Caveolin-1 Interacts with the Insulin Receptor and Can Differentially

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Caveolae may function as microdomains for signal- ing that help to determine specific biological actions mediated by the insulin receptor (IR). Caveolin-1, a.
Caveolin-1 Interacts with the Insulin Receptor and Can Differentially Modulate Insulin Signaling in Transfected Cos-7 Cells and Rat Adipose Cells

Fredrik H. Nystrom, Hui Chen, Li-Na Cong, Yunhua Li, and Michael J. Quon Hypertension-Endocrine Branch National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland 20892

Caveolae may function as microdomains for signaling that help to determine specific biological actions mediated by the insulin receptor (IR). Caveolin-1, a major component of caveolae, contains a scaffolding domain (SD) that binds to a caveolin-1 binding motif in the kinase domain of the IR in vitro. To investigate the potential role of caveolin-1 in insulin signaling we overexpressed wild-type (Cav-WT) or mutant (CavMut; F92A/V94A in SD) caveolin-1 in either Cos-7 cells cotransfected with IR or rat adipose cells (low and high levels of endogenous caveolin-1, respectively). Cav-WT coimmunoprecipitated with the IR to a much greater extent than Cav-Mut, suggesting that the SD is important for interactions between caveolin-1 and the IR in intact cells. We also constructed several IR mutants with a disrupted caveolin-1 binding motif and found that these mutants were poorly expressed and did not undergo autophosphorylation. Interestingly, overexpression of Cav-WT in Cos-7 cells significantly enhanced insulin-stimulated phosphorylation of Elk-1 (a mitogen-activated protein kinase-dependent pathway) while overexpression of Cav-Mut was without effect. In contrast, in adipose cells, overexpression of either Cav-WT or Cav-Mut did not affect insulin-stimulated phosphorylation of a cotransfected ERK2 (but did significantly inhibit basal phosphorylation of ERK2). Furthermore, we also observed a small inhibition of insulin-stimulated translocation of GLUT4 when either Cav-WT or CavMut was overexpressed in adipose cells. Thus, interaction of caveolin-1 with IRs may differentially modulate insulin signaling to enhance insulin action in Cos-7 cells but inhibit insulin’s effects in adipose cells. (Molecular Endocrinology 13: 2013–2024, 1999)

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INTRODUCTION The insulin receptor is a member of a large family of ligand-activated tyrosine kinases that share a number of postreceptor signaling pathways in common (1–3). Thus, it is of interest to elucidate mechanisms contributing to signal specificity after insulin activates its receptor. Caveolae are flask-shaped invaginations of the plasma membrane that are abundant in insulin targets such as muscle and adipose cells and that may contribute to specificity by creating microdomains for insulin signaling (4–6). Caveolins are scaffold-like proteins that form homo- and heterooligomers that are principal components of caveolae (7, 8). Indeed, overexpression of caveolin-1 is sufficient to drive formation of caveolae (9). Interestingly, insulin receptors are enriched in caveolae (5, 10), and the expression of caveolin, as well as the number of caveolae, increases dramatically when 3T3-L1 fibroblasts differentiate into adipocytes (11, 12). Furthermore, phosphorylation of caveolin in response to insulin stimulation occurs only in differentiated 3T3-L1 adipocytes but not in undifferentiated fibroblasts, suggesting that caveolin may play a role in specific metabolic insulin-signaling pathways present in adipocytes (13, 14). Previous peptide binding studies have shown that the scaffolding domain of caveolin-1 (residues 82–101) binds to a FXFXXXXF motif in the tyrosine kinase domain of the insulin receptor b-subunit (where F represents aromatic amino acids at positions 1193, 1195, and 1200 of the insulin receptor) (15). In addition, scaffolding domain peptides derived from caveolin-1 or -3 can increase tyrosine kinase activity of the insulin receptor (10). Some patients with syndromes of extreme insulin resistance have mutations of the insulin receptor in which this caveolin binding motif is disrupted (e.g. W1193L and W1200S) (16–21). The W1193L mutant undergoes accelerated degradation resulting in decreased cell surface expression of receptors and also has an impair2013

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Fig. 1. Overexpression of Caveolin-1 in Rat Adipose Cells and Cos-7 Cells Cells were transiently transfected with a control plasmid (C 5 pCIS2 for adipose cells and pCIS-eGFP for Cos-7 cells), or expression vectors for myc-tagged caveolin-1 (Cav-WT or Cav-Mut). For Cos-7 cells, 1 mg DNA/well was used while for adipose cells 4 mg DNA/cuvette were used. Cell lysates derived from each group (30 mg total protein) were subjected to immunoblotting with an anticaveolin-1 antibody. Recombinant myc-tagged caveolin-1 migrated more slowly on the gel than endogenous caveolin. Transfection efficiencies were approximately 5% for adipose cells and approximately 20% for Cos-7 cells.

ment in autophosphorylation (18–21). Although the W1200S mutant seems to be expressed at higher levels on the cell surface than W1193L, autophosphorylation of this mutant is severely impaired as well (16, 17). Taken together, both in vitro and in vivo studies suggest that caveolin may interact with the insulin receptor and play an important role in insulin signaling. However, the functional consequences of interactions between caveolin and insulin receptors are not well understood. In the present study, we used transient transfection techniques to overexpress wild-type and mutant forms of caveolin-1 in Cos-7 cells (low levels of endogenous caveolin-1) and rat adipose cells (high levels of endogenous caveolin-1) to investigate the role of caveolin-1 in insulin signaling. In addition, we constructed and characterized several insulin receptor mutants with a disrupted caveolin-1 binding motif. We demonstrate that the interaction between insulin receptors and caveolin-1 in intact cells is dependent upon an intact caveolin-1 scaffolding domain. Intriguingly, we found that overexpression of caveolin-1 enhanced insulin signaling in Cos-7 cells while inhibiting insulin action in adipose cells.

