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Endocrinology Vol. 144, No. 12 5497-5503
Copyright © 2003 by The Endocrine Society

Specificity of Insulin-Like Growth Factor I and Insulin on Shc Phosphorylation and Grb2 Recruitment in Caveolae

Claudia Biedi, Danilo Panetta, Daniela Segat, Renzo Cordera and Davide Maggi

Department of Endocrinology and Metabolism, University of Genova, 16132 Genova, Italy; and Department of Biology, University of Padova (D.S.), 35121 Padova, Italy

Address all correspondence and requests for reprints to: Dr. Renzo Cordera, Di.S.E.M., Viale Benedetto XV 6, 16132 Genova, Italy. E-mail: record{at}unige.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Caveolae are lipid raft microdomains that regulate endocytosis and signal transduction. IGF-I receptor (IGF-IR) localizes in caveolae and tyrosine phosphorylates caveolin 1, supporting a role for these subcellular regions in the compartmentalization of IGF-I signaling. Src homology 2/{alpha}-collagen related protein (Shc) is the main mediator of IGF-I mitogenic action, coupling IGF-IR phosphorylation to Ras-MAPK activation. Here we show that IGF-I induces Shc tyrosine phosphorylation in the caveolae with a time course significantly different from that observed in the nonraft cellular fractions. In the same time, IGF-I recruits growth factor receptor bound protein 2 (Grb2) to caveolae and activates p42/p44 MAPKs in these microdomains. Src family kinases regulate IGF-I action through an Shc-dependent mechanism. In R-IGF-IRWT cells, IGF-I causes Fyn enrichment in the caveolae with a time course consistent with Shc phosphorylation and Grb2 recruitment in these regions. Finally, we have observed that after IGF-I stimulation, IGF-IR and Fyn colocalize in lipid raft caveolin 1-enriched microdomains. As insulin and IGF-I share common substrates, the effect of insulin on these cellular processes was measured. Here we show that insulin also induces Shc phosphorylation and Grb2 recruitment to caveolae, but with a significantly different time course compared with IGF-I. Our results suggest that 1) IGF-I causes the colocalization of signaling proteins in caveolae through a phosphorylation-regulated mechanism; and 2) the time course of phosphorylation and recruitment of substrates in caveolae by insulin receptor and IGF-IR could determine the specific actions of these receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CAVEOLAE ARE PLASMA membrane regions originated from lipid rafts by oligomerization of caveolins (1). Several signaling proteins are enriched in the caveolae and interact with caveolin 1 that act as a scaffolding protein and regulate their activation (2). Also, a number of growth factor receptors reside in the caveolae, suggesting a role for these regions in both the compartmentalization as well as the cross-talk between signaling pathways (2). It has also been shown that growth factors cause phosphorylation of caveolin 1 at the level of tyrosine 14 and regulate function of the caveolae (3). A role of lipid rafts/caveolae in the action of insulin has been proposed (4) based on the following experimental results: insulin receptor (IR) as well as IR substrates have been detected in the caveolae (5); caveolin 1 is an activator of insulin signaling (6), and insulin causes caveolin 1 tyrosine phosphorylation in adipocytes and muscle cells (7, 8, 9, 10).

