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Address correspondence to Jeffrey E. Pessin, Department of Physiology and Biophysics, University of Iowa, Iowa City, IA 52242. Tel.: (319) 335-7823. Fax: (319) 335-7886. E-mail: jeffrey-pessin{at}uiowa.edu
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Abstract |
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Key Words: TC10; GLUT4; insulin; lipid rafts; compartmentalization
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Introduction |
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Of the many functions of insulin, one of the most important is to increase glucose uptake in striated muscle and adipose tissues (Czech, 1995; Summers et al., 1999). In the basal state, the insulin-responsive glucose transporter (GLUT)4* cycles slowly between the plasma membrane and one or more intracellular compartments, with the vast majority of the transporter residing within the cell interior (Martin et al., 1996a; Millar et al., 1999; Simpson et al., 2001). Insulin triggers a large increase in the rate of GLUT4 vesicle exocytosis, resulting in the accumulation of the transporter on the cell surface and a concomitant increase in glucose uptake (Rea and James, 1997; Pessin et al., 1999).
It has been well established that phosphatidylinositol 3 (PI-3) kinase activity is necessary for insulin-stimulated GLUT4 translocation (Cheatham et al., 1994; Okada et al., 1994; Martin et al., 1996b; Sharma et al., 1998; Czech, 2000). However, a clear demonstration for a sufficient role for PI-3 kinase has not been forthcoming. Moreover, several lines of evidence suggest that one or more PI-3 kinaseindependent signals may be required for insulin-stimulated GLUT4 translocation. For example, two naturally occurring insulin receptor mutations were unable to induce GLUT4 translocation and glucose uptake, yet were fully capable of activating PI-3 kinase (Krook et al., 1997). Activation of PI-3 kinase activity through the interleukin 4 receptor or engagement of integrin receptors also failed to enhance glucose uptake and GLUT4 translocation (Isakoff et al., 1995; Guilherme and Czech, 1998). Furthermore, a cell-permeable PI(3,4,5)P3 analogue had no effect on glucose uptake (Jiang et al., 1998). Together, these data suggest that additional insulin signaling pathways may exist that function independently of the pathway defined by the PI-3 kinase.
The Cbl protooncogene is specifically tyrosine phosphorylated by the insulin receptor in adipocytes through the adapter proteins Cbl-associated protein (CAP) and APS (Ribon et al., 1998; Ahmed et al., 2000). CAP directly interacts with Cbl and appears to be important in insulin signaling, since expression of a dominant-interfering CAP mutant (CAPSH3) markedly inhibited insulin-stimulated glucose uptake and GLUT4 translocation (Baumann et al., 2000). More recently, we have observed that the CAP/Cbl pathway is necessary for the activation of TC10, a Rho family GTPase that is highly expressed in muscle and adipose tissues (Neudauer et al., 1998; Imagawa et al., 1999; Chiang et al., 2001). Here we demonstrate that TC10 localizes to caveolin-enriched lipid microdomains at the plasma membrane. Furthermore, the compartmentalization of TC10 within lipid microdomains is essential for insulin-dependent activation of TC10 and GLUT4 translocation. These data provide a molecular basis for the specificity of insulin signaling with respect to glucose transport.
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Results |
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Syntaxin 6 is a resident protein of the TGN (Bock et al., 1996; Watson and Pessin, 2000). In contrast to the results obtained above with p115, TC10 showed substantial perinuclear overlap with syntaxin 6 in both control cells and in cells treated with BFA (Fig. 1 B, af). Nocodazole treatment resulted in a punctate distribution of syntaxin 6 and the accumulation of TC10 at the cell surface (Fig. 1 B, gi). The transferrin receptor (TfR) cycles between the cell surface and endosome compartments, where it often shows strong perinuclear labeling (Fig. 1 C, a). In control and BFA-treated cells, TfR also displayed a strong overlap with TC10 (Fig. 1 C, af). As described above, in the presence of nocodazole TC10 was found predominantly at the plasma membrane, where it overlapped with TfR (Fig. 1 C, gi). Together, these results indicate that TC10 is largely present in the plasma membrane and the perinuclear-recycling endosome compartment, although we can not unambiguously distinguish between recycling endosomes and the TGN in adipocytes.
