Rapid Insulin-Dependent Endocytosis of the Insulin Receptor by Caveolae in Primary Adipocytes

Background

The insulin receptor is localized in caveolae and is dependent on caveolae or cholesterol for signaling in adipocytes. When stimulated with insulin, the receptor is internalized.

Methodology/Principal Findings

We examined primary rat adipocytes by subcellular fractionation to examine if the insulin receptor was internalized in a caveolae-mediated process. Insulin induced a rapid, t_1/2<3 min, endocytosis of the insulin receptor in parallel with receptor tyrosine autophosphorylation. Concomitantly, caveolin-1 was phosphorylated at tyrosine(14) and endocytosed. Vanadate increased the phosphorylation of caveolin-1 without affecting insulin receptor phosphorylation or endocytosis. Immunocapture of endosomal vesicles with antibodies against the insulin receptor co-captured caveolin-1 and immunocapture with antibodies against tyrosine(14)-phosphorylated caveolin-1 co-captured the insulin receptor, demonstrating that the insulin receptor was endocytosed together with tyrosine(14)-phosphorylated caveolin-1. By immunogold electron microscopy the insulin receptor and caveolin-1 were colocalized in endosome vesicles that resembled caveosomes. Clathrin was not endocytosed with the insulin receptor and the inhibitor of clathrin-coated pit-mediated endocytosis, chlorpromazine, did not inhibit internalization of the insulin receptor, while transferrin receptor internalization was inhibited.

Conclusion

It is concluded that in response to insulin stimulation the autophosphorylated insulin receptor in primary adipocytes is rapidly endocytosed in a caveolae-mediated process, involving tyrosine phosphorylation of caveolin-1.     

Cholesterol depletion disrupts caveolae and insulin receptor signaling for metabolic control via insulin receptor substrate-1, but not for mitogen-activated protein kinase control

Insulin exerts its cellular control through receptor binding in caveolae in plasmalemma of target cells (Gustavsson, J., Parpal, S., Karlsson, M., Ramsing, C., Thorn, H., Borg, M., Lindroth, M., Peterson, K. H., Magnusson, K.-E., and Str?lfors, P. (1999) FASEB. J. 13, 1961-1971). We now report that a progressive cholesterol depletion of 3T3-L1 adipocytes with beta-cyclodextrin gradually destroyed caveolae structures and concomitantly attenuated insulin stimulation of glucose transport, in effect making cells insulin-resistant. Insulin access to or affinity for the insulin receptor on rat adipocytes was not affected as determined by (125)I-insulin binding. By immunoblotting of plasma membranes, total amount of insulin receptor and of caveolin remained unchanged. Receptor autophosphorylation in response to insulin was not affected by cholesterol depletion. Insulin treatment of isolated caveolae preparations increased autophosphorylation of receptor before and following cholesterol depletion. Insulin-increased tyrosine phosphorylation of an immediate downstream signal transducer, insulin receptor substrate-1, and activation of the further downstream protein kinase B were inhibited. In contrast, insulin signaling to mitogenic control as determined by control of the extracellular signal-related kinases 1/2, mitogen-activated protein kinase pathway was not affected. Insulin did not control Shc phosphorylation, and Shc did not control extracellular signal-related kinases 1/2, whereas cholesterol depletion constitutively phosphorylated Shc. In conclusion, caveolae are critical for propagating the insulin receptor signal to downstream targets and have the potential for sorting signal transduction for metabolic and mitogenic effects.     

PC12 cells have caveolae that contain TrkA. Caveolae-disrupting drugs inhibit nerve growth factor-induced, but not epidermal growth factor-induced, MAPK phosphorylation