It has been recently shown that IGF-I receptor (IGF-IR) also localizes in the lipid raft caveolin 1-enriched fractions and phosphorylates caveolin 1 (11, 12, 13). As a consequence, phosphorylated caveolin 1 redistributes in the lipid raft microdomains. This effect is IGF-I-dependent, but not insulin-dependent, suggesting a specific compartmentalization of IGF-I signaling (11). It has also been shown that caveolae regulate IGF-I-induced survival in multiple myeloma cells where caveolin 1 directly interacts with IGF-IR in coimmunoprecipitation experiments (12), and lipid rafts/caveolae regulate IGF-I-induced preadipocyte differentiation (13). This process is characterized by the activation of intracellular IGF-IR substrates, such as caveolin 1 and Src homology 2/{alpha}-collagen related protein (Shc) (13). Upon IGF-I stimulation, phosphorylated Shc binds growth factor receptor bound protein 2 (Grb2)-Son of sevenless (Sos) complex, activating the Ras-MAPK pathway (14). Src family kinases also regulate IGF-I action both in vitro (15) as well as in vivo (16, 17, 18). In particular, Fyn, which phosphorylates caveolin 1 (8) and is activated by IGF-I (18), regulates IGF-I actions by a Shc-dependent mechanism (18). These data suggest the relevance of caveolae-resident proteins in the regulation of IGF-I action at the plasma membrane. However, little is known about the time course and specificity of IGF-IR substrate phosphorylation and recruitment in caveolae. The aims of this work were to investigate the possible effects of IGF-I on 1) Shc phosphorylation in the lipid rafts caveolin 1-enriched fractions, 2) the recruitment of Grb2 and Fyn to these domains, and 3) the activation of the MAPK pathway in caveolae. Here we show that 1) Shc isoforms are present in the caveolae of R- fibroblasts that overexpress wild-type IGF-IR (R-IGF-IRWT) cells, and p52 is the most represented; 2) the time course of IGF-I-induced Shc phosphorylation in caveolae is different from that observed in nonraft cellular fractions; 3) Shc phosphorylation recruits Grb2 to the caveolae and activates the MAPK pathway in this compartment; 4) IGF-I induces the colocalization of IGF-IR and Fyn in the caveolae by Fyn enrichment in these regions; 5) Shc phosphorylation and Grb2 recruitment closely resemble Fyn enrichment in caveolae; and 6) the time course of Shc phosphorylation and Grb2 recruitment after IGF-I stimulation is significantly different from that caused by insulin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
R- fibroblasts, provided by Dr. R. Baserga (Thomas Jefferson University, Philadelphia, PA), derive from embryo mice with a targeted disruption of the IGF-IR gene and do not express IGF-IRs on the cell surface (19). Anti-IGF-IR ß-subunit, anti-Fyn, and anti Grb2 antibodies as well as enhanced chemiluminescence (ECL) reagents were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-Shc and antiphosphotyrosine (PY20) antibodies were obtained from Transduction Laboratories (Lexington, KY). Anti-p42/p44 MAPKs and anti phospho-p42/p44 (Thr202/Tyr204) MAPK antibodies were purchased from Cell Signaling Technology (Beverly, MA). Peroxidase-conjugated cholera toxin B subunit, antiplacental alkaline phosphatase (anti-PLAP), antirabbit and antimouse Ig horseradish peroxidase-linked, were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Donkey antimouse and antirabbit FITC, antimouse, and antirabbit tetramethyl rhodamine isothiocyanate (TRITC) antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). cDNA coding for IGF-IRWT was provided by Dr. Domenico Accili (Columbia University, New York, NY). The pCMV (cytomegalovirus) vector containing cDNA coding for human insulin receptor (HIR) was previously described (20).

Cell culture and transfection
R- cells were grown in DMEM supplemented with 2 mM glutamine, 10% fetal calf serum, 100 U/ml penicillin G, and 100 µg/ml streptomycin sulfate. Subconfluent cells were stably transfected with pcDNA3-IGF-IR (11), coding for human wild-type IGF-IR, pCMV-HIR, coding for human wild-type IR (20), or empty pcDNA3 vector by calcium phosphate precipitation. Cell clones overexpressing about 3 x 105 IGF-IRs (R-IGF-IRWT) or IRs (R-IRWT) were selected by addition of G418, and clonal cell lines were obtained by the limiting dilution subcloning technique. Receptors number was measured by [125I]IGF-I or [125I]insulin binding experiments.

Sucrose gradient centrifugation
Lipid raft-enriched domains were purified from cultured cells using a modified carbonate method (21). Serum-starved R-IGF-IRWT or R-IRWT cells were stimulated with IGF-I or insulin (20 nM), respectively, for 5 and 15 min before harvest. Then cells were lysed in 2 ml 0.5 M Na2CO3 (pH 11), containing protease inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM Na3VO4, and 1 mM phenylmethylsulfonylfluoride), incubated for 30 min on ice, scraped, and homogenized sequentially with a tightly fitting Dounce homogenizer (40 strokes; Kontes Co., Vineland, NJ) and three 10-sec bursts of a sonicator Soniprep 150. Equal amounts of cell lysates were then adjusted to 40% sucrose by mixing with 2 ml 80% sucrose prepared in 25 mM 2-[N-morpholino]ethanesulfonic acid], (pH 6.5), and 150 mM NaCl. This suspension was placed at the bottom of an ultracentrifuge tube and overlayed with a 5–35% discontinuous sucrose gradient. After centrifugation at 32,000 rpm for 22 h at 4 C in a swinging bucket rotor SW41 (Beckman, Fullerton, CA), a total of 12 fractions (1 ml each) were collected from the top of each gradient.