Since TC10 is predicted to be both farnesylated and palmitoylated at the COOH-terminus (Fig. 2 A), TC10 most likely transits through the secretory membrane system en route to the plasma membrane, in an identical manner to that established for H-Ras and several other proteins that undergo posttranslational prenylation and palmitoylation (Choy et al., 1999; Resh, 1999; Apolloni et al., 2000; Michaelson et al., 2001). Indeed, the TC10 expression pattern was essentially indistinguishable from the subcellular distribution of H-Ras, both in control cells and cells treated with BFA or nocodazole (Fig. 1 D, ai). Furthermore, incubation of cells with cycloheximide subsequent to transfection resulted in the rapid and identical chase (<4 h) of the entire endomembrane pool of both H-Ras and TC10 to the plasma membrane (data not shown). Thus, TC10 appears to be processed through the secretory membrane system and perinuclearrecycling endosomes during its transit to the plasma membrane in a manner similar to that described recently for H-Ras (Prior and Hancock, 2001).
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To determine whether the inhibitory property of TC10 was dependent on subdomain compartmentalization, we next cotransfected the TC10/H-Ras or TC10/K-Ras chimeras with GLUT4-EGFP. Insulin stimulation resulted in the appearance of a strong plasma membrane GLUT4-EGFP rim fluorescence, indicative of translocation (Fig. 2 C, a and b). Similar to our previous findings, coexpression of wild-type TC10 strongly inhibited GLUT4 translocation, whereas H-Ras and K-Ras were without any significant effect (Fig. 2 C, ch). Although the TC10/H-Ras chimera potently inhibited GLUT4-EGFP translocation in a manner identical to wild-type TC10, the TC10/K-Ras chimera behaved like K-Ras and had no effect on GLUT4-EGFP translocation (Fig. 2 C, il). These data were quantitated by determining the number of cells displaying a continuous cell surface GLUT4-EGFP fluorescence (Fig. 2 D).
We next determined whether TC10 subdomain compartmentalization was important for its activation by insulin (Fig. 3) . Using a GST-Pak1 pull down assay to precipitate the GTP-bound TC10 protein, we observed that insulin produced a time-dependent activation of the expressed wild-type TC10 protein (Fig. 3 A, lanes 15). Similarly, insulin activated the TC10/H-Ras chimera over the same time frame (Fig. 3 B, lanes 15). However, insulin was unable to activate the TC10/K-Ras chimera under the identical conditions (Fig. 3 C, lanes 15). In parallel, immunoblots of cell lysates demonstrated equal amounts of TC10 expression under all these conditions. Quantitation of these data demonstrated that insulin activated TC10/WT and TC10/H-Ras 1.7 ± 0.4- and 1.7 ± 0.3-fold, respectively. In contrast, TC10/K-Ras was not significantly activated by insulin (0.9 ± 0.1). Thus, these data demonstrate that the upstream pathway required for insulin-dependent activation of TC10 is also confined to the same specific lipid raft microdomain defined by the COOH-terminal H-Ras and TC10 targeting sequences.
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Discussion |
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Although the Switch I and Switch II domains of TC10 have a high degree of sequence similarity with Rac, Rho, and Cdc42, in other respects TC10 is an unusual member of the Rho protein family. In particular, most Rho family members contain a single COOH cysteine residue in the appropriate sequence context for geranylgeranylation and interact with guanylnucleotide dissociation inhibitors (Nobes and Hall, 1995; Bar-Sagi and Hall, 2000). In contrast, the COOH-terminal region of TC10 contains sequences similar to that of H-Ras, encoding for both farnesylation and palmitoylation (Hancock et al., 1989; Seabra, 1998). Recent studies have established that these COOH-terminal posttranslational modifications provide key trafficking determinants that may underlie signaling specificity (Sternberg and Schmid, 1999). For example, after farnesylation, proteolytic trimming, and carboxylmethylation, the subsequent palmitoylation of H-Ras allows for its insertion and trafficking through the secretory membrane system (Choy et al., 1999; Apolloni et al., 2000). This trafficking pattern ultimately results in the specific targeting of H-Ras to the caveolin-enriched plasma membrane microdomains (Dupree et al., 1993; Li et al., 1996; Song et al., 1996). In contrast, COOH-terminal K-Ras sequence does not contain any upstream palmitoylated cysteines, but instead has a polybasic motif that prevents its interaction with the Golgi apparatus and directs its association with the noncaveolin regions of the plasma membrane (Hancock et al., 1990; Choy et al., 1999; Roy et al., 1999; Apolloni et al., 2000).
