Localization of the insulin receptor in caveolae of adipocyte plasma membrane.

The insulin receptor is a transmembrane protein of the plasma membrane, where it recognizes extracellular insulin and transmits signals into the cellular signaling network. We report that insulin receptors are localized and signal in caveolae microdomains of adipocyte plasma membrane. Immunogold electron microscopy and immunofluorescence microscopy show that insulin receptors are restricted to caveolae and are colocalized with caveolin over the plasma membrane. Insulin receptor was enriched in a caveolae-enriched fraction of plasma membrane. By extraction with beta-cyclodextrin or destruction with cholesterol oxidase, cholesterol reduction attenuated insulin receptor signaling to protein phosphorylation or glucose transport. Insulin signaling was regained by spontaneous recovery or by exogenous replenishment of cholesterol. beta-Cyclodextrin treatment caused a nearly complete annihilation of caveolae invaginations as examined by electron microscopy. This suggests that the receptor is dependent on the caveolae environment for signaling. Insulin stimulation of cells prior to isolation of caveolae or insulin stimulation of the isolated caveolae fraction increased tyrosine phosphorylation of the insulin receptor in caveolae, demonstrating that insulin receptors in caveolae are functional. Our results indicate that insulin receptors are localized to caveolae in the plasma membrane of adipocytes, are signaling in caveolae, and are dependent on caveolae for signaling.     

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.     

Colocalization of beta-adrenergic receptors and caveolin within the plasma membrane.

The rapid amplification of beta-adrenergic receptor signaling involves the sequential activation of multiple signaling molecules ranging from the receptor to adenylyl cyclase. The prevailing view of the agonist-induced interaction between signaling molecules is based on random collisions between proteins that diffuse freely in the plasma membrane. The recent identification of G protein alpha- and betagamma-subunits in caveolae and their functional interaction with caveolin suggests that caveolae may participate in G protein-coupled signaling. We have investigated the potential interaction of beta-adrenergic receptors with caveolin under resting conditions. beta1- and beta2-adrenergic receptors were recombinantly overexpressed in COS-7 cells. Caveolae were isolated using the detergent-free sucrose gradient centrifugation method. beta1- and beta2-adrenergic receptors were localized in the same gradient fractions as caveolin, where Gsalpha- and betagamma-subunits were detected as well. Immunofluorescence microscopy demonstrated the colocalization of beta-adrenergic receptors with caveolin, indicating a nonrandom distribution of beta-adrenergic receptors in the plasma membrane. Using polyhistidine-tagged recombinant proteins, beta-adrenergic receptors were copurified with caveolin, suggesting that they were physically bound. Our results suggest that, in addition to clathrin-coated pits, caveolae may act as another plasma membrane microdomain to compartmentalize beta-adrenergic receptors.     

The caveolae dress code: structure and signaling

Over the past decade, interest in caveolae biology has peaked. These small bulb-shaped plasma membrane invaginations of 50–80 nm diameter present in most cell types have been upgraded from simple membrane structures to a more complex bona fide organelle. However, although caveolae are involved in several essential cellular functions and pathologies, the underlying molecular mechanisms remain poorly defined. Following the identification of caveolins and cavins as the main caveolae constituents, recent studies have brought new insight into their structural organization as a coat. In this review, we discuss how these new data on caveolae can be integrated in the context of their role in signaling and pathophysiology.     

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.     

Reduction of caveolin 1 gene expression in lung carcinoma cell lines

Caveolae are plasma membrane microdomains that have been implicated in organizing and concentrating certain signaling molecules. Caveolins, constitute the main structural proteins of caveolae. Caveolae are abundant in terminally differentiated cell types. However, caveolin-1 is down-regulated in transformed cells and may have a potential tumor suppressor activity. In the lung, caveolae are present in the endothelium, smooth muscle cells, fibroblasts as well as in type I pneumocytes. The presence of caveolae and caveolin expression in the bronchial epithelium, although probable, has not been investigated in human. We were interested to see if the bronchial epithelia express caveolins and if this expression was modified in cancer cells. We thus tested for caveolin-1 and -2 expression several bronchial epithelial primary cell lines as well as eight lung cancer cell lines and one larynx tumor cell line. Both caveolin-1 and -2 are expressed in all normal bronchial cell lines. With the exception of Calu-1 cell line, all cancer cell lines showed very low or no expression of caveolin-1 while caveolin-2 expression was similar to the one observed in normal bronchial epithelial cells.     