Immunoblot
Twenty-five microliters from each fraction were separated on 12% SDS-PAGE, transferred to nitrocellulose in Towbin buffer (20 mM Tris, 150 mM glycine, 20% methanol, and 0.02% sodium dodecyl sulfate) overnight at 100 mA. Nitrocellulose filters were blocked in Tris-buffered saline (TBS)/0.1% Tween 20/5% dry milk for 1 h at room temperature. Filters were incubated with primary antibodies for 2 h at room temperature on a rocking platform and washed extensively in TBS/0.1% Tween 20, and secondary horseradish peroxidase-linked antibodies were added for 1 h at room temperature. After a final wash in TBS/0.1% Tween 20, bound antibodies were detected using the ECL lighting system, according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Arlington Heights, IL). When indicated, different exposure times were used for lipid rafts/caveolae and nonraft cellular fractions. Bands of interest were quantitated by densitometry using NIH Image software.

To detect ganglioside GM1, 2 µl of each fraction, obtained by ultracentrifugation, were spotted on nitrocellulose and dried on air. Filter was then blocked for 1 h in TBS/0.1% Tween/10% dry milk at room temperature and incubated with peroxidase-linked cholera toxin B subunit for 30 min at room temperature. After extensive washing in TBS/0.1% Tween, ganglioside GM1 was detected by ECL.

Immunofluorescence
R- mock and R-IGF-IRWT cells were seeded on 10-mm glass coverslips and grown adherent for 2 d. Then cells were serum-starved for 24 h and treated with IGF-I (20 nM) for 5 and 15 min at 37 C. Finally, cells were washed once with ice-cold PBS, fixed for 30 min with 3% paraformaldehyde at room temperature, rinsed in ice-cold PBS, incubated with 30 mM NH4Cl, and permeabilized with PBS/0.025% saponin. Cells were subsequently incubated either with anti-Fyn (1:50) or anti-IGF-IR ß-subunit (1:50) antibodies for 45 min at room temperature. After washing with PBS, cells were incubated with antimouse FITC conjugate (1:100) and antirabbit TRITC conjugate (1:200). All antibodies were diluted in PBS/0.025% saponin. Coverslips were mounted using Moviol (Sigma-Aldrich Corp., St. Louis, MO). Analyses were performed with an epifluorescence inverted microscope (Olympus Optical Co. Ltd., Nagano, Japan). Images were stored using a chilled CCD b/w camera (Hamamatsu, Hamamatsu City, Japan) and processed with IP-LAB and Photoshop software (Adobe Systems, Mountain View, CA).

