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Division of Endocrinology and Metabolism, Department of Medicine, Veterans Administration Medical Center, University of California-San Diego (M.C., P.V., N.N., S.M., J.M.O.), La Jolla, California 92093; and the Program in Molecular Medicine and Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center (J.K., M.P.C.), Worcester, Massachusetts 01605
Address all correspondence and requests for reprints to: Dr. Jerrold M. Olefsky, Department of Medicine, University of California-San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-GRP1-AB significantly
inhibited insulin- and IGF-I-stimulated actin rearrangement in an
insulin receptor-overexpressing cell line (HIRcB) compared with that in
control experiments. Coinjection of GRP1-Sec7 along with constitutively
active Rac (Q67L) did not inhibit Rac-induced actin rearrangement.
Furthermore, GRP1 is not able to bind and act as a nucleotide exchange
factor for the small GTP-binding proteins of the Rho family. As GRP1
acts as a guanine nucleotide exchange factor for ARF6 proteins, we
propose a signaling pathway distinct from the small GTP-binding protein
Rac, connecting PtdIns(3,4,5)P3 via GRP1 to ARF6, leading to insulin-
and IGF-I-induced actin rearrangement. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Another signaling pathway involving PtdIns(3, 4, 5)P3, generated through PI 3-kinase activation after growth factor stimulation, is actin rearrangement. It has been shown that insulin induces membrane ruffling in several cell types, and more recently that this pathway is dependent on PtdIns(3, 4, 5)P3 generation through PI 3-kinase (7, 16, 17, 18, 19). The current model of growth factor-mediated actin reorganization involves a coordinated response of tyrosine phosphorylation, phosphoinositide modification, and activation of small GTP binding proteins (Rac, Rho, and ARF6).
Recently, Klarlund et al. have identified, using an expression cloning approach with a PtdIns(3, 4, 5)P3 probe, another potential effector protein, named general receptor for phosphoinositides-1 (GRP1) (20). GRP1 is a PH domain-containing protein that binds PtdIns(3, 4, 5)P3 with high affinity, but not PtdIns(3, 4)P2 or PtdIns(3)P, potentially making it a target for PI 3-kinase. GRP1 consists of an NH2-terminally located coiled coil domain followed by a Sec7 domain and a COOH-terminal PH domain. Furthermore, GRP1 acts as a nucleotide exchange factor for the small GTP proteins of the ARF family (21).
As the diversity of these cellular functions regulated by the 3'-polyphosphoinositides suggests that multiple effector proteins might operate to mediate these processes, we investigated the biological actions of GRP1 on cytoskeletal rearrangement, such as membrane ruffling and stress fiber breakdown, GLUT4 translocation, and DNA synthesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-GRP1
antibody, raised against full-length GRP1, was provided by Jes
Klarlund. Porcine insulin was provided by Eli Lilly & Co., Co. IGF-I was purchased from Life Technologies (Gaithersburg, MD). Polyclonal anti-GLUT4 antibody
(F349) was described previously (22). -Rac1, -RhoA, and -RhoB
antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). TRITC-phalloidin and all other reagents
were purchased from Sigma Chemical Co. (St. Louis, MO).
The glutathione-S-transferase (GST)-Rac construct was a gift
from Dr. A. Hall. It was purified by standard procedures from bacteria
by binding to glutathione beads.
Cell culture and microinjection
Rat-1 fibroblasts overexpressing wild-type human insulin
receptors were maintained in DMEM-Ham’s F-12 medium (Life Technologies) supplemented with 10% FCS and gentamicin
(Gemini Biological Products, Calabasas, CA), 2
mM Glutamax (Life Technologies), and 500
nM methotrexate (Sigma Chemical Co.). Cells
grown on glass coverslips were rendered quiescent by starvation for
36 h in serum-free DMEM. -GRP1 antibody (-GRP1-AB; 6.4
mg/ml) or GST fusion proteins (10 mg/ml) were injected into the
cytoplasm of cells using glass capillary needles. Preimmune rabbit IgG
(10 mg/ml) was added to the GST fusion protein to allow detection of
injected cells. After a recovery period of 1 h, cells were
stimulated with either 10 or 100 ng/ml insulin (1.7 and 17
nM) or 100 ng/ml IGF-I for 3 min to activate actin
rearrangement and with 100 ng/ml insulin for 18 h to assess DNA
synthesis. The cells were then fixed for staining.