RESULTS Overexpression of Caveolin-1 in Cos-7 Cells and Rat Adipose Cells To assess overexpression of recombinant caveolin-1 constructs, lysates derived from transiently transfected Cos-7 cells and rat adipose cells were immunoblotted with an antibody against caveolin-1 (Fig. 1). Recombinant caveolin-1 could be distinguished from the endogenous protein because myc-tagged caveolin-1 migrated more slowly on the gel. We detected abundant endogenous caveolin-1 in adipose cells while very little of the endogenous protein was observed in Cos-7 cells. Under our experimental conditions, transfection efficiencies were approximately 5% for adipose cells (22) and approximately 30% for Cos-7 cells as determined by expression of green fluorescent protein (data not shown). Therefore, by

comparing the density of bands representing endogenous and recombinant caveolin-1, we estimated levels of overexpression of approximately 20-fold in both Cos-7 cells and adipose cells. A mutant caveolin-1 construct (Cav-Mut) containing F92A and V94A point mutations in the scaffolding domain of caveolin-1 was expressed at levels comparable to the wild-type construct (Cav-WT). Characterization of Insulin Receptor Constructs We designed a number of insulin receptor mutants to disrupt the FXFXXXXF caveolin-1 binding motif present in the kinase domain of the receptor b-subunit to evaluate potential interactions between caveolin-1 and the insulin receptor. In the W1200T mutant, the distal aromatic amino acid in the binding motif has been changed to threonine, a residue found in a homologous region of c-src (1). The F1195G mutation disrupts the binding motif at the central aromatic residue while the triple mutation (W1193G/F1195G/ W1200G) replaces all three aromatic amino acids with glycine. The K1030A mutant is a kinase-inactive receptor resulting from disruption of the canonical ATP binding site. High level expression of the mature 95-kDa insulin receptor b-subunit was observed in lysates derived from Cos-7 cells transfected with either the wild-type or K1030A receptors (Fig. 2A). However, expression levels for the caveolin-binding motif mutants were substantially lower. Of the three binding motif mutants, the highest level of expression was observed with W1200T. Interestingly, levels of expression for the 210-kDa proinsulin receptor were similar for all of the insulin receptor constructs evaluated. Since the extracellular a-subunit was not altered in any of our constructs, we performed [125I]insulin binding studies in parallel with our immunoblotting experiments to determine the relative numbers of recombinant receptors present on the cell surface. The results of our binding studies were consistent with our immunoblotting experiments (Fig. 2, A and C). That is, the relative number of insulin receptors present on the cell surface was proportional to the amount of 95-kDa b-subunit de-

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Fig. 2. Overexpression of Insulin Receptor Constructs in Cos-7 Cells Cells were transiently transfected with a control plasmid (pCIS-eGFP) or expression vectors for wild-type (WT) or various mutant human insulin receptor constructs (1 mg DNA/well). W1200T, F1195G, and the triple-mutant (W1193G/F1195G/W1200G) are point mutants in the insulin receptor b-subunit that disrupt the caveolin-1 binding motif. The K1030A mutant is a kinaseinactive receptor. Some cells were used for [125I]insulin binding studies while other cells were treated without or with insulin (100 nM, 2 min) and cell lysates were subjected to SDS-PAGE followed by immunoblotting. A, Antiinsulin receptor b-subunit immunoblot. B, Antiphosphotyrosine immunoblot. The wild-type insulin receptor b-subunit undergoes autophosphorylation in response to insulin stimulation while the various mutant insulin receptors do not. C, Cell surface tracer [125 I]insulin binding at 4 C. Data shown are the mean 6 SEM of triplicate determinations from a single experiment using samples that correspond to the experiment shown in panel B. These experiments were repeated independently at least five times.

tected by immunoblotting. When we immunoblotted cell lysates from experiments shown in Fig. 2A with an antiphosphotyrosine antibody we observed normal autophosphorylation of the wild-type insulin receptor, but we could not detect significant autophosphorylation with any of the mutant insulin receptors (Fig. 2B). Interaction between Caveolin-1 and Insulin Receptors in Intact Cells To determine whether interactions between caveolin-1 and insulin receptors can occur in intact cells, we performed coimmunoprecipitation experiments using lysates derived from Cos-7 cells transiently cotrans-

fected with both myc-tagged caveolin-1 and insulin receptors (Fig. 3). In these cells, Cav-WT was easily detected in insulin receptor immunoprecipitates (Fig. 3, lane 3). In contrast, the interaction of the scaffolding-domain mutant (Cav-Mut) with the wild-type insulin receptor appeared to be significantly reduced (Fig. 3, lane 4). Interestingly, significant coimmunoprecipitation of Cav-WT with the W1200T insulin receptor mutant was observed even though this receptor has a disrupted FXFXXXXF caveolin-1 binding motif (Fig. 3, lane 5). Similar to our results with the wild-type insulin receptor, coimmunoprecipitation of Cav-Mut with W1200T was significantly impaired (Fig. 3, lane 6). We also observed significant coimmunoprecipitation of

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Fig. 3. Coimmunoprecipitation of Caveolin-1 and Insulin Receptors in Transfected Cos-7 Cells Cells were transiently cotransfected with various combinations of myc-tagged caveolin constructs and insulin receptor constructs (1 mg of each plasmid/well). Whole cell lysates were immunoprecipitated with an antibody against the b-subunit of the insulin receptor followed by immunoblotting with either an anti-myc antibody to detect coimmunoprecipitation of caveolin-1 (upper panel, lanes 1–6), or an antiinsulin receptor b-subunit antibody to verify comparable immunoprecipitation efficiency (lower panel, lanes 1–6). Lanes 7–12 show the levels of caveolin and insulin receptor expression present in cell lysates before immunoprecipitation.