Nerve growth factor (NGF) induces survival and differentiation of the neural crest-derived PC12 cell line. Caveolae are cholesterol-enriched, caveolin-containing plasma membrane microdomains involved in vesicular transport and signal transduction. Here we demonstrate the presence of caveolae in PC12 cells and their involvement in NGF signaling. Our results showed the expression of caveolin-1 by Western blot and confocal immuno-microscopy. The presence of plasma membrane caveolae was directly shown by rapid-freeze deep-etching electron microscopy. Moreover, combined deep-etching and immunogold techniques revealed the presence of the NGF receptor TrkA in the caveolae of PC12 cells. These data together with the cofractionation of Shc, Ras, caveolin, and TrkA in the caveolae fraction supported a role for these plasma membrane microdomains in NGF signaling. To approach this hypothesis, caveolae were disrupted by treatment of PC12 cells with cholesterol binding drugs. Either filipin or cyclodextrin treatment increased basal levels of MAPK phosphorylation. In contrast, pretreatment of PC12 cells with these drugs inhibited the NGF- but not the epidermal growth factor-induced MAPK phosphorylation without affecting the TrkA autophosphorylation. Taken together, our results demonstrate the presence of caveolae in PC12 cells, which contain the high affinity NGF receptor TrkA, and the specific involvement of these cholesterol-enriched plasma membrane microdomains in the propagation of the NGF-induced signal.     

Initiation of BMP2 signaling in domains on the plasma membrane

Bone morphogenetic protein 2 (BMP2) is a potent growth factor crucial for cell fate determination. It directs the differentiation of mesenchymal stem cells into osteoblasts, chondrocytes, adipocytes, and myocytes. Initiation of BMP2 signaling pathways occurs at the cell surface through type I and type II serine/threonine kinases housed in specific membrane domains such as caveolae enriched in the caveolin-1 beta isoform (CAV1β, caveolae) and clathrin-coated pits (CCPs). In order for BMP2 to initiate Smad signaling it must bind to its receptors on the plasma membrane resulting in the phosphorylation of the BMP type Ia receptor (BMPRIa) followed by activation of Smad signaling. The current model suggests that the canonical BMP signaling pathway, Smad, occurs in CCPs. However, several recent studies suggested Smad signaling may occur outside of CCPs. Here, we determined; (i) The location of BMP2 binding to receptors localized in caveolae, CCPs, or outside of these domains using AFM and confocal microscopy. (ii) The location of phosphorylation of BMPRIa on the plasma membrane using membrane fractionation, and (iii) the effect of down regulation of caveolae on Smad signaling. Our data indicate that BMP2 binds with highest force to BMP receptors (BMPRs) localized in caveolae. BMPRIa is phosphorylated in caveolae and the disruption of caveolae-inhibited Smad signaling in the presence of BMP2. This suggests caveolae are necessary for the initiation of Smad signaling. We propose an extension of the current model of BMP2 signaling, in which the initiation of Smad signaling is mediated by BMPRs in caveolae.     

Immunopurification and characterization of rat adipocyte caveolae suggest their dissociation from insulin signaling

Adipocytes play an important role in the insulin-dependent regulation of organismal fuel metabolism and express caveolae at levels as high or higher than any other cell type. Recently, a link between insulin signaling and caveolae has been suggested; nevertheless, adipocyte caveolae have been the subject of relatively few studies, and their contents have been minimally characterized. With the aid of a new monoclonal antibody, we developed a rapid procedure for the immunoisolation of caveolae derived from the plasma membrane of adipocytes, and we characterized their protein content. We find that immunopurified adipocyte caveolae have a relatively limited protein composition, and they lack the raft protein, flotillin, and insulin receptors. Immunogold labeling and electron microscopy of the adipocyte plasma membrane confirmed the lack of insulin receptors in caveolae. In addition to caveolins, the structural components of caveolae, their major protein constituents, are the semicarbazide-sensitive amine oxidase and the scavenger lipoprotein receptor CD36. The results are consistent with a role for caveolae in lipid flux in and of adipocytes.     

Caveolae regulate smad signaling as verified by novel imaging and system biology approaches

The contribution of caveolae in Bone Morphogenetic Protein 2 (BMP2) activated Smad signaling was quantified using a system biology approach. BMP2 plays crucial roles during processes such as hematopoiesis, embryogenesis, and skeletal development. BMP2 signaling is tightly regulated on the plasma membrane by its receptors. The localization of BMP receptors in caveolae and endocytosis through clathrin‐coated pits are thought to regulate the signaling; however the conclusions in the current literature are inconsistent. Therefore published literature was used to establish a mathematical model that was validated using confocal AFM (atomic force microscopy), confocal microscopy, and sucrose density centrifugation followed by Western blots, and reporter gene assays. The model and experiments confirmed that both caveolae and CCPs regulate the Smad‐dependent signaling pathway, however caveolae are centers at the plasma membrane where receptor–ligand interaction is crucial, Smad phosphorylation occurs, and a high degree of Smad signaling is regulated. This demonstrates a role for caveolae that needs to be considered and further studied. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Role of caveolin and caveolae in insulin signaling and diabetes