3T3-L1 cells were maintained in DMEM-high glucose (Life Technologies) supplemented with 10% FCS and penicillin
G/streptomycin (Omega Scientific, Cambridge, MA) and
differentiated into adipocytes as described previously, then reseeded
onto glass coverslips (23). Cells were serum starved 2 h before
microinjection for GLUT4 detection. All reagents for microinjection
were dissolved in microinjection buffer (5 mM
NaPO4 and 100 mM KCl, pH 7.4). All reagents,
except -GRP1-AB, were coinjected with preimmune sheep IgG (10 mg/ml)
to allow detection of injected cells. After a recovery period of 1
h, the cells were stimulated with 3 or 10 ng/ml insulin for 20 min,
then fixed for staining.
GST fusion protein preparation
Molecular cloning of the GST fusion proteins out of specific
sequences within the GRP1 structure, i.e. GST fusion
proteins containing residues 239–399 (PH domain), residues 52–260
(Sec7 domain), residues 5–71, or residues 5–399, has been described
previously (20). All fusion proteins were produced from
Escherichia coli by IPTG
(isopropyl-1-thio-ß-galactopyranoside) induction and purified on
glutathione-agarose beads (24). Eluted proteins were concentrated and
exchanged into microinjection buffer using a Centricon-30 filter
(Amicon, Beverly, MA).
Preparation of antibodies for microinjection
-GRP1-AB was concentrated and exchanged into microinjection
buffer (5 mM NaPO4 and 100 mM KCl,
pH 7.4) using a Centricon-30 filter (Amicon).
Immunofluorescence and cell quantification
Actin localization. One hour after cytoplasmic injections,
cells were stimulated with or without insulin (10 or 100ng/ml) or IGF-I
(100 ng/ml) for 3 min and fixed with 3.7% formaldehyde in PBS for 10
min at room temperature. Cells were permeabilized in 0.2% Triton X-100
for 5 min, washed in PBS, and incubated at room temperature for 45 min
with rhodamine-phalloidin (0.125 mg/ml) to visualize the location of
polymerized actin at the cell membrane (membrane ruffles) or stress
fibers and with FITC-conjugated antirabbit to detect injected cells.
After staining, coverslips were washed successively in PBS for 5 min
and mounted in PBS containing 15% Gelvatol (Monsanto Co., St. Louis,
MO) (polyvinyl alcohol), 33% glycerol, and 0.1% sodium
azide.
GLUT4 staining. Cells were serum starved 2 h before stimulation with or without 3 or 10 ng/ml insulin for 20 min. Immunostaining of GLUT4 was performed essentially as previously described (23). The cells were fixed in 3.7% formaldehyde in PBS for 10 min at room temperature. After washing, the cells were permeabilized and blocked with 0.1% Triton X-100 and 2% FCS in PBS for 10 min. Cells were then incubated with F349 (1 µg/ml) in PBS with 2% FCS overnight at 4 C. After washing, GLUT4 and injected IgG were detected by incubation with FITC-conjugated donkey antirabbit IgG antibody and AMCA-conjugated donkey antisheep or antirabbit IgG antibody, respectively, followed by observation under immunofluorescence microscope. Each coverslip was examined with the observer blinded to the experimental conditions, and the aminomethylcouarin-positive microinjected cells on each coverslip were evaluated for the presence of plasma membrane-associated GLUT4 staining.
BrdU incorporation. One hour after injection cells were stimulated with or without insulin (100 ng/ml) for 18 h. BrdU (10 µM/ml) was then added during the last 6 h of stimulation to allow its incorporation into newly synthesized DNA. Cells were fixed for 10 min in 3.7% formaldehyde-PBS, washed with PBS, and incubated for 1 h at room temperature with rat anti-BrdU antibody (1:500 in PBS containing 10 mM MgCl2, 20 U/ml deoxyribonuclease I, and 0.5% Nonidet P-40). Coverslips were washed with PBS and incubated for an additional hour with TRITC-anti rat and FITC-antirabbit or FITC-antimouse antibodies. All secondary antibodies were used at a 1:100 dilution in PBS-0.5% Nonidet P-40. Coverslips were washed and mounted in Gelvatol as before.
