATPase a-subunit
(clone M7-PB-E9) and polyclonal antibodies against calnex-
B. Desbuquois et al. Insulin-induced translocation of Grb14 in rat liver
FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS 4373
in were from Sigma-Aldrich (St Louis, MO, USA). GST–
Grb14 and rabbit polyclonal antibodies against Grb14 have
been described previously [6]. Horseradish peroxidase-con-
jugated goat anti-(rabbit IgG) and goat anti-(mouse IgG)
were from Biorad (Hercules, CA, USA). bpV(phen) was a
gift from B. Posner (McGill University, Montreal, Quebec,
Canada). Reduced and carboxyamidomethylated lysozyme
was a gift from R. Kohanski (Mount Sinai School of Medi-

cine, New York, NY, USA). [
125
I]TyrA14 insulin, receptor
grade, was from Perkin Elmer Life Sciences (Waltham,
MA, USA). Nitrocellulose membranes were from BioRad
and Schleicher and Schu
¨
ll (Du
¨
ren, Germany). The enhanced
chemiluminescence detection kit was from Amersham-Phar-
macia (Little Chalfont, UK) and Pierce Biotechnology Inc.
(Rockford, IL, USA). WGA–Sepharose 6MB (WGA–
Sepharose), protein G–Sepharose, protein A–Sepharose and
other chemicals were purchased from Sigma-Aldrich.
Animals and injections
Animal studies were performed according to the French
Guidelines for Use and Care of Experimental Animals.
Male Sprague–Dawley rats (body weight, 190–210 g) were
obtained from Elevage Janvier (Le Genest Saint Isle,
France). Animals were housed in an animal facility with
12 h light cycles, fed ad libitum, and fasted overnight
(16–18 h) before study. After pentobarbital anesthesia, rats
received an intravenous injection (penis vein) of insulin or
insulin analogs (30 lg unless otherwise stated), EGF
(100 lg) or bpV(phen) (1.2 lmol), diluted in 0.5 mL of
NaCl ⁄ P
i
containing 0.1% BSA. At the indicated times, the
liver was rapidly excised through a median incision, and

minced in ice-cold 0.25 m sucrose.
Subcellular fractionation
Livers were homogenized in 0.25 m sucrose containing
2.5 mm NaF, 1 mm Na
3
VO
4
,1mm benzamidine and
0.5 mm phenylmethanesulfonyl fluoride (about 5 mL per
gram of tissue) using a Dounce homogenizer (12 up-and-
down strokes of the loose-fitting pestle). Following dilution
to about 70 mL and removal of fibrous and undisrupted
tissue by centrifugation at 250 g
av
for 5 min, the homoge-
nate was subjected to differential centrifugation, generating
nuclear (1500 g
av
for 10 min), mitochondrial (3200 g
av
for
10 min), light mitochondrial–microsomal (150 000 g
av
for
45 min) and supernatant fractions. In some experiments, an
additional centrifugation step was introduced after sedimen-
tation of the mitochondrial fraction, generating separate
light mitochondrial (25 000 g
av
for 10 min) and microsomal

(150 000 g
av
for 45 min) fractions. Flotation through dis-
continuous sucrose density gradients was performed to iso-
late plasma membranes from the nuclear fraction
(1.42 ⁄ 0.25 m sucrose interface) and Golgi⁄ endosomes from
the light mitochondrial–microsomal fraction (1 ⁄ 0.25 m
sucrose interface), essentially as described in [38] and [18],
respectively. Fractions were assayed for protein content
according to the method of Lowry et al. [39], using BSA as
standard. Measurement of organelle marker enzymes
showed that enrichments of the plasma membrane fraction
in 5¢-nucleotidase and alkaline phosphodiesterase (plasma
membrane markers) and of the Golgi ⁄ endosome fraction in
galactosyltransferase (Golgi marker), ATP-dependent acidi-
fication (endosomal marker) and in vivo internalized
[
125
I]insulin were as described in previous reports
[17,38,40,41]. Fractions were analyzed for Grb14, IRb and
phosphorylated IRb by western blotting. In some experi-
ments, the microsomal fraction was subfractionated by ana-
lytical centrifugation (85 000 g
av
for 15 h) through a linear
sucrose density gradient (1.03–1.25 gÆmL
)1
). Subfractions
were assayed for density by refractometry and for content
of the following organelle markers: calnexin (ER);

Na
+
⁄ K
+
-ATPase (plasma membrane); and EEA1
(endosomes).
Extraction and immunoprecipitation of
membrane-associated Grb14 and insulin receptor
Subcellular fractions prepared as described above were
resuspended in 25 mm Hepes buffer (pH 7.6) containing
1mm Na
3
VO
4
, 2.5 mm NaF, 1 mm phenylmethanesulfonyl
fluoride, 1 mm benzamidine, 1 lgÆmL
)1
pepstatin A,
2 lgÆmL
)1
leupeptin, 5 lgÆmL
)1
aprotinin, and, when indi-
cated, 1–2 m KCl, 1 m NaCl, 4 m urea or 0.1 m Na
2
CO
3
(pH 11.5). After 60–75 min at 4 °C, suspensions were cen-
trifuged at 100 000 g for 45 min. Pellets were resuspended
into Laemmli sample buffer and analyzed for Grb14 and