Membrane microdomains,caveolae, and caveolar endocytosis of sphingolipids (Review)

Caveolae are flask-shape membrane invaginations of the plasma membrane that have been implicated in endocytosis, transcytosis, and cell signaling. Recent years have witnessed the resurgence of studies on caveolae because they have been found to be involved in the uptake of some membrane components such as glycosphingolipids and integrins, as well as viruses, bacteria, and bacterial toxins. Accumulating evidence shows that endocytosis mediated by caveolae requires unique structural and signaling machinery (caveolin-1, src kinase), which indicates that caveolar endocytosis occurs through a mechanism which is distinct from other forms of lipid microdomain-associated, clathrin-independent endocytosis. Furthermore, a balance of glycosphingolipids, cholesterol, and caveolin-1 has been shown to be important in regulating caveolae endocytosis.     

Membrane microdomains, caveolae, and caveolar endocytosis of sphingolipids

Caveolae are flask-shape membrane invaginations of the plasma membrane that have been implicated in endocytosis, transcytosis, and cell signaling. Recent years have witnessed the resurgence of studies on caveolae because they have been found to be involved in the uptake of some membrane components such as glycosphingolipids and integrins, as well as viruses, bacteria, and bacterial toxins. Accumulating evidence shows that endocytosis mediated by caveolae requires unique structural and signaling machinery (caveolin-1, src kinase), which indicates that caveolar endocytosis occurs through a mechanism which is distinct from other forms of lipid microdomain-associated, clathrin-independent endocytosis. Furthermore, a balance of glycosphingolipids, cholesterol, and caveolin-1 has been shown to be important in regulating caveolae endocytosis.     

Differential targeting of beta -adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to functionally regulate the cAMP signaling pathway

Differential modes for beta(1)- and beta(2)-adrenergic receptor (AR) regulation of adenylyl cyclase in cardiomyocytes is most consistent with spatial regulation in microdomains of the plasma membrane. This study examines whether caveolae represent specialized subdomains that concentrate and organize these moieties in cardiomyocytes. Caveolae from quiescent rat ventricular cardiomyocytes are highly enriched in beta(2)-ARs, Galpha(i), protein kinase A RIIalpha subunits, caveolin-3, and flotillins (caveolin functional homologues); beta(1)-ARs, m(2)-muscarinic cholinergic receptors, Galpha(s), and cardiac types V/VI adenylyl cyclase distribute between caveolae and other cell fractions, whereas protein kinase A RIalpha subunits, G protein-coupled receptor kinase-2, and clathrin are largely excluded from caveolae. Cell surface beta(2)-ARs localize to caveolae in cardiomyocytes and cardiac fibroblasts (with markedly different beta(2)-AR expression levels), indicating that the fidelity of beta(2)-AR targeting to caveolae is maintained over a physiologic range of beta(2)-AR expression. In cardiomyocytes, agonist stimulation leads to a marked decline in the abundance of beta(2)-ARs (but not beta(1)-ARs) in caveolae. Other studies show co-immunoprecipitation of cardiomyocytes adenylyl cyclase V/VI and caveolin-3, suggesting their in vivo association. However, caveolin is not required for adenylyl cyclase targeting to low density membranes, since adenylyl cyclase targets to low buoyant density membrane fractions of HEK cells that lack prototypical caveolins. Nevertheless, cholesterol depletion with cyclodextrin augments agonist-stimulated cAMP accumulation, indicating that caveolae function as negative regulators of cAMP accumulation. The inhibitory interaction between caveolae and the cAMP signaling pathway as well as domain-specific differences in the stoichiometry of individual elements in the beta-AR signaling cascade represent important modifiers of cAMP-dependent signaling in the heart.     

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.     

Caveolin; different roles for insulin signal?

Caveolae, discovered by electron microscope in the 1950s, are membrane invaginations that accommodate various molecules that are involved in cellular signaling. Caveolin, a major protein component of caveolae identified in 1990s, has been known to inhibit the function of multiple caveolar proteins, such as kinases, which are involved in cell growth and proliferation, and thus considered to be a general growth signal inhibitor. Recent studies using transgenic mouse models have suggested that insulin signal may be exempted from this inhibition, which rather requires the presence of caveolin for proper signaling. Caveolin may stabilize insulin receptor protein or directly stimulate insulin receptors. Other studies have demonstrated that caveolae provide the TC10 complex with cellular microdomains for glucose transportation through Glut4. These findings suggest that caveolin plays an important role in insulin signal to maintain glucose metabolism in intact animals. However, the role of caveolin in insulin signal may differ from that in other transmembrane receptor signals.     