The data presented here demonstrate that TC10 traffics in a manner analogous to that of H-Ras. Like H-Ras, TC10 appears to transiently associate with the secretory membrane system while en route to the plasma membrane and cycle through the perinuclear-recycling endosome compartment. This was based on the ability of cycloheximide to efficiently chase TC10 out of the endomembrane system, the strong colocalization of TC10 with the transferrin receptor, and the complete loss of perinuclear localization in the presence of nocodazole. It should be noted that the apparent absence of TC10 in the perinuclear region after nocodazole treatment could reflect either a rapid exit out of the Golgi apparatus or the presence of an alternative trafficking pathway to the plasma membrane, as has been reported for Ras2 in yeast (Bartels et al., 1999). Nevertheless, in adipocytes, TC10 and H-Ras displayed indistinguishable intracellular distributions and trafficking patterns that are similar to that recently reported for TC10 in fibroblasts (Michaelson et al., 2001).
At present, the downstream effector proteins that link activated TC10 with GLUT4 translocation remain undefined. Nevertheless, the data presented here clearly demonstrate that the specific COOH-terminal targeting sequences of TC10 are essential for the insulin-dependent activation of TC10 and regulation of GLUT4 translocation. Indeed, chimeric proteins in which the 19 COOH-terminal residues of TC10 were replaced with the corresponding amino acids from H-Ras (TC10/H-Ras) resulted in functional targeting in terms of insulin-stimulated TC10 activation and inhibition of GLUT4 translocation. In contrast, the corresponding TC10/K-Ras chimera was completely refractory to insulin stimulation and had no significant inhibitory effect on GLUT4 translocation. Although we cannot completely rule out the possibility that TC10 may function within endomembrane structures, the ability of the TC10/H-Ras chimera to recapitulate wild-type TC10 function is more consistent with a model wherein TC10 operates within lipid microdomain compartments at the plasma membrane. This conclusion is further supported by the strong colocalization of TC10 and the TC10/H-Ras chimera with plasma membrane caveolae and the fact that the TC10/K-Ras chimera was specifically excluded from this plasma membrane subdomain compartment. In addition, we have demonstrated previously that the insulin-stimulated assembly of the signaling complex that activates TC10 is also localized to plasma membrane lipid raft microdomains (Chiang et al., 2001).
One interesting morphological feature of the adipocyte plasma membrane is the presence of both multiple individual caveolae and clusters of caveolae organized into large ring-shaped structures that are visualized at both the electron microscopic and light microscopy levels (Chlapowski et al., 1983; Severs, 1988; Voldstedlund et al., 1993; Gustavsson et al., 1999). Previous studies have observed that insulin-stimulated glucose uptake is inhibited by ß-CD (Gustavsson et al., 1999; Parpal et al., 2001) and our data demonstrate clearly that these higher-order caveolin and TC10 aggregates are sensitive to cholesterol extraction. Consistent with a necessary role for lipid raft microdomains, expression of the dominant-interfering caveolin 3 mutant (Cav3/DGV) inhibited insulin-stimulated GLUT4 translocation and activation of TC10. Thus, these data demonstrate that lipid raft compartmentalization is necessary for TC10 function with respect to insulin-stimulated GLUT4 translocation. Importantly, these data also demonstrate that the compartmentalized insulin signals leading to TC10 activation are independent from those regulating activation of the PI-3 kinase pathway.
One issue inherent to our understanding of intracellular signaling is the mechanism by which distinct cellular responses can result from the engagement and activation of similar arrays of downstream effector proteins. Numerous models have been proposed to account for this phenomenon, including the strength of signal generation, combinatorial diversity of different effector subsets, and/or differences in the temporal and spatial activation of downstream targets. In the case of insulin action, this problem has been complicated by numerous studies demonstrating a variety of signals capable of activating the PI-3 kinase pathway, yet unable to stimulate GLUT4 translocation or glucose transport (Weise et al., 1995; Krook et al., 1997; Guilherme and Czech, 1998; Jiang et al., 1998). In contrast, the activation of TC10 by insulin in adipocytes is not reproduced by other growth factors and requires the assembly of established signaling components in a spatially restricted subcompartment of the plasma membrane. Thus, insulin receptor signaling specificity, at least for GLUT4 translocation, appears to result from a combination of both general activation of a PI-3 kinase signal in conjunction with a spatially restricted and cell type-specific effector activation.
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Materials and methods |
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Cell culture and transient transfection of 3T3L1 adipocytes
Murine 3T3L1 preadipocytes were purchased from the American Type Tissue Culture Collection. Cells were cultured in DME supplemented with 25 mM glucose and 10% calf serum at 37°C with 8% C02. Cells were differentiated and transfected by electroporation as described previously (Watson and Pessin, 2000). The cells were then plated on glass coverslips and stimulated with 100 nM insulin for 30 min.