Cells, treated as described above, were double-immunostained either with anti-PLAP (1:300) or anti-Fyn (1:50) antibodies and incubated at room temperature for 45 min. Cells were processed as described above and after washing with PBS were incubated with antirabbit FITC conjugate (1:100) and antimouse TRITC conjugate (1:200). Immunofluorescence were analyzed as described.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
As previously shown, IGF-IRs are highly enriched in the caveolae of R-IGF-IRWT cells (11). First, we investigated the subcellular distribution of Shc in this cell line. As shown in Fig. 1Go, in R-IGF-IRWT cells, all three Shc proteins are present in the caveolae (fractions 6–8), identified by the colocalization of ganglioside GM1 and caveolin 1. These fractions are also highly enriched in caveolin 2 and flotillin (data not shown). p52 appears the most expressed Shc isoform in the lipid raft caveolin 1-enriched microdomains. Then we determined the time course of Shc phosphorylation in the raft fractions in the presence of IGF-I. IGF-I (20 nM) caused Shc phosphorylation in the caveolae, and this effect reached a peak after 15 min (Fig. 2Go), whereas in the nonraft fractions, the peak of this effect was observed after 5 min. To determine whether IGF-I recruits Shc to caveolae, the same filters probed with PY20 antibody were stripped and immunoblotted with an anti-Shc antibody. IGF-I did not determine any increase in the amount of Shc in caveolae (Fig. 3Go, left panel) compared with nonraft cellular fractions 9–12 (Fig. 3Go, right panel). However, IGF-I caused a redistribution of Shc isoforms in the lipid raft caveolin 1-enriched fractions 6–8 (Fig. 3Go, left panel). Grb2 binds to tyrosine-phosphorylated Shc and activates the MAPK pathway (22). In basal conditions the major amount of Grb2 was detected in nonraft fractions (Fig. 4AGo), whereas after IGF-I stimulation, a 3-fold enrichment of Grb2 was observed in the caveolae. This recruitment reached the maximum after 15 min (Fig. 4CGo), a time consistent with Shc phosphorylation in the same fraction. IGF-I also caused p42/p44 MAPK phosphorylation in the caveolae with a time course superimposable with those of Shc phosphorylation and Grb2 recruitment in this compartment (Fig. 5CGo).



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  		FIG. 1. Shc isoforms are associated with the lipid raft caveolin 1-enriched microdomains in R-IGF-IRWT cells. Total cell lysate in 0.5 M Na2CO3 (pH 11) was separated by ultracentrifugation on a discontinuous sucrose gradient, and a total of 12 fractions were collected, as described in Materials and Methods. The positions of the lipid rafts were identified by dot blot of GM1 using horseradish peroxidase-conjugated cholera toxin B subunit as a probe (A). Twenty-five microliters from each fraction were separated on 12% SDS-PAGE, transferred on nitrocellulose, blotted with an antibody directed against caveolin 1 (B) or against Shc (C), and developed by ECL. Coomassie blue staining (D) shows the relative protein enrichment in each fraction. All samples derive from the same ultracentrifugation experiment.

 


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  		FIG. 2. IGF-I causes Shc tyrosine phosphorylation in the caveolae of R-IGF-IRWT cells. Upper panel, Serum-starved R-IGF-IRWT cells (A) were stimulated with 20 nM IGF-I for 5 (B) and 15 (C) min. Proteins from each fraction were separated on 12% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with an antiphosphotyrosine antibody. On the left of each panel, molecular weight markers are shown. The immunoblots presented are representative of three separate experiments. Lower panel, Bands corresponding to phosphorylated Shc isoforms were quantified by densitometry using NIH Image software, and results were expressed as tyrosine-phosphorylated Shc amount (PY-Shc)/total Shc ratio in the lipid rafts/caveolae (continuous line) and nonraft cellular fractions (dotted line).

 


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  		FIG. 3. Shc distribution in lipid rafts/caveolae compared with nonraft cellular fractions of R-IGF-IRWT cells. Serum-starved R-IGF-IRWT cells (A) were stimulated with IGF-I (20 nM) for 5 (B) and 15 (C) min. Then cells were lysed, and 12 fractions were obtained as described in Materials and Methods. Proteins from each fraction, separated on 12% SDS-PAGE, were probed with an anti-Shc antibody. A longer exposure time was used for lipid rafts/caveolae (fractions 6–8) compared with nonraft fractions 9–12. The data presented are representative of three separate experiments. On the left of each panel, molecular weight markers are shown.

 


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  		FIG. 4. Grb2 recruitment to the caveolae in IGF-I-stimulated R-IGF-IRWT cells. Total cell lysates were obtained from serum-starved R-IGF-IRWT cells in the absence of IGF-I (A) or after IGF-I (20 nM) stimulation for 5 (B) and 15 (C) min. A total of 12 fractions were collected from the top of each gradient after ultracentrifugation of total cell lysates. Twenty-five microliters from each fraction were separated on 12% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with an anti-Grb2 antibody. The data presented are representative of three separate experiments.

 


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  		FIG. 5. IGF-I induces p42/p44 MAPK activation in caveolae. The same filters used for the anti-Shc immunoblot were stripped and reprobed with an antiphospho-p42/p44 MAPK antibody. The immunoblots presented are representative of three separate experiments. On the left of each panel, molecular weight markers are shown.