Reggie-1 and reggie-2 localize in non-caveolar rafts in epithelial cells: cellular localization is not dependent on the expression of caveolin proteins

Reggie-1 and reggie-2 are highly conserved and widely expressed proteins associated with membrane rafts. The molecular function of reggies remains to be clarified, but recent data indicate that they are involved in various cellular processes such as insulin signaling, phagocytosis and actin remodeling. However, there is discrepancy in the literature if reggies are associated with caveolae or non-caveolar rafts. Reggies are expressed and raft associated also in many cells which do not contain caveolae, such as neurons and lymphocytes. However, it is not clear if the function or localization of reggies are dependent on the presence of caveolae and expression of caveolin-1 protein. In this study, we directly addressed this question in epithelial cells. We could show that ectopic expression of caveolin-1 does not result in any change in the cellular localization of reggie-1, which is present at the plasma membrane also in the absence of caveolin-1. On the other hand, caveolin-2, which localizes in caveolae, is dependent on caveolin-1 expression in order to be localized at the plasma membrane. Although reggie-1 and reggie-2 strongly interact with each other, we did not detect a direct interaction between caveolin-1 and reggies by means of a yeast two-hybrid assay, nor could reggies be co-immunoprecipitated with caveolin-1. Furthermore, endogenous reggie-1 and -2 were found not to colocalize with caveolin-1 in epithelial cells. Thus, our data indicate that reggies are localized in microdomains different from caveolae, and the function of reggies is different from and independent of caveolin-1.     

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.     

Differential impact of caveolae and caveolin-1 scaffolds on the membrane raft proteome

Caveolae, a class of cholesterol-rich lipid rafts, are smooth invaginations of the plasma membrane whose formation in nonmuscle cells requires caveolin-1 (Cav1). The recent demonstration that Cav1-associated cavin proteins, in particular PTRF/cavin-1, are also required for caveolae formation supports a functional role for Cav1 independently of caveolae. In tumor cells deficient for Golgi β-1,6N-acetylglucosaminyltransferase V (Mgat5), reduced Cav1 expression is associated not with caveolae but with oligomerized Cav1 domains, or scaffolds, that functionally regulate receptor signaling and raft-dependent endocytosis. Using subdiffraction-limit microscopy, we show that Cav1 scaffolds are homogenous subdiffraction-limit sized structures whose size distribution differs from that of Cav1 in caveolae expressing cells. These cell lines displaying differing Cav1/caveolae phenotypes are effective tools for probing the structure and composition of caveolae. Using stable isotope labeling by amino acids in cell culture, we are able to quantitatively distinguish the composition of caveolae from the background of detergent-resistant membrane proteins and show that the presence of caveolae enriches the protein composition of detergent-resistant membrane, including the recruitment of multiple heterotrimeric G-protein subunits. These data were further supported by analysis of immuno-isolated Cav1 domains and of methyl-β-cyclodextrin-disrupted detergent-resistant membrane. Our data show that loss of caveolae results in a dramatic change to the membrane raft proteome and that this change is independent of Cav1 expression. The proteomics data, in combination with subdiffraction-limit microscopy, indicates that noncaveolar Cav1 domains, or scaffolds are structurally and functionally distinct from caveolae and differentially impact on the molecular composition of lipid rafts.     

Localization of apolipoprotein E receptor 2 to caveolae in the plasma membrane

The LDL receptor (LDL-R) promotes the specific endocytosis and lysosomal delivery of extracellular lipoprotein ligands via clathrin-coated pits. It was widely assumed that other closely related members of the LDL-R gene family would have similar functions, but recent experimental evidence has revealed that one such protein, apolipoprotein E receptor 2 (apoER2), has a critical role as an "outside-in" signal transducer in the brain. ApoER2 signaling appears to require interaction between its cytoplasmic domain and adapter molecules such as Dab1, JIP 1 and JIP 2, and PSD-95. Many of the receptors for other signaling pathways affected by such adapter molecules are compartmentalized into specialized microdomains within the plasma membrane termed caveolae. Here, we show that apoER2, but not LDL-R, is localized to caveolae, supporting the concept that its physiological role is in cell signaling, rather than in endocytosing ligands.     

Cavin proteins: New players in the caveolae field

Caveolae are specialized lipid microdomains, forming small invaginations in the plasma membrane, known to be implicated in multiple functions including lipid storage, cell signaling and endocytosis. Formation of these wide flask-shaped invaginations is dependent on the expression of a caveolar coat protein, namely caveolin. Until now, the accepted paradigm was that caveolin was the sole and only structural protein of caveolae since its expression was necessary and sufficient to drive caveolae biogenesis. The recent characterizations of PTRF/cavin-1 and subsequently other cavin family members in caveolae formation have highlighted additional levels of complexity in the biogenesis of these plasma membrane invaginations. In this review, recent advances on the role of the different cavin family members in the regulation of caveolae structures as well as potential new functions will be discussed.     