Cell quantification. Slides were analyzed on a Zeiss Axiophot immunofluorescence microscope (Zeiss, New York, NY). AMCA-positive 3T3-L1 adipocytes on each coverslip were evaluated for the presence of plasma membrane-associated GLUT4 as previously described (23). HIRcB cells, FITC positive for cytoplasmic injections, displaying parallel actin fibers that colocalize with the nucleus were scored as positive for stress fibers (results are given as the percentage of stress fibers). HIRcB cells that showed actin staining at the periphery were scored as positive for membrane ruffles. HIRcB cells positive for coinjected rabbit IgG were counted for the presence of incorporated BrdU (results are given as the percentage of BrdU incorporation). The observer was blinded to the experimental conditions.
Imaging. The cells were inspected with a Zeiss Axiophot fluorescence microscope. Images were captured using a CCD camera from Photometrics (Tucson, AZ) and were saved using Isee software from Inovision (Durham, NC) to be subsequently used for prints.
GST-GRP1 fusion protein pull-downs of small GTP-binding
proteins
HIRcB cells were lysed (100 mM HEPES, 2% Triton
X-100, 20 mM EDTA, 300 mM NaCl, 4
mM phenylmethylsulfonylfluoride, 20% glycerol, 8
mM NaVO4, 400 mM NaF, and 40
mM NaPyroPO4), and one of each GST fusion
protein (GST-GRP1, GST-Sec7, GST-PH, and GST-N-terminal) was added
to 500 µl cell lysates (200 µg protein), diluted to a final
concentration of 1 µM, and incubated for 4 h at 4 C.
Then, 50 µl of a 50% slurry solution of glutathione-Sepharose beads
(Promega Corp., Madison, WI) were added and incubated at 4
C for 2 h. Beads were collected by spinning in a microfuge at
10,000 x g for 5 min. Pellets were washed in lysis
buffer three times and boiled, and the supernatant was separated on
12.5% SDS-PAGE and transferred to Immobilon-P (Millipore Corp., Bedford, MA) by electroblotting. For immunoblotting,
membranes were blocked (Tris-buffered saline, Tween, and 5% nonfat dry
milk) and then blotted with Rac1, RhoA, and RhoB antibodies
(Santa Cruz Biotechnology) at a 1:1000 dilution. Blots
were incubated with horseradish peroxidase-linked secondary antibody
(Amersham) at a 1:2000 dilution, followed by detection
with enhanced chemiluminescence according to the manufacturer’s
instruction (Amersham).
Rac GDP/GTP nucleotide exchange assay
Bacteria containing Rac cloned in a pGEX vector were grown to
OD600 of approximately 0.6 in Luria Bertoni broth. To
induce production of the fusion product, IPTG was added to a
concentration of 0.25 mM. After 3–4 h, the bacteria were
harvested by centrifugation and lysed, and the fusion protein was bound
to glutathione agarose as previously described (21). The beads were
transferred to assay buffer [50 mM HEPES (pH 7.5), 100
mM KCl, and 1 mM dithiothreitol], and assays
were performed exactly as previously described (21).
Statistics
Statistical significance was assessed by Student’s t
test for paired data.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). HIRcB cells grown on coverslips at
low density were serum starved to induce quiescence and then stimulated
with either insulin (10 or 100 ng/ml) or 100 ng/ml IGF-I for 3 min. The
cells were then fixed, and the effect of growth factor treatment on the
actin cytoskeleton was visualized by fluorescence microscopy of cells
stained with TRITC-phalloidin, a fluorescent compound that binds
selectively to filamentous actin (26). Microinjection of -GRP1-AB
inhibited the ability of insulin and IGF-I to induce membrane ruffling,
and this is depicted in Fig. 2. As can be
seen, uninjected cells (arrows) show easily detectable
membrane ruffles after insulin stimulation. In contrast, the cells
injected with -GRP1-AB, detected by FITC staining (lower
panel), do not display membrane ruffles. Quantification of
phalloidin-stained cells was conducted by scoring individual cells for
the presence or absence of membrane ruffles, and these results are
summarized in bar graph form in Fig. 3.