Cav-WT with a kinase-inactive insulin receptor mutant (with an intact caveolin binding motif) that was expressed at levels comparable to the wild-type receptor (data not shown). We performed similar coimmunoprecipitation experiments in rat adipose cells transiently transfected with Cav-WT or Cav-Mut (Fig. 4). Since adipose cells express high levels of endogenous insulin receptors, it was not necessary to cotransfect the cells with recombinant insulin receptors. Consistent with our results in Cos-7 cells, Cav-WT coimmunoprecipitated with insulin receptors much better than Cav-Mut (Fig. 4, lanes 3–6). In addition, insulin stimulation did not alter the ability of Cav-WT to coimmunoprecipitate with the insulin receptor (Fig. 4, lanes 3–4). We observed similar coimmunoprecipitation of endogenous insulin receptors with endogenous caveolin-1 in freshly isolated untransfected adipose cells (data not shown). Taken together, our results from both Cos-7 cells and adipose cells are consistent with the hypothesis that interactions between caveolin-1 and insulin receptors in intact cells depend upon the caveolin-1 scaffolding domain. Functional Consequences of Overexpression of Caveolin-1 in Cos-7 Cells We used the Path-Detect kit from Stratagene (La Jolla, CA) to assess effects of overexpression of caveolin-1 on insulin-induced Elk-1 phosphorylation in Cos-7 cells. In this assay, phosphorylation of a transfected GAL4 binding domain/Elk-1 activation domain fusion protein binds and activates a cotransfected GAL4 binding sequence/luciferase reporter plasmid result-

ing in increased luciferase expression. We observed a 4-fold increase in Elk-1 phosphorylation upon insulin stimulation of control cells overexpressing wild-type insulin receptors (Fig. 5A). Interestingly, co-overexpression of Cav-WT significantly enhanced insulinstimulated phosphorylation of Elk-1 to levels approximately twice those observed in the control cells. In contrast, overexpression of Cav-Mut did not significantly affect phosphorylation of Elk-1, suggesting that the ability of Cav-WT to modulate insulin signaling is dependent upon an intact scaffolding domain. In some experiments [125 I]insulin binding studies and anti-myc immunoblots were performed in parallel to confirm comparable overexpression of recombinant insulin receptors and caveolin constructs in all groups (data not shown). In contrast to cells overexpressing wild-type insulin receptors, Elk-1 phosphorylation did not significantly increase upon insulin stimulation of cells overexpressing the K1030A kinase-inactive insulin receptor mutant (Fig. 5B). Co-overexpression of Cav-WT or Cav-Mut with the K1030A receptor did not significantly change Elk-1 phosphorylation in either the absence or the presence of insulin. We obtained similar results from cells overexpressing the W1200T mutant insulin receptor as might be expected from a receptor that does not undergo autophosphorylation (data not shown). To further investigate the effects of caveolin-1 on insulin signaling in Cos-7 cells we assessed insulinstimulated extracellular signal-regulated kinase-2 (ERK2) phosphorylation in cells cotransfected with insulin receptors, hemagglutinin (HA)-tagged ERK2, and either Cav-WT or Cav-Mut (Fig. 6). Similar to our re-

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Fig. 4. Coimmunoprecipitation of Insulin Receptors and Caveolin-1 from Rat Adipose Cells Cells were transiently transfected with a control plasmid (pCIS2) or myc-tagged caveolin-1 constructs (Cav-WT or Cav-Mut) (4 mg DNA/cuvette). After transfection, cells were treated without or with insulin (100 nM, 2 min), and membrane fractions were subjected to immunoprecipitation with an antibody against the b-subunit of the insulin receptor. Samples were then immunoblotted with either an anti-myc antibody to detect coimmunoprecipitation of caveolin-1 (upper panel, lanes 1–6) or an antiinsulin receptor b-subunit antibody to verify comparable immunoprecipitation efficiency (lower panel, lanes 1–6). Lanes 7–12 show the levels of caveolin-1 and insulin receptor present in cell membrane fractions before immunoprecipitation.

sults with Elk-1, insulin stimulated a significant increase in ERK-2 phosphorylation in control cells (Fig. 6A, lanes 1–2). However, in contrast to our results with Elk-1, overexpression of Cav-WT blocked the ability of insulin to stimulate ERK2 phosphorylation (Fig. 6A, lanes 3–4). Nevertheless, this effect of Cav-WT appeared to depend upon an intact scaffolding domain because insulin-stimulated phosphorylation of ERK-2 in cells overexpressing Cav-Mut was similar to that of the control cells (Fig. 6A, lanes 5–6). Functional Consequences of Overexpression of Caveolin-1 in Adipose Cells Adipose cells cotransfected with caveolin-1 constructs and an HA-tagged ERK2 were treated without or with insulin, and phosphorylation of recombinant ERK2 was assessed by immunblotting anti-HA immunoprecipitates with an anti-phospho-mitogen-activated protein kinase (MAPK) antibody (Fig. 7A, upper panel). In the absence of insulin, ERK2 was phosphorylated at a detectable level in control cells cotransfected with an empty expression vector (pCIS2) and HA-ERK2. Upon insulin stimulation, we observed an approximately 3-fold increase in ERK2 phosphorylation (Fig. 7A, lanes 1–2; Fig. 7C). Co-overexpression of either Cav-WT or Cav-Mut resulted in a significant approximately 3-fold decrease in basal ERK2 phosphorylation (in the absence of insulin) (Fig. 7A, lanes 3 and 6; Fig. 7C). No significant differences in the level of ERK2 phosphorylation were observed between any of the groups in the presence of insulin (Fig. 7A, lanes 2, 4, and 6; Fig. 7C). Note that cell lysates used in these experiments did not include the nuclear fraction so that the amount of ERK2 recovered in anti-HA immunoprecipitates was slightly less in the samples derived from insulin-stimulated cells (presumably because some of the ERK2 underwent translocation to the nu-