Caveolae are specialized membrane microdomains present within the plasma membrane of the vast majority of cell types. They have a unique composition in that they are highly enriched in cholesterol, sphingolipids, and their coat proteins the caveolins (-1, -2, and -3). In recent years it has been recognized that caveolae act as signaling platforms, serving as a concentrating point for numerous signaling molecules, as well as regulating flux through many distinct signaling cascades. Although caveolae are found in a variety of cell types, they are most abundant in adipose tissue. This fact has led to the intense study of the function of these organelles in adipocytes. It has now become apparent that effective insulin signaling in the adipocyte may be strictly dependent on localization of at least two insulin-responsive elements to caveolae (insulin receptor and GLUT4), as well as on a direct functional interaction between caveolin-1 and the insulin receptor. We present a critical discussion of these recent findings.     

Nanoscale Imaging of Epidermal Growth Factor Receptor Clustering: EFFECTS OF INHIBITORS*

The development of some solid tumors is associated with overexpression of the epidermal growth factor receptor (EGFR) and often correlates with poor prognosis. Near field scanning optical microscopy, a technique with subdiffraction-limited optical resolution, was used to examine the influence of two inhibitors (the chimeric 225 antibody and tyrosine phosphorylation inhibitor AG1478) on the nanoscale clustering of EGFR in HeLa cells. The EGFR is organized in small clusters, average diameter of 150 nm, on the plasma membrane for both control and EGF-treated cells. The numbers of receptors in individual clusters vary from as few as one or two proteins to greater than 100. Both inhibitors yield an increased cluster density and an increase in the fraction of clusters with smaller diameters and fewer receptors. Exposure to AG1478 also decreases the fraction of EGFR that colocalizes with both rafts and caveolae. EGF stimulation results in a significant loss of the full-length EGFR from the plasma membrane with the concomitant appearance of low molecular mass proteolytic products. By contrast, AG1478 reduces the level of EGFR degradation. Changes in receptor clustering provide one mechanism for regulating EGFR signaling and are relevant to the design of strategies for therapeutic interventions based on modulating EGFR signaling.     

Insulin second messengers

The molecular pathways for insulin's signal transduction from its cell surface receptor to the cell's interior metabolic machinery remain in many ways uncharted. Lately two molecules have been proposed as second messengers transducing the insulin signal into the target cell. One is a phospholigosaccharide/inositolphosphoglycan and the other is diacylglycerol, both deriving from the same plasma membrane glycolipid, which is hydrolysed in response to insulin treatment. The phospho-oligosaccharide appears to mediate many metabolic effects of insulin through control of the phosphorylation state of key regulatory metabolic enzymes. Diacylglycerol may mediate insulin's stimulation of glucose transport over the plasma membrane. The glycolipid precursor of these putative second messengers, as well as the receptor for insulin, appear to be localized in caveolae microdomains of the plasma membrane, and glucose transporters accumulate in caveolae in response to insulin treatment, suggesting a focal role for caveolae in insulin signalling.     

Characterization of the stimulatory action of insulin on insulin-like growth factor II binding to rat adipose cells. Differences in the mechanism of insulin action on insulin-like growth factor II receptors and glucose transporters