-GRP1 antibody injected at 6.4 mg/ml caused an inhibition of insulin
(Fig. 3A, 100 ng/ml; Fig. 3B, 10 ng/ml)- and IGF-I (Fig. 3C)-induced
membrane ruffling by 35 ± 3.5%, 37 ± 4%, and 25 ±
3.7%, respectively (mean ± SE; P <
0.05 for all). Full-length GRP1 fusion protein (10 mg/ml) tended to
cause membrane ruffling by 20% in unstimulated HIRcB cells compared
with a basal level of 3% in IgG-injected cells, but this effect did
not reach statistical significance. The injected GST-PH domain reduced
IGF-I (100 ng/ml; Fig. 3C)- and insulin (10 ng/ml; Fig. 3B)-induced
membrane ruffling by 37.6 ± 6.7% (P < 0.05) and
36 ± 4.5% (P < 0.05; mean ±
SE), respectively. After stimulation with 100 ng/ml insulin
(Fig. 3A), we observed only a slight, but not significant, effect of
the PH domain. This may be due to the high amount of insulin receptors
in HIRcB cells (106/cell), which could mask the inhibitory
effect at high insulin concentrations. The GST-Sec7 domain inhibited
insulin (10 and 100 ng/ml)- and IGF-I (100 ng/ml)-stimulated membrane
ruffling by 40 ± 5.4%, 47 ± 5.4%, and 31 ± 5.2%,
respectively (mean ± SE; P < 0.05
for all). The N-terminal coiled coil domain had no effect on membrane
ruffling in basal, insulin-stimulated, or IGF-I-stimulated states. GST
alone did not affect insulin- and IGF-I-induced cytoskeletal
rearrangement.
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). Rac (Q67L) alone had a marked effect
to induce membrane ruffling. The GST-Sec7 domain was not able to block
Rac (Q67L)-induced membrane ruffling. The effect of insulin (100 ng/ml)
to induce membrane ruffling was not influenced by microinjection of
active Rac. However, when active Rac and GST-Sec7 were coinjected into
insulin-stimulated cells, membrane ruffling was reduced by about 20%.
These data indicate that Rac and GRP1 act on distinct pathways leading
to membrane ruffling.
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-GRP1-AB on growth
factor-induced stress fiber breakdown in HIRc-B cells-GRP1-AB had an effect on
ligand-induced stress fiber breakdown (Fig. 5).
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-GRP1-AB on
insulin-induced GLUT4 translocation in 3T3-L1 adipocytes and on DNA
synthesis in HIRcB cells-GRP1-AB had any effect on GLUT4 distribution in either basal
or insulin-stimulated cells (Fig. 6B).
The same GRP1 reagents were microinjected into Rat-1 fibroblast (HIRcB)
cells that were stimulated with insulin (100 ng/ml) and then stained
for BrdU incorporation. None of the GRP1 reagents had any effect on
basal or insulin-stimulated DNA synthesis (Fig. 6A).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-GRP1-AB into 3T3-L1 adipocytes had no
effect on insulin-induced GLUT4 translocation or on DNA synthesis in
HIRcB cells. The actin cytoskeleton is implicated in many cellular functions, and an understanding of how actin filament organization is orchestrated is a central question in cell biology. Growth factor-stimulated membrane ruffling is caused through activation of PI 3-kinase and thereby generation of PtdIns(3, 4, 5)P3 as well as activation of the small GTP-binding protein Rac, which itself activates the serine/threonine kinase PAK and POR1 (27). The mechanism by which D3 phosphoinositides signal to the small GTP-binding proteins Rac and Rho is still unknown, although recently Tiam1, Vav, and Sos were shown to display in part PI 3-kinase-dependent GTP-GDP exchange activity on Rac (28, 29, 30). However, inhibition of growth factor-induced actin rearrangement by the PH domain, the Sec7 domain, and the antibody in our study cannot be explained by an interaction with Rac itself, as we did not observe guanine nucleotide exchange factor activity of GRP1 on Rac in guanine nucleotide exchange assays or the presence of one of the small GTP-binding proteins of the Rho family (Rac1, RhoA, and RhoB) in GRP1-GST fusion protein precipitations. This observation is supported by a recent report showing that the overall structure of the Sec7 domain of ARNO, which is structurally closely related to GRP1 and is a guanine nucleotide exchange factor for ARF proteins, is unrelated to the catalytic domains of nucleotide exchange factors for Ras and Rho proteins (31). One possible, although unlikely, explanation for our findings would be an effect of GRP1 on a still unknown guanine nucleotide exchange factor for Rac, thereby blocking growth factor-induced signaling to membrane ruffling. On the other hand, GRP1 and to a lesser extent its Sec7 domain were recently shown to act as guanine nucleotide exchange factors on ARF proteins, where the exchange activity for ARF1 and -5 of full-length GRP1 was 6-fold enhanced by adding PtdIns(3, 4, 5)P3 (recently, an even greater exchange activity on ARF6 was found in vivo by the same investigators; unpublished observation) (21). ARF6 itself was recently shown to act as an inducer of actin rearrangement (27) that could not be blocked by a dominant negative mutant of Rac1 (S17N), indicating a Rac-independent pathway to actin rearrangement. Furthermore a downstream protein involved in membrane ruffling named POR1, which binds to Rac-1 and ARF6 in a GTP-dependent manner, has recently been identified. Truncated versions of POR1 inhibit the induction of membrane ruffling by an activated mutant of Rac1 (V12Rac1) and ARF6 (27, 32). The nucleotide exchange activity of GRP1 on ARF6 and our microinjection results suggest an involvement of GRP1 in this ARF6-mediated signaling pathway to actin polymerization, acting downstream of PI 3-kinase and independent of Rac1. The inhibition of growth factor stimulated actin rearrangement by the antibody against GRP1 and the trend of wild-type GRP1 to induce membrane ruffling points toward stimulatory properties of GRP1. The inhibition of GRP1’s PH domain on insulin- and IGF-I-induced actin redistribution is mediated by binding to PtdIns(3, 4, 5), thereby blocking the intracellular pathways downstream of this phosphoinositide. We postulate that the inhibition by the Sec7 domain is mediated through binding to ARF6, thereby obstructing recruitment of endogenous GRP1 to ARF6. Furthermore, PtdIns 3,4,5P3 may mediate specific subcellular localizations of ARF to its preferred site of action.