cleus upon insulin stimulation) (Fig. 7A, lower panel). As a control experiment, we verified comparable expression of the various transgenes in each experimental group by immunoblotting cell lysates with antibodies against either the HA epitope (Fig. 7B, upper panel) or the Myc epitope (Fig. 7B, lower panel). Interestingly, when anti-HA immunoprecipitates were immunblotted with an antibody against myc, we detected comparable coimmunoprecipitation of Cav-WT or Cav-Mut with HA-ERK2 that was unaffected by treatment of the cells with insulin (Fig. 8). These results suggested that interactions between ERK2 and caveolin-1 do not depend on the caveolin-1 scaffolding domain (in contrast to interactions with the insulin receptor). Thus, interactions of caveolin-1 with ERK2 may help to explain why overexpression of either Cav-WT or Cav-Mut inhibits basal phosphorylation of ERK2 in adipose cells. We examined the effects of overexpressing caveolin-1 on actions of insulin that are unrelated to MAPKdependent pathways by studying insulin-stimulated translocation of GLUT4 in transfected rat adipose cells (Fig. 9). We observed an approximately 2-fold recruitment of GLUT4 to the cell surface in response to insulin stimulation in control cells cotransfected with pCIS2 and GLUT-HA. Interestingly, overexpression of either Cav-WT or Cav-Mut caused a small, but statistically significant, decrease in the insulin doseresponse curves (when compared with the control).

DISCUSSION Characterization of Caveolin-1 and Insulin Receptors in Intact Cells We studied interactions between insulin receptors and overexpressed Cav-WT or Cav-Mut in Cos-7 cells and rat adipose cells. The F92A and V94A mutations intro-

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Fig. 5. Insulin-Stimulated Phosphorylation of Elk-1 in Cos-7 Cells Overexpressing Caveolin-1 and Insulin Receptors The PathDetect kit was used to investigate effects of caveolin-1 on Elk-1 phosphorylation as described in Materials and Methods. Cells were transiently cotransfected with pFAElk (0.025 mg DNA/well), pFR-Luc (0.5 mg DNA/well), a caveolin construct (0.5 mg DNA/well), and an insulin receptor construct (0.5 mg DNA/well). After overnight serum starvation, cells were treated without or with insulin (100 nM, 6 h) and the luciferase activity in each sample was determined. A, Coexpression of wild-type, but not mutant, caveolin-1 with wildtype insulin receptor significantly enhanced insulin-stimulated phosphorylation of Elk-1 (P , 0.007). Results shown are the mean 6 SEM of nine independent experiments performed in triplicate (normalized to the group transfected with wildtype insulin receptors and stimulated with insulin). B, Insulin did not stimulate Elk-1 phosphorylation in Cos-7 cells overexpressing the kinase-inactive insulin receptor mutant K1030A. Coexpression of wild-type or mutant caveolin-1 with the K1030A insulin receptor mutant had no effect on Elk-1 phosphorylation. Results shown are the mean 6 SEM of five independent experiments performed in triplicate. Data were normalized to the group transfected with wild-type insulin receptors and stimulated with insulin and also corrected for the level of cell surface insulin binding.

duced into our Cav-Mut construct were designed to disrupt the binding of caveolin-1 to FXFXXXXF caveolin-1 binding motifs. The choice of these mutations was based upon alanine scanning mutagenesis studies of interactions between caveolin-1 peptides and the epidermal growth factor receptor (15). Previous peptide binding studies demonstrated that the

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Fig. 6. Insulin-Stimulated Phosphorylation of ERK2 in Transfected Cos-7 Cells Cells were cotransfected with constructs for HA-tagged ERK2, human insulin receptor, and either a control plasmid or caveolin constructs. After transfection, cells were treated without or with insulin (100 nM, 4 min), and whole-cell lysates were prepared. A, Lysates were immunoprecipitated with anti-HA antibody (HA-11) followed by immunoblotting with antiphospho-MAPK antibodies (upper panel) or HA-11 (lower panel). A representative blot is shown from an experiment that was performed independently twice. B, As a control experiment to verify comparable expression of the caveolin constructs, lysates were immunoblotted with anti-myc antibodies.

scaffolding domain of caveolin-1 (residues 82–101) interacts specifically with the caveolin binding motif present in the b-subunit of the insulin receptor as well as a number of other receptor tyrosine kinases and downstream signaling molecules (8, 10, 15). In addition, the insulin receptor is enriched in subcellular fractions containing caveolae (23, 24). In keeping with these previous studies, our coimmunoprecipitation experiments showed, for the first time, that the fulllength insulin receptor and caveolin-1 can interact in intact cells. While insulin stimulation did not result in any detectable change in this interaction, the markedly impaired ability of Cav-Mut to coimmunoprecipitate with the insulin receptor suggests that interactions between insulin receptors and caveolin-1 depend upon the caveolin-1 scaffolding domain. Our results are consistent with a direct interaction in intact cells between the scaffolding domain of caveolin-1 and the caveolin binding motif in the insulin receptor. As a complementary approach to studying interactions between the insulin receptor and caveolin-1, we

Caveolin Modulates Insulin Signaling

Fig. 7. Insulin-Stimulated Phosphorylation of ERK2 in Transfected Rat Adipose Cells Cells were cotransfected with an HA-tagged ERK2 construct (1 mg DNA/cuvette) and either a control plasmid or caveolin constructs (4 mg DNA/cuvette). After transfection, cells were treated without or with insulin (100 nM, 4 min), and whole-cell lysates were prepared. A, Lysates were immunoprecipitated with anti-HA antibody (HA-11) followed by immunoblotting with HA-11 or antiphosphoMAPK antibodies. A representative blot is shown from an experiment that was performed independently five times. B, As a control experiment to verify comparable expression of the various constructs, lysates were immunoblotted with antibodies against either HA (to detect recombinant ERK2) or Myc (to detect recombinant caveolin). C, Results of quantifying phosphorylated HA-tagged ERK2 from five independent experiments (means 6 SEM) similar to the one shown in panel A. The basal level of ERK2 phosphorylation was significantly decreased in cells overexpressing either Cav-WT or Cav-Mut when compared with the control group (P , 0.05). There was no significant difference between groups in the levels of insulin-stimulated ERK2 phosphorylation.