 
Then the effect of IGF-I on the cellular distribution of Fyn was investigated. In R-IGF-IRWT cells, Fyn was barely detectable in total cell lysates (data not shown). After IGF-I stimulation, a significant enrichment of Fyn in the caveolae, with a peak at 15 min, was detected (Fig. 6CGo). Moreover, Fyn translocation was exclusively IGF-IR dependent. In fact, in R- mock cells, IGF-I did not induce Fyn enrichment in caveolae (data not shown).



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  		FIG. 6. IGF-I induces Fyn recruitment to the lipid raft-enriched fractions in R-IGF-IRWT. Serum-starved R-IGF-IRWT cells (A) were stimulated with IGF-I (20 nM) for 5 (B) and 15 (C) min. Twenty-five microliters from each fraction, obtained by ultracentrifugation on discontinuous sucrose gradient, were separated on 12% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with an anti-Fyn antibody. On the left of each panel, molecular weight markers are shown. The immunoblots presented are representative of three separate experiments.

 
To investigate whether IGF-IRs and Fyn colocalize upon IGF-I stimulation, double-immunofluorescence experiments were performed in permeabilized R-IGF-IRWT cells and compared with R- mock cells using anti-Fyn and anti-IGF-IR antibodies. In the absence of IGF-I, Fyn staining was absent on plasma membrane (Fig. 7AGo), whereas in the presence of the IGF-I (20 nM), we detected Fyn aggregation in the caveolae of R-IGF-IRWT cells, with a peak at 15 min (Fig. 7Go, B and C). At 15 min, the staining obtained with anti IGF-IR ß-subunit antibody is superimposable (Fig. 7CGo), suggesting an IGF-I-induced colocalization of Fyn and IGF-IR. As expected, Fyn aggregation is absent in R- mock cells (data not shown). These results suggest that this effect is IGF-IR dependent. Moreover, to confirm Fyn enrichment in lipid rafts in the presence of IGF-I, we performed double immunofluorescence using a glycosylphosphatidylinositol-anchored protein, PLAP, that is highly enriched in the detergent-resistant microdomains (23). Permeabilized R-IGF-IRWT and R- mock cells were immunostained with either anti-PLAP or anti-Fyn antibodies in the absence or presence of IGF-I (20 nM) for 5 and 15 min. As demonstrated above, staining of Fyn was very weak in the absence of IGF-I. On the contrary, after 5 and 15 min of IGF-I stimulation, staining was increased, and almost all cells aggregated and colocalized with PLAP (Fig. 7Go, D–F), confirming the role of IGF-I to induce Fyn enrichment in plasma membrane and its colocalization with IGF-IR.



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  		FIG. 7. Fyn, in the presence of IGF-I, forms membrane patches and colocalizes with PLAP and IGF-IR. R-IGF-IRWT cells were plated on glass coverslips and further cultured for 2 d. Serum-starved cells (A and D) were stimulated with IGF-I at 37 C for 5 (B and E) and 15 (C and F) min. Then cells were fixed, permeabilized, double-immunostained with anti-Fyn (green) and anti-IGF-IR ß-subunit (red) antibodies, and detected by antimouse FITC conjugate and antirabbit TRITC conjugate (A–C). Cells processed as described above were double-immunostained with anti-PLAP (green) and anti-Fyn (red) antibodies and detected by antirabbit FITC conjugate and antimouse TRITC conjugate antibodies (D–F).

 
IR and IGF-IR share common intracellular substrates, but show different biological effects. It has been shown that the time courses of Shc and IRS protein phosphorylation are significantly different between insulin- and IGF-I-treated cells (22). These results suggest that a different early compartmentalization of IR and IGF-IR signaling could regulate the specific effects of these receptors. Then we measured the time course of insulin-induced Shc phosphorylation in the caveolae of R-IRWT cells. As predicted, insulin determined Shc tyrosine phosphorylation in these microdomains (Fig. 8Go). However, the time course of this effect was significantly different from that of IGF-I in R-IGF-IRWT cells. In fact, it was faster, reaching a peak in 5 min. Grb2 was also recruited to the caveolae in the R-IRWT cells, with a time course superimposable with that of Shc phosphorylation in these regions (Fig. 9Go).