Another growth factor-induced effect involving cytoskeletal
rearrangement is stress fiber breakdown (7, 19). Injection of GRP1, its
domains, and the -GRP1-AB had no effect on insulin- and
IGF-I-induced stress fiber breakdown. This is consistent with our
observation that overexpression of p150 SH2-domain containing inositol
phosphatase, a 5'-phosphatase that converts PtdIns(3, 4, 5)P3
to PtdIns(3, 4)P2, does not inhibit this signaling cascade
(Vollenweider, P., et al., unpublished observations).
Indeed, previous reports suggest that stress fiber formation in
different cell types is regulated by the small GTP-binding protein Rho
(33). Generation of stress fibers by the Rho protein parallels its
ability to stimulate the formation of 4,5-phosphorylated
phosphatidylinositol [PtdIns(4, 5)P2] (34). Dissociation of the focal
adhesion proteins, -actinin and vinculin from PtdIns(4, 5)P2 upon
stimulation with growth factors may lead to stress fiber breakdown (35, 36). As we (19) and others have shown that stress fiber breakdown is
dependent on PI3-kinase, we speculate that PI3-kinase stimulates stress
fiber breakdown by phosphorylating the D-3 position of PtdIns(4, 5)P2,
which causes its release from focal adhesion-localized proteins.
Without this anchoring effect of PtdIns(3, 4)P2, -actinin and
vinculin can no longer localize to focal adhesions, and this would lead
to stress fiber breakdown. As GRP1 binds specifically to
PtdIns(3, 4, 5)P3 and not to PtdIns(3, 4)P2, it is not surprising that
insulin- and IGF-I-induced stress fiber breakdown is not affected by
GRP1 in our cell system.
Insulin stimulates the translocation of GLUT4 from an intracellular storage compartment to the cell surface, and activation of PI3-kinase is an essential step of this pathway. However, injections of the various GRP1 reagents into 3T3-L1 adipocytes had no effect on basal or insulin-stimulated GLUT4 distributions. Within the limits of this experimental system, these results suggest that GRP1 may not be necessary in this insulin effect.
PtdIns(3, 4, 5)P3 generation after growth factor stimulation is also critical for mitogenic signaling (6, 37, 38). As GRP1 binds with high specificity to PtdIns(3, 4, 5)P3, we investigated possible effects of GRP1 on DNA synthesis. As we could not observe any inhibitory or stimulatory effects, GRP1 may not be involved in mitogenic signaling. These data also demonstrate that GRP1 reagents do not have any nonspecific toxic effects in our cell systems.
Our data are consistent with a signaling pathway, where GRP1 seems to serve as a signaling protein connecting PtdIns(3, 4, 5)P3 to ARF6, leading to actin rearrangement, but this interaction does not appear to be necessary for insulin-mediated GLUT4 translocation or DNA synthesis.
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Footnotes |
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Received June 1, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunits. Cell 77:83–93[CrossRef][Medline]
-Actinin and vinculin are PIP2-binding proteins involved in
signaling by tyrosine kinase. J Biol Chem 269:1518–1522-actinin function. Nature 359:150–152[CrossRef][Medline]
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