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generated several insulin receptor mutants with a disrupted caveolin binding motif (residues 1193–1200; WSFGVVLW). This region of the receptor is likely to be important for signaling because it is located just distal to the three critical tyrosine phosphorylation sites in the activation loop of the kinase domain (residues 1158, 1162, and 1163) (25) and is also well conserved in homologous regions of many other tyrosine kinases (2). We were unable to detect insulin-stimulated autophosphorylation of any of our mutant receptors. Moreover, it was difficult to evaluate interactions of caveolin-1 with the W1193G receptor mutant and the W1193G/F1195G/W1200G triple mutant because mature insulin receptors were poorly expressed on the cell surface for these constructs (although the proreceptor seemed to be present). Thus, the region of the insulin receptor containing the caveolin-1 binding motif may be necessary for the catalytic function of the kinase domain and may also be important for normal posttranslational receptor processing. Interestingly, these results are consistent with previous characterizations of a similar naturally occurring human insulin receptor mutation (W1193L) that has impaired autophosphorylation, undergoes accelerated degradation of the proreceptor (due to binding heat shock protein 90), and has markedly decreased cell surface expression (18, 19, 21). To avoid potential problems with abnormal folding that may be occurring with the W1193G receptor and the triple mutant, we designed a W1200T mutant that disrupts the caveolin binding motif by mimicking the homologous region of c-src (WSFGILLT in region IX of the kinase domain). Although the WSFGILLT sequence in c-src does not perfectly match the canonical caveolin binding motif (15), the scaffolding domain of caveolin-1 is known to interact directly with c-src (26). It remains possible that this imperfect motif may also bind to the scaffolding domain of caveolin-1 since the region of c-src that binds to caveolin-1 has not been elucidated. For example, the reductase domain of endothelial nitric oxide synthase (eNOSr) also does not contain a known caveolin binding motif, but caveolin-1 binds to eNOSr and the scaffolding domain of caveolin-1 inhibits eNOSr activity (27). We observed significant coimmunoprecipitation of Cav-WT with the W1200T insulin receptor mutant while interaction with Cav-Mut was significantly impaired. Thus, similar to interactions between wild-type insulin receptors and caveolin-1, the scaffolding domain of caveolin-1 is important for binding to the W1200T receptor. Our results also suggest that either the canonical caveolin-1 binding motif is not strictly required for the insulin receptor to bind caveolin-1 or that caveolin-1 is capable of binding to another region of the insulin receptor. Although the W1200T receptor was not expressed as well as the wild-type receptor, it did achieve higher levels of expression than the W1193G mutant or triplemutant. Similar to our other two receptor mutants, W1200T also had impaired autophosphorylation. This was intriguing because a threonine residue in the ho-

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Fig. 8. Coimmunoprecipitation of ERK2 and Caveolin-1 in Rat Adipose Cells Cells were cotransfected with HA-ERK2 (1 mg DNA/cuvette) and a control plasmid (pCIS2), Cav-WT, or Cav-Mut (4 mg DNA/cuvette). After transfection, cells were treated without or with insulin (100 nM, 4 min) and whole-cell lysates were immunoprecipitated with HA-11 followed by immunoblotting with HA-11 or Myc antibodies (lanes 1–6). As a control experiment to verify comparable expression of the various constructs, lysates were immunoblotted with antibodies against either HA (to detect recombinant ERK2), or Myc (to detect recombinant caveolin) (lanes 7–12).

mologous position of c-src does not interfere with catalytic activity. Nevertheless, a similar naturally occurring insulin receptor mutation (W1200S) that causes severe insulin resistance in a dominant fashion is also kinase inactive (16, 17). Functional Consequences of Overexpression of Caveolin-1 We chose to explore the functional role of caveolin-1 in insulin signaling in both Cos-7 cells that express little endogenous caveolin-1 as well as in rat adipose cell that express high levels of caveolin-1. Terminally differentiated cells including classical insulin targets such as skeletal muscle and adipose cells often have abundant caveolae and high levels of endogenous caveolin (8). In contrast, undifferentiated or dedifferentiated transformed cells usually have few or no caveolae and lower levels of caveolin (11, 12, 28). Oncogenic transformation by activated Neu lowers the level of endogenous caveolin-1 in fibroblasts (29). Moreover, downregulation of caveolin-1 by stable transfection of antisense constructs in NIH 3T3 cells results in increased cellular transformation (30). Conversely, overexpression of recombinant caveolin-1 in transformed cells results in reversion of this phenotype (31). This negative correlation between caveolin-1 expression and cellular transformation suggests that caveolin-1 may have an inhibitory effect on growth factor or oncogenic signaling. Interestingly, caveolin-1 only binds to wild-type Ras and c-src but not to activated Ras or v-src, and overexpression of caveolin-1 inhibits c-src autophosphorylation in transfected 293T cells (26, 32). Furthermore, numerous studies suggest that interactions between caveolin and a variety of signaling molecules, including G proteins, src family kinases, and growth factor receptors, may serve to sequester inactive signaling molecules and inhibit signaling (for review see Ref. 8). Thus, caveolin-1 seems to generally function in an inhibitory role in most studies. However, there is some evidence that the role of caveolins in cellular signaling may be cell type dependent and