Insulin is known to increase the number of cell surface insulin-like growth factor II (IGF-II) receptors in isolated rat adipose cells through a subcellular redistribution mechanism similar to that for the glucose transporter. The effects of insulin on these two processes, therefore, have now been directly compared in the same cell preparations. 1) Insulin increases the steady state number of cell surface IGF-II receptors by 7-13-fold without affecting receptor affinity; however, insulin stimulates glucose transport activity by 25-40-fold. 2) The insulin concentration required for half-maximal stimulation of cell surface IGF-II receptor number is approximately 30% lower than that for the stimulation of glucose transport activity. 3) The half-time for the achievement of insulin's maximal effect at 37 degrees C is much shorter for IGF-II receptor number (approximately 0.8 min) than for glucose transport activity (approximately 2.6 min). 4) Reversal of insulin's action at 37 degrees C occurs more rapidly for cell surface IGF-II receptors (t1/2 congruent to 2.9 min) than for glucose transport activity (t1/2 congruent to 4.9 min). 5) When the relative subcellular distribution of IGF-II receptors is examined in basal cells, less than 10% of the receptors are localized to the plasma membrane fraction indicating that most of the receptors, like glucose transporters, are localized to an intracellular compartment. However, in response to insulin, the number of plasma membrane IGF-II receptors increases only approximately 1.4-fold while the number of glucose transporters increases approximately 4.5-fold. Thus, while the stimulatory actions of insulin on cell surface IGF-II receptors and glucose transport activity are qualitatively similar, marked quantitative differences suggest that the subcellular cycling of these two integral membrane proteins occurs by distinct processes.     

Cholesterol depletion inhibits epidermal growth factor receptor transactivation by angiotensin II in vascular smooth muscle cells: role of cholesterol-rich microdomains and focal adhesions in angiotensin II signaling

Angiotensin II (Ang II) induces transactivation of the epidermal growth factor (EGF) receptor (EGF-R), which serves as a scaffold for various signaling molecules in vascular smooth muscle cells (VSMCs). Cholesterol and sphingomyelin-enriched lipid rafts are plasma membrane microdomains that concentrate various signaling molecules. Caveolae are specialized lipid rafts that are organized by the cholesterol-binding protein, caveolin, and have been shown to be associated with EGF-Rs. Angiotensin II stimulation promotes a rapid movement of AT(1) receptors to caveolae; however, their functional role in angiotensin II signaling has not been elucidated. Here we show that cholesterol depletion by beta-cyclodextrin disrupts caveolae structure and concomitantly inhibits tyrosine phosphorylation of the EGF-R and subsequent activation of protein kinase B (PKB)/Akt induced by angiotensin II. Similar inhibitory effects were obtained with other cholesterol-binding agents, filipin and nystatin. In contrast, EGF-R autophosphorylation and activation of Akt/PKB in response to EGF are not affected by cholesterol depletion. The early Ang II-induced upstream signaling events responsible for transactivation of the EGF-R, such as the intracellular Ca(2+) increase and c-Src activation, also remain intact. The EGF-R initially binds caveolin, but these two proteins rapidly dissociate following angiotensin II stimulation during the time when EGF-R transactivation is observed. The activated EGF-R is localized in focal adhesions together with tyrosine-phosphorylated caveolin. These findings suggest that 1) a scaffolding role of caveolin is essential for EGF-R transactivation by angiotensin II and 2) cholesterol-rich microdomains as well as focal adhesions are important signal-organizing compartments required for the spatial and temporal organization of angiotensin II signaling in VSMCs.     

p42/p44 MAP kinase activation is localized to caveolae-free membrane domains in airway smooth muscle

Caveolae are abundant plasma membrane invaginations in airway smooth muscle that may function as preorganized signalosomes by sequestering and regulating proteins that control cell proliferation, including receptor tyrosine kinases (RTKs) and their signaling effectors. We previously demonstrated, however, that p42/p44 MAP kinase, a critical effector for cell proliferation, does not colocalize with RTKs in caveolae of quiescent airway myocytes. Therefore, we investigated the subcellular sites of growth factor-induced MAP kinase activation. In quiescent myocytes, though epidermal growth factor receptor (EGFR) was almost exclusively found in caveolae, p42/p44 MAP kinase, Grb2, and Raf-1 were absent from these membrane domains. EGF induced concomitant phosphorylation of caveolin-1 and p42/p44 MAP kinase; however, EGF did not promote the localization of p42/p44 MAP kinase, Grb2, or Raf-1 to caveolae. Interestingly, stimulation of muscarinic M(2) and M(3) receptors that were enriched in caveolae-deficient membranes also induced p42/p44 MAP kinase phosphorylation, but this occurred in the absence of caveolin-1 phosphorylation. This suggests that the localization of receptors to caveolae and interaction with caveolin-1 is not directly required for p42/p44 MAP kinase phosphorylation. Furthermore, we found that EGF exposure induced rapid translocation of EGFR from caveolae to caveolae-free membranes. EGFR trafficking coincided temporally with EGFR and p42/p44 MAP kinase phosphorylation. Collectively, this indicates that although caveolae sequester some receptors associated with p42/p44 MAP kinase activation, the site of its activation is associated with caveolae-free membrane domains. This reveals that directed trafficking of plasma membrane EGFR is an essential element of signal transduction leading to p42/p44 MAP kinase activation.     