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  		FIG. 8. Effect of insulin on Shc tyrosine phosphorylation in the caveolae of R-IRWT cells. Upper panel, Serum-starved R-IRWT cells (A) were stimulated with insulin (20 nM) for 5 (B) and 15 (C) min and lysed in 0.5 M Na2CO3 (pH 11). A total of 12 fractions were collected from the top of each gradient after ultracentrifugation of total cell lysates. Twenty-five microliters from each fraction were separated on 12% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with an antiphosphotyrosine antibody. On the left of each panel, molecular weight markers are shown. The immunoblots presented are representative of three separate experiments. Lower panel, Bands of interest were quantified by densitometry using NIH Image software, and results were expressed as the tyrosine-phosphorylated Shc (PY-Shc)/total Shc ratio in the lipid rafts/caveolae (continuous line) and nonraft cellular fractions (dotted line).

 


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  		FIG. 9. Insulin induced Grb2 enrichment in the caveolae of R-IRWT cells. Total cell lysates were obtained from serum-starved R-IRWT cells (A) stimulated with insulin for 5 (B) and 15 (C) min. The experiments were conducted as described in Fig. 4Go. The data presented are representative of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Caveolae are a plasma membrane compartment that plays a role in the intracellular trafficking of signaling proteins, possibly by recruiting or excluding membrane receptors and their intracellular substrates in a time-dependent manner (1). Both IR and IGF-IR, as well as their intracellular substrates, localize in the caveolae and tyrosine phosphorylate caveolin 1. It has been shown that caveolae regulate both insulin and IGF-I signaling (5, 6, 7, 8, 9, 10, 11, 12, 13). Previously, we have shown that IGF-I, but not insulin, induces phosphocaveolin 1 redistribution in the lipid raft microdomains and formation of membrane patches (11), regions highly enriched in tyrosine-phosphorylated proteins (24). This finding suggests that caveolin 1 function is differentially regulated by IR or IGF-IR. Shc is expressed as three different isoforms (25). Here we have shown that Shc is highly enriched in the caveolae of R-IGF-IRWT cells, and p52 is the most represented isoform. Several data have suggested a specific role for p52 Shc in the IGF-I action: 1) p52 is the main tyrosine-phosphorylated Shc isoform in IGF-I-induced 3T3-L1 cell proliferation (26); 2) in the IGF-IR knockout mouse, p52-Grb2 interaction and MAPK pathway activation are greatly impaired (27); 3) the transfection of a p52 Shc-dominant mutant blocks IGF-I-induced Shc phosphorylation and impairs IGF-I mitogenic action in SH-SY5Y cells (28). Our results on p52 enrichment in the caveolae further extend these data, suggesting a specific role for p52 tyrosine phosphorylation in this compartment. After IGF-I binds, IGF-IRs autophosphorylate and, in turn, tyrosine phosphorylate intracellular substrates, such as insulin receptor substrate proteins and Shc (29). Based on the evidence that IGF-IRs are highly enriched in the caveolae (11, 12, 13), these regions could represent a specific compartment in substrate activation. Shc phosphorylation in the caveolae shows a specific time course. In fact, IGF-I caused Shc phosphorylation in the lipid raft caveolin 1-enriched fractions, with a peak at 15 min, whereas in nonraft fractions 9–12, this effect was detectable after 5 min. IGF-I did not cause a significant enrichment of total Shc in caveolae, suggesting the presence of a resident pool of this protein already available for IGF-I signaling. Tyrosine-phosphorylated Shc binds Grb2, and this effect is necessary for full activation of the Ras-MAPK pathway by IGF-I. In R-IGF-IRWT cells, IGF-I caused the recruitment of Grb2 to the caveolae with a time superimposable to the Shc phosphorylation time course. This effect was IGF-IR dependent. In fact, it was absent in mock-transfected R- cells. Also, p42/p44 MAPKs are phosphorylated in the caveolae upon IGF-I stimulation with a time course that strongly correlates with Shc phosphorylation and Grb2 recruitment in this compartment, further supporting a role for caveolae in IGF-IR signal transduction, probably through Ras activation. In fact, a subset of cellular Ras associates with caveolae (30). Thus it could be hypothesized that Shc phosphorylation in these microdomains activates a specific Ras-MAPK pathway, further compartmentalizing IGF-I action.