more diverse than previously appreciated. For example, the development of multidrug resistance in some tumor cell lines has been associated with up-regulation of caveolin-1 and caveolae (33, 34), while suppression of caveolin-1 in androgen-insensitive metastatic murine prostate cancer cells leads to a more differentiated phenotype that responds to androgens by undergoing apoptosis (35). In Cos-7 cells transiently transfected with insulin receptors, overexpression of Cav-WT resulted in a significant increase in the ability of insulin to phosphorylate Elk-1 (while overexpression of Cav-Mut was without effect). This was dependent upon the presence of a catalytically active insulin receptor since cells expressing the kinase-inactive mutants K1030A or W1200T did not phosphorylate Elk-1 in response to insulin (in either the absence or presence of recombinant caveolin-1). Although our results do not fit with the paradigm of caveolin acting as an inhibitor of signaling, they are in agreement with a recent report demonstrating that overexpression of caveolin-3 may activate the insulin receptor kinase in HEK293T cells (10). That study also showed that scaffolding-domain peptides from caveolin-1 or -3 can bind and activate the insulin receptor kinase in vitro. Taken together with our demonstration that the insulin receptor binds to caveolin-1 in intact cells, it seems likely that our Elk-1 phosphorylation results are explained, at least in part, by interaction of caveolin-1 with the insulin receptor. It is also possible that the scaffolding domain of caveolin-1 mediates interactions with other insulin-signaling proteins to enhance Elk-1 phosphorylation. Indeed, previous studies have demonstrated that signaling molecules related to Elk-1 phosphorylation, such as Ras, Raf, MAPK/ERK kinase, and ERK2, are associated with caveolae (36) and caveolin-1 can bind to Ras (32) and ERK2 (this study). However, these interactions seem less likely to explain our results because the binding of caveolin-1 to these molecules may sequester inactive forms and tend to inhibit signaling (8). Indeed, in Cos-7 cells, overexpression of Cav-WT sup-

Caveolin Modulates Insulin Signaling

Fig. 9. Insulin-Stimulated Recruitment of GLUT4-HA to the Cell Surface of Adipose Cells Overexpressing Cav-WT or Cav-Mut Data are expressed as a percentage of cell surface GLUT4 in the presence of a maximally effective insulin concentration for the control group. A, Recruitment of epitope-tagged GLUT4 to the cell surface of cells cotransfected with CavWT/GLUT4-HA (4 mg and 1 mg DNA/cuvette, respectively) (F) was decreased compared with the control group (E). Results shown are the means 6 SEM of six independent experiments. The best-fit curve for the control group had an ED50 5 0.07 nM. The best-fit curve for the Cav-WT group had an ED50 5 0.12 nM. The difference in the two curves was statistically significant by multivariate ANOVA (P , 1 3 1025). B, Recruitment of epitope-tagged GLUT4 to the cell surface of cells cotransfected with Cav-Mut/GLUT4-HA (F) was decreased compared with the control group (E). The difference in the two curves was statistically significant by multivariate ANOVA (P , 1 3 1026). The best-fit curve for the control group had an ED50 5 0.10 nM. The best-fit curve for the Cav-Mut group had an ED50 5 0.05 nM. Results are the means 6 SEM of seven independent experiments.

pressed insulin-stimulated phosphorylation of ERK-2 while overexpression of Cav-Mut was without effect on ERK2 phosphorylation. To study effects of overexpression of caveolin-1 on MAPK-dependent pathways in rat adipose cells, we examined phosphorylation of a cotransfected ERK2 in response to insulin stimulation. In contrast to our results in Cos-7 cells, overexpression of either Cav-WT or Cav-Mut decreased the basal level of ERK2 phosphorylation (in the absence of insulin) but was without

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effect on insulin-stimulated phosphorylation. It is unlikely that these results are explained by interactions of caveolin-1 with the insulin receptor since overexpression of Cav-Mut (which does not bind well to the insulin receptor) yielded similar results. Somewhat surprisingly, we found that both Cav-WT and Cav-Mut coimmunoprecipitated with ERK2 in adipose cells. Furthermore, this interaction was not affected by treating the cells with insulin. When we scanned the amino acid sequence of human ERK2, we could not identify any known caveolin scaffolding-domain binding motifs. Taken together, these findings suggest that an intact scaffolding domain may not be necessary for interactions of caveolin-1 with ERK2 in adipose cells. When we investigated effects of overexpression of caveolin-1 on a MAPK-independent function in rat adipose cells, we found that both Cav-WT and Cav-Mut caused a small decrease in insulin-induced translocation of GLUT4. Since both the wild-type and mutant caveolin mediated a similar effect, it is unlikely that this was caused by interactions of the caveolin-scaffolding domain with the insulin receptor. Downstream effectors of insulin action that have been implicated in mediating recruitment of GLUT4 to the cell surface such as PKC-z (37) are also known to interact with caveolin. However, these interactions are also unlikely to explain our data because the scaffolding domain of caveolin-1 inhibits PKC-z activity (38), and our effects are observed with both Cav-WT and Cav-Mut. It is interesting that in adipose cells, overexpression of either Cav-WT or Cav-Mut had inhibitory effects on both MAPK-dependent and -independent pathways while in Cos-7 cells, Cav-WT enhanced some insulinstimulated MAPK-dependent pathways. This differential modulation of signaling may be due to differences in endogenous levels of caveolin-1. For example, since caveolin peptides have been shown to enhance insulin receptor tyrosine kinase activity (10), overexpression of caveolin in cells that have low endogenous levels of caveolin might be predicted to enhance insulin signaling as we observed. However, if the cell already has high endogenous levels of caveolin, its interaction with the insulin receptor may be saturated, and overexpression of caveolin may interfere with other downstream effectors. In addition, it is possible that other proteins that are important for the ability of caveolin to modulate signaling may be expressed in a cell type-dependent fashion. For example, insulin stimulation results in tyrosine phosphorylation of caveolin-1 by fyn only in 3T3-L1 adipocytes but not in undifferentiated preadipocytes (13, 39). A kinase that is induced upon adipocyte differentiation has been implicated in phosphorylation of c-cbl, which can then activate fyn leading to phosphorylation of caveolin (13). Conclusions Interactions between caveolin-1 and insulin receptors in intact cells are dependent upon the caveolin-1 scaf-