Tyrosine phosphorylation of the insulin receptor during insulin-stimulated internalization in rat hepatoma cells

We have studied the phosphorylation state of the insulin receptor during receptor-mediated endocytosis in the well-differentiated rat hepatoma cell line Fao. Insulin induced the rapid internalization of surface-iodinated insulin receptors into a trypsin-resistant compartment, with a 3-fold increase in the internalization rate over that seen in the absence of insulin. Within 20 min of insulin stimulation, 30-35% of surface receptors were located inside the cell. This redistribution was half-maximal by 10.5 min. Similar results were obtained when the loss of surface receptors was measured by 125I-insulin binding. Tyrosyl phosphorylation of internalized insulin receptors was measured by immunoprecipitation with antiphosphotyrosine antibody. Immediately after insulin stimulation, 70-80% of internalized receptors were tyrosine phosphorylated. Internalized receptors persisted in a phosphorylated state after the dissociation of insulin but were dephosphorylated prior to their return to the plasma membrane. After 45-60 min of insulin stimulation, the tyrosine phosphorylation of the internal receptor pool decreased by 45%, whereas the phosphorylation of surface receptors was unchanged. These data suggest that insulin induces the internalization of phosphorylated insulin receptors into the cell and that the phosphorylation state of the internal receptor pool may be regulated by insulin.     

Spatial organization of EGF receptor transmodulation by PDGF.

Even though the modulation of EGF receptors by PDGF is well documented, it is not known where on the cell surface cross-talk between the two receptor systems takes place. The recent finding that both populations of receptors are concentrated in cell surface caveolae suggestes that the confinement of the two receptors to this space might facilitate their interaction. Here we show that stimulation of PDGF receptors in caveolae with PDGF causes a subpopulation of EGF receptors in the same membrane fraction to become phosphorylated on tyrosine. Coincident with tyrosine phosphorylation, the binding of EGF to its receptor markedly declines. Loss of EGF binding is partially blocked by tyrosine kinase inhibitors. Despite the close proximity of the two receptors in caveolae, we saw no evidence that EGF could stimulated PDGFR tyrosine phosphorylation. These results suggest that these two receptor systems are highly organized in caveolae.     

Dynamic association of human insulin receptor with lipid rafts in cells lacking caveolae

Cholesterol-sphingolipid rich plasma membrane domains, known as rafts, have emerged as important regulators of signal transduction. The adipocyte insulin receptor (IR) is localized to and signals via caveolae that are formed by polymerization of caveolins. Caveolin binds to IR and stimulates signalling. We report that, in liver-derived cells lacking caveolae, autophosphorylation of the endogenous IR is dependent on raft lipids, being compromised by acute cyclodextrin-mediated cholesterol depletion or by antibody clustering of glycosphingolipids. Moreover, we provide evidence that IR becomes recruited to detergent-resistant domains upon ligand binding and that clustering of GM2 ganglioside inhibits IR signalling apparently by excluding the ligand-bound IR from these domains. Our results indicate that, in cells derived from liver, an important insulin target tissue, caveolae are not required for insulin signalling. Rather, the dynamic recruitment of the ligand-bound IR into rafts may serve to regulate interactions in the initiation of the IR signalling cascade.     