Triacylglycerol is synthesized in a specific subclass of caveolae in primary adipocytes

A principal metabolic function of adipocytes is to synthesize triacylglycerol (TG) from exogenous fatty acids. The level of fatty acids has to be tightly controlled in the adipocyte, as they can act as detergents that rapidly dissolve the plasma membrane, causing cell lysis if allowed to accumulate. Fatty acids therefore have to be efficiently converted to TG and stored in the central lipid droplet. We report that in intact primary adipocytes exogenous oleic acid was taken up and directly converted to TG in the plasma membrane, in a novel subclass of caveolae that specifically contains the protein perilipin. Isolated caveolae catalyzed de novo TG synthesis from oleic acid and glycerol 3-phosphate. Electron microscopy revealed the presence of caveolin and perilipin in caveolae and in lipid-laden bulbs in the plasma membrane, and fluorescence microscopy demonstrated colocalization of fatty acids/TG with caveolin and perilipin at the plasma membrane. A second caveolae fraction was isolated, which lacked perilipin and the triacylglycerol synthesizing enzymes. Both caveolae fractions contained caveolin-1 and the insulin receptor. The findings demonstrate that specific subclasses of caveolae carry out specific functions in cell metabolism. In particular, triacylglycerol is synthesized at the site of fatty acid entry in one of these caveolae classes.     

Dissociation of insulin receptor expression and signaling from caveolin-1 expression

The presence of cell surface caveolin/caveolae has been postulated to influence the localization, expression levels, and kinase activity of numerous receptors, including the insulin receptor. However, there are conflicting data concerning the effects of caveolin on insulin receptor expression and function. To help clarify this issue, we created a gain of function situation by expressing caveolin-1 at various levels in HEK-293 cells where the endogenous level of caveolin-1 is very low. We generated four permanent lines of this cell expressing amounts of caveolin-1 ranging from 10 to 40 times that of parental cells. The amount of caveolin-1 in the human embryonic kidney cells expressing the highest caveolin levels is comparable with that of adipocytes, cells that naturally express one of the highest levels of caveolin-1. We measured insulin receptor amount and insulin-dependent receptor autophosphorylation as well as insulin receptor substrate 1 (IRS1) tyrosine phosphorylation as an index of insulin signaling. We found that the insulin receptor level was essentially the same in the parental and all four derived cell lines. Likewise, we determined that insulin-dependent insulin receptor and IRS1 tyrosine phosphorylation was not significantly different in the four cell lines representing parental, low, medium, and high levels of caveolin-1 expression. We conclude that insulin receptor expression and ligand-dependent signaling is independent of caveolin-1 expression.     

Caveolins, caveolae, and lipid rafts in cellular transport, signaling, and disease.

Caveolae were initially described some 50 years ago. For many decades, they remained predominantly of interest to structural biologists. The identification of a molecular marker for these domains, caveolin, combined with the possibility to isolate such cholesterol- and sphingolipid-rich regions as detergent-insoluble membrane complexes paved the way to more rigorous characterization of composition, regulation, and function. Experiments with knock-out mice for the caveolin genes clearly demonstrate the importance of caveolin-1 and -3 in formation of caveolae. Nonetheless, detergent-insoluble domains are also found in cells lacking caveolin expression and are referred to here as lipid rafts. Caveolae and lipid rafts were shown to represent membrane compartments enriched in a large number of signaling molecules whose structural integrity is essential for many signaling processes. Caveolin-1 is an essential structural component of cell surface caveolae, important for regulating trafficking and mobility of these vesicles. In addition, caveolin-1 is found at many other intracellular locations. Variations in subcellular localization are paralleled by a plethora of ascribed functions for this protein. Here, more recent data addressing the role of caveolin-1 in cellular signaling and the development of diseases like cancer will be preferentially discussed.     

Insulin signaling in microdomains of the plasma membrane

Although the effects of insulin on glucose and lipid metabolism are well documented, gaps remain in our understanding of the precise molecular mechanisms of signal transduction. Recent evidence suggests that compartmentalization of signaling molecules and metabolic enzymes may explain the unique cellular effects of the hormone. Signal initiation from the insulin receptor is restricted in part to caveolae microdomains of the plasma membrane. A fraction of the insulin receptor directly interacts with caveolin, thus directing the protein to caveolae. Following its activation by insulin, the receptor recruits a series of adapter proteins, resulting in the activation of the G protein TC10, which also resides in caveolae. TC10 can influence a number of cellular processes, including changes in the actin cytoskeleton, recruitment of effector including the adapter protein CIP4, and assembly of the exocyst complex. These events play crucial roles in the trafficking, docking and fusion of vesicles containing the insulin-responsive glucose transporter Glut4 at the plasma membrane.