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folding domain. Point mutations disrupting the caveolin-1 binding motif in the insulin receptor result in markedly decreased cell surface receptor expression and a defect in receptor autophosphorylation consistent with a functionally important role for this region of the insulin receptor. Indeed, a number of patients with syndromes of extreme insulin resistance have mutations in this very region. Finally, our data suggest that caveolin may have both inhibitory and stimulatory roles in insulin signaling that depend upon the cellular context.

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against the HA epitope (HA-11) were obtained from BabCO (Berkeley, CA). Transient Transfection of Cos-7 Cells Cos-7 cells were cultured in DMEM supplemented with 25 mM glucose, 20 mM glutamine, 100 U/ml penicillin G, 100 mg/ml streptomycin, and 10% FCS at 37 C, 5% CO2. Lipofectamine Plus (Life Technologies, Inc., Gaithersburg, MD) was used to transfect cells at approximately 80% confluency in six-well dishes according to the manufacturers protocol. Transient Transfection of Rat Adipose Cells and Assay for Cell Surface Epitope-Tagged GLUT4

MATERIALS AND METHODS Expression Plasmids pCIS2: Cytomegalovirus (CMV)-based parent expression vector that generates high levels of expression in adipose cells and Cos-7 cells (40, 41). Cav-WT: HindIII/BamH I fragment (;600-bp) containing cDNA for myc-tagged canine caveolin-1 [obtained from M. Lisanti (7, 42)] was blunt-ended and ligated in the sense orientation into the HpaI site of pCIS2. Cav-Mut: F92A and V94A point mutations in the scaffolding domain of caveolin-1 were introduced into Cav-WT using the mutagenic oligonucleotide 59-CTT CAC CAC CGC CAC TGC GAC AAA ATA C-39 and the Morph mutagenesis kit (5Prime33Prime; Boulder, CO). This mutagenesis also causes a loss of a Tsp 45 I restriction site. hIR-WT: pCIS2 vector containing the cDNA for the human insulin receptor (22). hIR-K1030A: kinase-inactive point mutant of hIR-WT (obtained from S.I. Taylor) hIR-W1200T: W1200T point mutant of hIR-WT generated using the mutagenic oligonucleotide 59-GGC GTG GTC CTT ACC GAA ATC ACT AGC TTG GC-39. This oligonucleotide also introduced a silent mutation that created an extra restriction site for Bfa I. hIR triple-mutant: mutant of hIR-WT containing W1193G, F1195G, and W1200G mutations to disrupt FXFXXXXF caveolin binding motif. The mutagenic oligonucleotide 59CAC TTC TTC TGA CAT GGG GTC CGG TGG CGT GGT CCT TGG GGA AAT CAC-39 used also causes loss of a Bsplu 11 restriction site. hIR-F1195G: mutant derived from hIR-triple mutant using mutagenic oligonucleotide 59-CTG ACA TGT GGT CCG GTG GCG TGG TCC TTT GGG AAA TCA CCA GC-39 which changes back F1193 and W1200 and also restores the restriction site for Bsplu 11. PCIS2-GLUT4 HA: the expression vector for GLUT4-HA was constructed as described previously (43). HA-ERK2: expression vector for HA-tagged ERK2 (gift from M. Cobb). The presence of correct mutations in the various constructs was verified by direct sequencing. Antibodies Murine monoclonal antibodies directed against phosphotyrosine (4G10) were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal antibodies against the myc epitope and the insulin receptor b-subunit were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal antibodies against caveolin-1 (no. 13620) were obtained from Transduction Laboratories, Inc. (Lexington, KY). Phospho-MAPK antibodies were obtained from New England Biolabs, Inc. (Beverly, MA) and monoclonal antibodies

Isolated adipose cells from epididymal fat pads of male rats (170–200 g, CD strain) were transfected by electroporation as described previously (41, 44). To assess effects of insulin on translocation of GLUT4, groups of cells were transfected with an empty expression vector pCIS2 alone or cotransfected with GLUT4-HA and pCIS2, Cav-WT, or Cav-Mut. Twenty hours after electroporation, adipose cells were treated with insulin at 37 C for 30 min. Cell surface epitope-tagged GLUT4 was quantified by using the anti-HA mouse monoclonal antibody HA-11 in conjunction with 125I-labeled sheep antimouse IgG as described (22). Immunoprecipitation and Immunoblotting For Cos-7 cells, lysates were prepared by washing cells with PBS and then scraping the cells in ice-cold RIPA buffer on ice (20 mM Tris-HCl, pH 7.4, 2.5 mM EDTA, 50 mM NaF, 1 mM sodium orthovanadate, 1% Triton X-100 and 0.1% SDS, and a protease inhibitor cocktail (Complete Tablet, Boehringer Mannheim, Mannheim, Germany)). For some experiments, transfected cells were serum starved overnight and then treated without or with insulin (100 nM, 2 min) followed by freezing on liquid nitrogen. Cell lysates were then prepared as described above. Lysates were centrifuged at 10,000 3 g for 10 min to pellet cellular debris. For immunoprecipitation with the antiinsulin receptor b-subunit antibody, 0.6 mg antibody and prewashed protein A-agarose (5% of the total volume) were mixed with each sample (300 mg total protein) at 4 C on a rotating wheel overnight. The immune complexes were washed three times with RIPA buffer, and samples were pelleted by centrifugation and eluted by boiling in Laemmli sample buffer for 5 min followed by SDS-PAGE and immunoblotting with various antibodies. For adipose cells, whole cell lysates and membrane fractions were prepared as previously described (44). Immunoprecipitation with the antiinsulin receptor b-subunit antibody was carried out on membrane fractions as described above. For immunoprecipitation of HA-ERK2, whole cell lysates (400 mg total protein) were incubated with 7 mg HA-11 antibody and buffer supplemented with 100 nM of okadaic acid at 4 C on a rotating wheel. After 2 h, protein G-agarose was added (5% of total volume) and the samples were incubated overnight at 4 C on a rotating wheel. Samples were then treated as described above and immunoblotted with antibodies against the HA-tag or phospho-MAPK. Quantification of phospho-ERK2 was performed using a laser scanning densitometer (Molecular Dynamics, Inc., Sunnyvale, CA). 125