Separation and characterization of caveolae subclasses in the plasma membrane of primary adipocytes; segregation of specific proteins and functions

Caveolae are nearly ubiquitous plasma membrane domains that in adipocytes vary in size between 25 and 150 nm. They constitute sites of entry into the cell as well as platforms for cell signalling. We have previously reported that plasma membrane-associated caveolae that lack cell surface access can be identified by electron microscopy. We now report the identification, after density gradient ultracentrifugation, of a subclass of very high-density apparently closed caveolae that were not labelled by cell surface protein labelling of intact cells. These caveolae contained caveolin-1 and caveolin-2. Another class of high-density caveolae contained caveolin-1, caveolin-2 and specifically fatty acid transport protein-1, fatty acid transport protein-4, fatty acyl-CoA synthetase, hormone-sensitive lipase, perilipin, and insulin-regulated glucose transporter-4. This class of caveolae was specialized in fatty acid uptake and conversion to triacylglycerol. A third class of low-density caveolae contained the insulin receptor, class B scavenger receptor-1, and insulin-regulated glucose transporter-4. Small amounts of these proteins were also detected in the high-density caveolae. In response to insulin, the insulin receptor autophosphorylation and the amount of insulin-regulated glucose transporter-4 increased in these caveolae. The molar ratio of cholesterol to phospholipid in the three caveolae classes varied considerably, from 0.4 in very high-density caveolae to 0.9 in low-density caveolae. There was no correlation between the caveolar contents of caveolin and cholesterol. The low-density caveolae, with the highest cholesterol concentration, were particularly enriched with the cholesterol-rich lipoprotein receptor class B scavenger receptor-1, which mediated cholesteryl ester uptake from high-density lipoprotein and generation of free cholesterol in these caveolae, suggesting a specific role in cholesterol uptake/metabolism. These findings demonstrate a segregation of functions in caveolae subclasses.     

Cross-talk between insulin and Wnt signaling in preadipocytes: role of Wnt co-receptor low density lipoprotein receptor-related protein-5 (LRP5)

Disturbed Wnt signaling has been implicated in numerous diseases, including type 2 diabetes and the metabolic syndrome. In the present study, we have investigated cross-talk between insulin and Wnt signaling pathways using preadipocytes with and without knockdown of the Wnt co-receptors LRP5 and LRP6 and with and without knock-out of insulin and IGF-1 receptors. We find that Wnt stimulation leads to phosphorylation of insulin signaling key mediators, including Akt, GSK3β, and ERK1/2, although with a lower fold stimulation and slower time course than observed for insulin. These Wnt effects are insulin/IGF-1 receptor-dependent and are lost in insulin/IGF-1 receptor double knock-out cells. Conversely, in LRP5 knockdown preadipocytes, insulin-induced phosphorylation of IRS1, Akt, GSK3β, and ERK1/2 is highly reduced. This effect is specific to insulin, as compared with IGF-1, stimulation and appears to be due to an inducible interaction between LRP5 and the insulin receptor as demonstrated by co-immunoprecipitation. These data demonstrate that Wnt and insulin signaling pathways exhibit cross-talk at multiple levels. Wnt induces phosphorylation of Akt, ERK1/2, and GSK3β, and this is dependent on insulin/IGF-1 receptors. Insulin signaling also involves the Wnt co-receptor LRP5, which has a positive effect on insulin signaling. Thus, altered Wnt and LRP5 activity can serve as modifiers of insulin action and insulin resistance in the pathophysiology of diabetes and metabolic syndrome.     

Depletion of plasma membrane cholesterol dampens hydrostatic pressure and shear stress-induced mechanotransduction pathways in osteoblast cultures

The preferential association of cholesterol and sphingolipids within plasma membranes forms organized compartments termed lipid rafts. Addition of caveolin proteins to this lipid milieu induces the formation of specialized invaginated plasma membrane structures called caveolae. Both lipid rafts and caveolae are purported to function in vesicular transport and cell signaling. We and others have shown that disassembly of rafts and caveolae through depletion of plasma membrane cholesterol mitigates mechanotransduction processes in endothelial cells. Because osteoblasts are subjected to fluid-mechanical forces, we hypothesize that cholesterol-rich plasma membrane microdomains also serve the mechanotransduction process in this cell type. Cultured human fetal osteoblasts were subjected to either sustained hydrostatic pressure or laminar shear stress using a pressure column or parallel-plate apparatus, respectively. We found that sustained hydrostatic pressure induced protein tyrosine phosphorylation, activation of extracellular signal-regulated kinase (ERK)1/2, and enhanced expression of c-fos in both time- and magnitude-dependent manners. Similar responses were observed in cells subjected to laminar shear stress. Both sustained hydrostatic pressure- and shear stress-induced signaling were significantly reduced in osteoblasts pre-exposed to either filipin or methyl--cyclodextrin. These mechanotransduction responses were restored on reconstitution of lipid rafts and caveolae, which suggests that cholesterol-rich plasma membrane microdomains participate in the mechanotransduction process in osteoblasts. In addition, mechanical force-induced phosphoproteins were localized within caveolin-containing membranes. These data support the concept that lipid rafts and caveolae serve a general function as cell surface mechanotransduction sites within the plasma membrane. lipid rafts; caveolae; extracellular signal-regulated kinase     