Immunofluorescence and image analysis
Transfected adipocytes were washed in PBS and fixed for 15 min in 4% paraformaldehyde containing 0.2% Triton X-100, washed in PBS, and blocked in 5% donkey serum plus 1% BSA (both from Sigma-Aldrich) for 1 h. Primary and secondary antibodies were used at 1:100 dilutions (unless otherwise indicated) in blocking solution and samples were mounted on glass slides with Vectashield (Vector Laboratories). Cells were imaged using confocal fluorescence microscopy. Images were then imported into Adobe Photoshop® (Adobe Systems, Inc.) for processing and composite files were generated.
Preparation and processing of plasma membrane sheets
Adipocyte plasma membrane sheets were prepared as described previously (Robinson et al., 1992). The membrane sheets were fixed in 2% paraformaldehyde at room temperature for 20 min and blocked with 5% donkey serum. Membrane sheets were incubated with rabbit polyclonal TC10 antibody (10 µg/ml) and caveolin 2 monoclonal antibody (1:10) for 90 min at 37°C. Primary antibodies were detected with Texas red dyeconjugated donkey antimouse antibody and FITC-conjugated donkey antirabbit antibody for 2 h at room temperature. To detect expressed TC10 and TC10/Ras chimeras in plasma membrane sheets, the electroporated cells were plated on collagen-coated coverslips and allowed to recover for 18 h in complete media. Cells were then osmotically swelled in hypotonic buffer and fixed in 2% paraformaldehyde/PBS for 10 min before sonication. Membrane sheets were then processed as described above and the expressed TC10 constructs were detected with an HA antibody (1:100) and a caveolin 1 antibody (1:250).
Cholesterol extraction
Methyl-ß-cyclodextrin was added directly to serum-free DME at a final concentration of 10 mM and the cells were incubated at 37°C for various times. Plasma membrane sheets were prepared as described above.
Electron microscopy
Plasma membrane sheets were prepared as described above except that they were fixed with a combination of 4% paraformaldehyde and 0.05% glutaraldehyde for 30 min. The samples were then incubated with a monoclonal caveolin 1 antibody followed by a 10-nm gold-conjugated rabbit antimouse antibody. The sheets were then extensively washed and fixed a second time with 2.5% glutaraldehyde and 0.1 M sodium cacodylate, pH 7.2, followed by staining with 1% osmium tetraoxide, 1.5% potassium ferrocyanide, and 0.1 M sodium cacodylate, pH 7.2. The plasma membrane sheets were dehydrated by serial extracts with ethanol, embedded in eponate-12, sliced on a microtome, and visualized by transmission electron microscopy.
TC10/Ras chimeric constructs and DNA cloning
The TC10/H-Ras and TC10/K-Ras chimeras were generated using the PCR-based overlap extension method as described (Horton et al., 1993). In brief, the COOH-terminal 19 amino acids of TC10 (KHTVKKRIGSRCINCCLIT) were replaced with either the final 19 amino acids of H-Ras (LNPPDESGPGCMSCKCVLS) or the final 19 amino acids of K-Ras (MSKDGKKK-KKKSKTKCVIM). The mouse caveolin-3 cDNA was purchased from the American Type Culture Collection and subcloned into the pcDNA3.1/Myc-HIS vector (Invitrogen). To generate the Cav3/DGV mutant, PCR was used to remove the NH2-terminal 54 amino acids from caveolin 3 (Roy et al., 1999).
Affinity precipitation of TC10 using GST-Pak1/CRIB domain
Precipitation of activated (GTP-bound) TC10 using the GST-Pak1/CRIB domain fusion protein was performed as described previously (Baumann et al., 2000). In brief, cell lysates in a volume of 100 µl were incubated for 1 h at 4°C with 200 µl of binding buffer (25 mM Tris, pH 7.5, 1 mM DTT, 30 mM MgCl2, 40 mM NaCl, 0.5% NP-40) in the presence of 8 µg of GST-Pak1/CRIB coupled to glutathione-Sepharose 4B beads. The beads were then washed three times with 25 mM Tris, pH 7.5, 1 mM DTT, 30 mM MgCl2, 40 mM NaCl, 1% NP-40 and once with the same buffer without NP-40. The beads were suspended in 20 µl Laemmli sample buffer. Proteins were separated by 420% SDS-PAGE, transferred to nitrocellulose membrane and blotted with a HA monoclonal antibody, and detected by chemiluminescence (New England Nuclear).
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Footnotes |
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Acknowledgments |
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Submitted: 15 February 2001
Revised: 11 July 2001
Accepted: 12 July 2001
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References |
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