I-Labeled Insulin Binding

Tracer insulin binding to the cell surface of Cos-7 cells transfected with various insulin receptor constructs was assessed at 4 C as previously described (45).

Caveolin Modulates Insulin Signaling

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Phosphorylation of Elk-1 9.

The Path-Detect System (Stratagene) was used to assess the effects of overexpression of caveolin-1 on insulin-stimulated phosphorylation of Elk-1 in Cos-7 cells. In this assay, phosphorylation of a transfected GAL4 binding domain/Elk-1 activation domain fusion protein results in activation of a cotransfected GAL4 binding sequence/luciferase reporter plasmid resulting in increased luciferase expression. The luciferase activity was measured in cell lysates as previously described (46) and was assumed to correlate with the level of Elk-1 phosphorylation.

10.

Statistical Analysis

12.

Paired Student’s t-tests were used for comparing individual points where appropriate. P , 0.05 was considered to signify statistical difference. The insulin dose-response curves for GLUT4 translocation were fit using a nonlinear least squares method and were compared by multiple ANOVA (MANOVA) as described (44).

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11.

14.

Acknowledgments We thank M. Lisanti for supplying the caveolin-1 cDNA and M. Cobb for the HA-tagged ERK2. We also thank Simeon I. Taylor for helpful discussions and for providing us with the human insulin receptor K1030A mutant.

Received June 17, 1999. Revision received August 25, 1999. Accepted September 1, 1999. Address requests for reprints to: Michael J. Quon, M.D., Ph.D., Hypertension-Endocrine Branch, National Heart, Lung, and Blood Institute, NIH, Building 10, Room 8C-103, 10 Center Drive MSC 1754, Bethesda, Maryland 20892-1754. E-mail: [email protected].

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37. Bandyopadhyay G, Standaert ML, Kikkawa U, Ono Y, Moscat J, Farese RV 1999 Effects of transiently expressed atypical (z, l), conventional (a, b) and novel (d, e) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-z and C-l. Biochem J 337: 461–470 38. Oka N, Yamamoto M, Schwencke C, Kawabe J, Ebina T, Ohno S, Couet J, Lisanti MP, Ishikawa Y 1997 Caveolin interaction with protein kinase C. Isoenzyme-dependent regulation of kinase activity by the caveolin scaffolding domain peptide. J Biol Chem 272:33416–33421 39. Mastick CC, Brady MJ, Saltiel AR 1995 Insulin stimulates the tyrosine phosphorylation of caveolin. J Cell Biol 129: 1523–1531 40. Choi T, Huang M, Gorman C, Jaenisch R 1991 A generic intron increases gene expression in transgenic mice. Mol Cell Biol 11:3070–3074 41. Quon MJ, Zarnowski MJ, Guerre-Millo M, de la Luz Sierra M, Taylor SI, Cushman SW 1993 Transfection of DNA into isolated rat adipose cells by electroporation: evaluation of promoter activity in transfected adipose cells which are highly responsive to insulin after one day in culture. Biochem Biophys Res Commun 194:338–346 42. Scherer PE, Tang Z, Chun M, Sargiacomo M, Lodish HF, Lisanti MP 1995 Caveolin isoforms differ in their Nterminal protein sequence and subcellular distribution. Identification and epitope mapping of an isoformspecific monoclonal antibody probe. J Biol Chem 270: 16395–16401 43. Quon MJ, Butte AJ, Zarnowski MJ, Sesti G, Cushman SW, Taylor SI 1994 Insulin receptor substrate 1 mediates the stimulatory effect of insulin on GLUT4 translocation in transfected rat adipose cells. J Biol Chem 269: 27920–27924 44. Chen H, Wertheimer SJ, Lin CH, Katz SL, Amrein KE, Burn P, Quon MJ 1997 Protein-tyrosine phosphatases PTP1B and syp are modulators of insulin- stimulated translocation of GLUT4 in transfected rat adipose cells. J Biol Chem 272:8026–8031 45. Quon MJ, Cama A, Taylor SI 1992 Postbinding characterization of five naturally occurring mutations in the human insulin receptor gene: impaired insulin-stimulated c-jun expression and thymidine incorporation despite normal receptor autophosphorylation. Biochemistry 31: 9947–9954 46. Chen H, Srinivas PR, Cong LN, Li Y, Grunberger G, Quon MJ 1998 Alpha2-Heremans Schmid glycoprotein inhibits insulin-stimulated Elk-1 phosphorylation, but not glucose transport, in rat adipose cells. Endocrinology 139: 4147–4154