Cholesterol-dependent lipid assemblies regulate the activity of the ecto-nucleotidase CD39

CD39 (ecto-nucleoside triphosphate diphosphohydrolase-1; E-NTPDase1) is a plasma membrane ecto-enzyme that regulates purinergic receptor signaling by controlling the levels of extracellular nucleotides. In blood vessels this enzyme exhibits a thromboregulatory role through the control of platelet aggregation. CD39 is localized in caveolae, which are plasma membrane invaginations with distinct lipid composition, similar to dynamic lipid microdomains, called rafts. Cholesterol is enriched together with sphingolipids in both rafts and caveolae, as well as in other specialized domains of the membrane, and plays a key role in their function. Here, we examine the potential role of cholesterol-enriched domains in CD39 function. Using polarized Madin-Darby canine kidney (MDCK) cells and caveolin-1 gene-disrupted mice, we show that caveolae are not essential either for the enzymatic activity of CD39 or for its targeting to plasma membrane. On the other hand, flotation experiments using detergent-free or detergent-based approaches indicate that CD39 associates, at least in part, with distinct lipid assemblies. In the apical membrane of MDCK cells, which lacks caveolae, CD39 is localized in microvilli, which are also cholesterol and raft-dependent membrane domains. Interfering with cholesterol levels using drugs that either deplete or sequester membrane cholesterol results in a strong inhibition of the enzymatic and anti-platelet activity of CD39. The effects of cholesterol depletion are completely reversed by replenishment of membranes with pure cholesterol, but not by cholestenone. These data suggest a functional link between the localization of CD39 in cholesterol-rich domains of the membrane and its role in thromboregulation.     

Primary and essential role of the adaptor protein APS for recruitment of both c-Cbl and its associated protein CAP in insulin signaling

APS (adapter protein with Pleckstrin homology and Src homology 2 domains) is recruited by the autophosphorylated insulin receptor and is essential for Glut4 translocation. Although both APS and CAP (c-Cbl-associated protein) interact with c-Cbl during insulin signaling, the relative importance of each protein in recruiting c-Cbl has not been clear. We performed a side-by-side comparison by ectopic expression of APS or Src homology 2-Balpha (SH2-Balpha) and CAP in Chinese hamster ovary (CHO) cells. In cells co-expressing insulin receptor and CAP, without APS, no association of the insulin receptor and CAP could be detected and no insulin-stimulated phosphorylation of Cbl was observed. Insulin-stimulated Cbl phosphorylation was reconstituted when APS was co-expressed with insulin receptor, with or without CAP. APS or SH2-Balpha and CAP interacted in the basal state, and in the case of APS this interaction was mediated by the C terminus of APS. Insulin stimulation resulted in the dissociation of APS and CAP. Similarly, insulin stimulation also resulted in the dissociation of SH2-Balpha and CAP in CHO cells. CAP was localized to the membrane in the presence of APS. Insulin stimulation resulted in the re-localization of CAP to the cytosol only when APS was co-expressed. In 3T3-L1 adipocytes, small interfering RNA-mediated knockdown of the mouse APS gene abolished the insulin-stimulated phosphorylation of c-Cbl. Taken together, these results indicate that APS plays a central role in recruiting both CAP and c-Cbl to the insulin receptor after insulin stimulation and is necessary and sufficient for the insulin-stimulated phosphorylation of c-Cbl, whereas SH2-Balpha may provide an alternative pathway for the recruitment of CAP.