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
´

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

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

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

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

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

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
** *

** *
**
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-

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

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

+
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

)

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.

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

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-

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

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

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

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

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

*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

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.

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

(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);

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

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-

common signaling adapter. Front Biosci 9, 603–618.
3 Lim MA, Riedel H & Liu F (2004) Grb10: more than a
simple adaptor protein. Front Biosci 9, 387–403.
4 Shen TL & Guan JL (2004) Grb7 in intracellular signal-
ing and its role in cell regulation. Front Biosci 9, 192–200.
5 Holt LJ & Siddle K (2005) Grb10 and Grb14: enigmatic
regulators of insulin action – and more? Biochem J 388,
393–406.
6 Kasus-Jacobi A, Perdereau D, Auzan C, Clauser E,
Van Obberghen E, Mauvais-Jarvis F, Girard J &
Burnol A-F (1998) Identification of the rat adapter
Grb14 as an inhibitor of insulin actions. J Biol Chem
273, 26026–26035.
7 Bereziat V, Kasus-Jacobi A, Perdereau D, Cariou B,
Girard J & Burnol AF (2002) Inhibition of insulin
receptor catalytic activity by the molecular adapter
Grb14. J Biol Chem 277, 4845–4852.
8 Depetris RS, Hu J, Gimpelevich I, Holt LJ, Daly RJ &
Hubbard SR (2005) Structural basis for inhibition of
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 4375
the insulin receptor by the adaptor protein Grb14. Mol
Cell 20, 325–333.
9 Hemming R, Agatep R, Badiani K, Wyant K, Arthur
G, Gietz RD & Triggs-Raine B (2001) Human growth
factor receptor bound 14 binds the activated insulin
receptor and alters the insulin-stimulated tyrosine phos-
phorylation levels of multiple proteins. Biochem Cell
Biol 79, 21–32.
10 Cariou B, Perdereau D, Cailliau K, Browaeys-Poly E,

fication of a novel human tankyrase through its interac-
tion with the adaptor protein Grb14. J Biol Chem 276,
17172–17180.
16 Rajala RV, Chan MD & Rajala A (2005) Lipid–protein
interactions of growth factor receptor-bound protein 14
in insulin receptor signaling. Biochemistry 44, 15461–
15471.
17 Desbuquois B, Lopez S & Burlet H (1982) Ligand-
induced translocation of insulin receptors in intact rat
liver. J Biol Chem 257, 10852–10860.
18 Khan MN, Baquiran G, Brule C, Burgess J, Foster B,
Bergeron JJ & Posner BI (1989) Internalization and
activation of the rat liver insulin receptor kinase in vivo.
J Biol Chem 264, 12931–12940.
19 Burgess JW, Wada I, Ling N, Khan MN, Bergeron JJ
& Posner BI (1992) Decrease in beta-subunit phosp-
hotyrosine correlates with internalization and activation
of the endosomal insulin receptor kinase. J Biol Chem
267, 10077–10086.
20 Authier F, Merlen C, Amessou M & Danielsen GM
(2004) Use of high affinity insulin analogues to assess
the functional relationships between insulin receptor
trafficking, mitogenic signaling and mRNA expression
in rat liver. Biochimie 86, 157–166.
21 Faure R, Baquiran G, Bergeron JJ & Posner BI (1992)
The dephosphorylation of insulin and epidermal growth
factor receptors. Role of endosome-associated phosp-
hotyrosine phosphatase(s). J Biol Chem 267, 11215–
11221.
22 Bevan AP, Burgess JW, Drake PG, Shaver A, Bergeron

29 Balbis A, Baquiran G, Bergeron JJ & Posner BI (2000)
Compartmentalization and insulin-induced transloca-
tions of insulin receptor substrates, phosphatidylinositol
3-kinase, and protein kinase B in rat liver. Endocrinol-
ogy 141, 4041–4049.
30 Lotti LV, Lanfrancone L, Migliaccio C, Zompetta C,
Pelicci G, Salcini AE, Falini B, Pelicci PG & Torrisi
MR (1996) Shc proteins are localized on endoplasmic
reticulum membranes and are redistributed after tyro-
sine kinase receptor activation. Mol Cell Biol 16, 1946–
1954.
31 Drenan RM, Liu X, Bertram PG & Zheng XF (2004)
FKBP12-rapamycin-associated protein or mammalian
target of rapamycin (FRAP ⁄ mTOR) localization in the
endoplasmic reticulum and the Golgi apparatus. J Biol
Chem 279, 772–778.
32 Frangioni JV, Beahm PH, Shifrin V, Jost CA & Neel
BG (1992) The nontransmembrane tyrosine phospha-
tase PTP-1B localizes to the endoplasmic reticulum via
Insulin-induced translocation of Grb14 in rat liver B. Desbuquois et al.
4376 FEBS Journal 275 (2008) 4363–4377 ª 2008 The Authors Journal compilation ª 2008 FEBS
its 35 amino acid C-terminal sequence. Cell 68, 545–
560.
33 Baumann O & Walz B (2001) Endoplasmic reticulum of
animal cells and its organization into structural and
functional domains. Int Rev Cytol 205, 149–214.
34 Watt SA, Kular G, Fleming IN, Downes CP & Lucocq
JM (2002) Subcellular localization of phosphatidylinosi-
tol 4,5-bisphosphate using the pleckstrin homology
domain of phospholipase C delta1. Biochem J 363, 657–

activating and non-activating components of autophos-
phorylation of the insulin receptor protein kinase.
Biochem Biophys Res Commun 134, 1312–1318.
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 4377