IRb content by western blotting.
For immunoprecipitation, cell fractions were resuspended
in Hepes buffer containing 100 mm NaCl, phosphatase and
protease inhibitors as indicated above, and 0.5–1%
Triton X-100. After 20 min at 4 °C, suspensions were
centrifuged at 150 000 g for 60 min, and supernatants were
incubated for 16 h at 4 °C with polyclonal antibodies
against Grb14 or monoclonal antibodies against IRb.
Protein A–agarose or protein G–Sepharose, respectively,
was then added, and after rotatory shaking for 2 h, beads
were sedimented and washed four times in 25 mm Hepes
buffer, 100 mm NaCl, 1 mm Na
3
VO
4
and 0.1% Triton
X-100. Immunoprecipitated Grb14 and IRb were analyzed
by western blotting.
In vitro dephosphorylation of IR phosphorylated
in vivo
Plasma membrane and Golgi ⁄ endosome fractions of insulin-
injected rats (2 min postinjection) prepared in the absence of
phosphatase inhibitors were resuspended in 25 mm Hepes
buffer (pH 7.6) containing 0.15 m NaCl, 1 mm EDTA, 1 mm
dithiothreitol, 0.5 mm phenylmethanesulfonyl fluoride,
Insulin-induced translocation of Grb14 in rat liver B. Desbuquois et al.
4374 FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS
0.2 mgÆmL
)1
BSA, 0.05 mgÆmL

)1
bacitracin, 5 lgÆmL
)1
aprotinin, 5 lgÆmL
)1
leupeptin, and 2.5 lgÆmL
)1
peps-
tatin A. After incubation at 37 °C for 5–40 min in the
absence or presence of 0.2 mm bpV(phen), suspensions were
supplemented with KCl (0.5 m final concentration) at 4 °C
and immediately centrifuged at 150 000 g for 40 min. The
total incubation mixture and the pellet were analyzed for
phosphorylated IRb and Grb14 content by western blotting.
IR purification and quantitation
IR was partially purified from Triton X-100-soluble extracts
of cell fractions by adsorption on WGA–Sepharose.
Extracts (0.5–1 mL, 2–5 mg of protein) were incubated on
a rotatory shaker with WGA–Sepharose (1 : 10, v ⁄ v) for
2 h at 4 °C, and glycoproteins were eluted with 0.3 m
N-acetylglucosamine in 25 mm Hepes buffer (pH 7.6), 0.1%
Triton X-100, and 0.2 mm phenylmethanesulfonyl fluoride.
IR contents in crude extracts and WGA eluates were quan-
titated by measurements of specific [
125
I]insulin binding
(0.02 nm) as previously described [17].
Assay of IR tyrosine kinase activity
This assay was based on the ability of IR to phosphorylate
RCAM-lysozyme, a high-affinity substrate [42]. For assay

of in vitro insulin-stimulated kinase activity, WGA eluates
from a light mitochondrial–microsomal fraction prepared
from control rat liver (about 2 fmol of insulin bound) were
preincubated in the absence or presence of insulin (0.5 lm)
for 1 h at 23 °C. A phosphorylation mix (50 mm ATP,
1mm CTP, 4 mm MnCl
2
,8mm MgCl
2
) was then added in
the presence or absence of GST–Grb14 at the indicated
concentration. After 30 min at 23 °C, RCAM-lysozyme
(6 lm) was added and incubations were allowed to proceed
further for 10–40 min, until the reaction was stopped by
the addition of 4· Laemmli sample buffer. For assay of
in vivo insulin-stimulated kinase activity, intact Golgi ⁄ endo-
some fractions or WGA eluates of light mitochondrial–
microsomal and plasma membrane fractions prepared from
insulin-injected rat liver and containing equal amounts of
IR (0.5–2 fmol of insulin bound) were incubated with the
phosphorylation mix and RCAM-lysozyme added together
at the same concentrations as above for 10–40 min at
23 °C. RCAM-lysozyme phosphorylation was monitored by
western blotting using antibody against phosphotyrosine.
Western blotting
Equal amounts of cell fraction proteins (10 lg unless other-
wise indicated) were supplemented with Laemmli sample
buffer, subjected to SDS ⁄ PAGE analysis (15% acrylamide
for RCAM-lysozyme, 8–10% acrylamide for other
proteins), and immunodetected with the indicated antibod-

ies. Immunoreactive bands were revealed using an enhanced
chemiluminescence detection detection kit. The signals iden-
tified on the autoradiograms were quantitated by scanning
densitometry using a chemi genius 2 scan (Genesnap;
Syngene, Cambridge, UK).
Statistical analysis
Values of densitometric measurements were expressed as
means ± SEM and analyzed by ANOVA. When significant
differences were detected, a posteriori comparisons between
means were conducted using the Fisher LSD test. Correla-
tion analysis were also performed between phosphorylated
IR and Grb14. Calculations were carried out using the
statview program.
Acknowledgements
This work was supported by the Ministe
`
re de la Recher-
che (ACI no. 02 2 0537 ⁄ 8). We thank Dr Gillian Dan-
ielsen for her supply of superactive insulin analogs, Dr
Barry Posner for his gift of bpV(phen), and Dr Ronald
Kohanski for his gift of reduced and carboxyamidome-
thylated lysozyme. We also thank Dr Tarik Issad for
critically reading the manuscript, and Dr Maria-Ange-
les Ventura for her help in statistical analysis.
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