Insulin causes Shc phosphorylation in lipid raft caveolin 1-enriched fractions of R-IRWT cells, but the time course of this effect is significantly different from that observed in IGF-I-stimulated R-IGF-IRWT cells. In fact, insulin caused Shc phosphorylation as well as Grb2 recruitment in the caveolae, with a peak in 5 min.

IGF-IR binds c-Src protein kinase family members, such as Fyn and Src, in a ligand-dependent manner (15). The IGF-IR is constitutively phosphorylated in c-Src-transformed cells, and the Src-mediated transformation of mouse fibroblasts is IGF-IR dependent (16). More recently, a role for Src and Fyn has been determined in the 3T3-L1 differentiation to adipocytes induced by IGF-I through an Shc-dependent mechanism (18). Caveolin 1 is tyrosine phosphorylated in Src-transformed cells (31, 32). Therefore, Src family kinases could regulate IGF-I action by Shc tyrosine phosphorylation in caveolae, where caveolin 1 could act as an adaptor protein in a phosphotyrosine-dependent manner. In the integrin {alpha}-subunit activation, caveolin 1 allows Fyn-dependent Shc tyrosine phosphorylation that, in turn, recruits Grb2 (33, 34). By double immunofluorescence and cell fractionation, we observed that IGF-IR and Fyn colocalize in a PLAP-enriched fraction of plasma membrane in the presence of IGF-I. This effect reached a maximum after 15 min, as did Shc phosphorylation and Grb2 recruitment induced by IGF-I. Fyn translocation was IGF-IR dependent; in fact, it was absent in R- mock cells (data not shown). At least two hypotheses could be proposed to explain the mechanism of Shc activation by Fyn. IGF-I could directly phosphorylate Shc, thus releasing Fyn and activating its kinase activity. On the other hand, activated IGF-IRs could directly interact with Fyn, as previously shown for c-Src (17), and phosphorylate Shc. It has been shown that 4-amino-1-tert-butyl-3-(1'-naphthyl)pyrazole[3,4-d] pyrimidine (PP1), a highly specific inhibitor of Src kinase activity, impairs p52 Shc tyrosine phosphorylation and MAPK activation induced by IGF-I (18). In R-IGF-IRWT cells, PP2, a PP1-related Src kinase inhibitor compound (35), completely inhibits IGF-I induced Grb2 recruitment in the caveolae (Biedi, C., manuscript in preparation).

Interestingly, the time course of IGF-I-induced Shc phosphorylation in the caveolae seems to correlate with the time course of the IGF-I-IGF-IR complex internalization, which is slower than that of insulin-IR complex endocytosis (36). Moreover, it has been shown that the time course of insulin and IGF-I intracellular substrate activation correlates with the receptor internalization rate (22, 37).

In conclusion, we have shown that IGF-I determines the clustering of signaling proteins in caveolae in a phosphorylation-dependent manner. The different time courses of substrate phosphorylation and recruitment by IGF-IR and IR could regulate the early compartmentalization of IGF-I and the insulin signaling pathway and regulate the specificity of these hormones.


   Acknowledgments
 
We thank Mrs. Maria Rosa Dagnino for the skillful administrative assistance.


   Footnotes
 
This work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC), Italian Ministry for University and Research (to R.C.), Università di Genova.

Abbreviations: ECL, Enhanced chemiluminescence; Grb2, growth factor receptor bound protein 2; IGF-IR, IGF-I receptor; IR, insulin receptor; PLAP, placental alkaline phosphatase; R-IGF-IRWT, R- fibroblasts that overexpress wild-type IGF-IR; Shc, Src homology 2/{alpha}-collagen related protein; Sos, son of sevenless; TBS, Tris-buffered saline; TRITC, tetramethyl rhodamine isothiocyanate.

Received April 3, 2003.

Accepted for publication August 12, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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