Caveolin isoforms in resident and elicited rat peritoneal macrophages

Caveolin--an integral membrane protein--is the principal component of caveolae membranes in vivo. Multiple forms of caveolin have been identified: caveolin-1alpha, caveolin-1beta, caveolin-2 and caveolin-3. They differ in their specific properties and tissue distribution. When we studied the lysate of resident and elicited macrophages isolated from rat peritoneal cavity by Western blot analysis, we identified two different proteins (approximately 29 kDa and approximately 20 kDa) which were labelled with anti-caveolin antibodies. The approximately 20-kDa protein was labelled specifically only by anti-VIP21/caveolin-1, while the approximately 29-kDa protein was labelled by anti-VIP21/caveolin-1 and anti-caveolin-2. The presence of the approximately 29-kDa protein was characteristic of resident macrophages, and only a small amount of the approximately 20-kDa protein was detected in these cells. Elicitation resulted in a significant increase in the amount of the approximately 20-kDa protein labelled by anti-VIP21/caveolin-1 only. According to its molecular mass and antibody-specificity, this protein might be identical with the caveolin-1beta isoform. Our morphological (confocal and electron microscopical) studies have shown that in resident cells caveolin was present in the cytoplasm, in smaller vesicles and multivesicular bodies around the Golgi area. Only a very small amount of caveolae was found on the surface of these cells. In elicited macrophages, caveolae (labelled with the anti-VIP21/caveolin-1 antibody) appeared in large numbers on the cell surface, but caveolin detected by anti-caveolin-2 was also found in small vesicles and multivesicular bodies in the cytoplasm. According to these results, the absence of caveolae in resident cells can be explained by the absence of caveolin-1. The expression of the approximately 29-kDa (caveolin-related) protein in resident macrophages seems to be insufficient for caveolae formation. Elicitation significantly increased the expression of caveolin-1, and the increased amount of caveolin-1 resulted in caveolae formation on the cell surface.     

cav-p60 expression in rat muscle tissues

Caveolae are plasmalemmal invaginations of uncertain function. In view of the large number of hypotheses on caveolar functions, it is important to identify which components of caveolae are tissue specific and which are general. The only well-characterized major protein of caveolae is caveolin, which exists in three tissue-specific isoforms: caveolin-1, -2, and -3. Recently cav-p60 was characterized as a 60-kDa caveola-specific protein in adipocytes. The distributions of cav-p60 and caveolin isoforms in different rat muscle tissues were examined by immunofluorescence and immunoelectron microscopy. Cav-p60 was present in caveolae of skeletal and heart muscle, in vascular and intestinal smooth muscle, and in adipocyte caveolae. Furthermore cav-p60 was present in endothelial cells and cells of perineural sheaths. Caveolin-1 and -2 were present in adipocytes, endothelial cells, and cells of perineural sheaths. In all kinds of vascular and intestinal smooth muscle, caveolin-1 and -2 were present at high levels, whereas caveolin-3 expression was low or undetectable, depending on the specific smooth muscle subtype. High levels of caveolin-3 were found only in caveolae and T tubules of skeletal and heart muscle. We conclude that cav-p60 is a highly specific marker of caveolae in many if not all cell types having caveolae.     

Plasma membrane cyclic nucleotide phosphodiesterase 3B (PDE3B) is associated with caveolae in primary adipocytes

Caveolae, plasma membrane invaginations particularly abundant in adipocytes, have been suggested to be important in organizing insulin signalling. Insulin-induced activation of the membrane bound cAMP degrading enzyme, phosphodiesterase 3B (PDE3B) is a key step in insulin-mediated inhibition of lipolysis and is also involved in the regulation of insulin-mediated glucose uptake and lipogenesis in adipocytes. The aim of this work was to evaluate whether PDE3B is associated with caveolae. Subcellular fractionation of primary rat and mouse adipocytes demonstrated the presence of PDE3B in endoplasmic reticulum and plasma membrane fractions. The plasma membrane PDE3B was further analyzed by detergent treatment at 4 degrees C, which did not solubilize PDE3B, indicating an association of PDE3B with lipid rafts. Detergent-treated plasma membranes were studied using Superose-6 chromatography which demonstrated co-elution of PDE3B with caveolae and lipid raft markers (caveolin-1, flotillin-1 and cholesterol) at a Mw of >4000 kDa. On sucrose density gradient centrifugation of sonicated plasma membranes, a method known to enrich caveolae, PDE3B co-migrated with the caveolae markers. Immunoprecipitation of caveolin-1 using anti caveolin-1 antibodies co-immunoprecipitated PDE3B and immunoprecipitation of flag-PDE3B from adipocytes infected with a flag-PDE3B adenovirus resulted in co-immunoprecipitation of caveolin-1. Studies on adipocytes with disrupted caveolae, using either caveolin-1 deficient mice or treatment of adipocytes with methyl-beta-cyclodextrin, reduced the membrane associated PDE3B activity. Furthermore, inhibition of PDE3 in primary rat adipocytes resulted in reduced insulin stimulated glucose transporter-4 translocation to caveolae, isolated by immunoprecipitation using caveolin-1 antibodies. Thus, PDE3B, a key enzyme in insulin signalling, appears to be associated with caveolae in adipocytes and this localization seems to be functionally important.     

Role of Src-induced dynamin-2 phosphorylation in caveolae-mediated endocytosis in endothelial cells

Albumin transcytosis, a determinant of transendothelial permeability, is mediated by the release of caveolae from the plasma membrane. We addressed the role of Src phosphorylation of the GTPase dynamin-2 in the mechanism of caveolae release and albumin transport. Studies were made in microvascular endothelial cells in which the uptake of cholera toxin subunit B, a marker of caveolae, and (125)I-albumin was used to assess caveolae-mediated endocytosis. Albumin binding to the 60-kDa cell surface albumin-binding protein, gp60, induced Src activation (phosphorylation on Tyr(416)) within 1 min and resulted in Src-dependent tyrosine phosphorylation of dynamin-2, which increased its association with caveolin-1, the caveolae scaffold protein. Expression of kinase-defective Src mutant interfered with the association between dynamin-2, which caveolin-1 and prevented the uptake of albumin. Expression of non-Src-phosphorylatable dynamin (Y231F/Y597F) resulted in reduced association with caveolin-1, and in contrast to WT-dynamin-2, the mutant failed to translocate to the caveolin-rich membrane fraction. The Y231F/Y597F dynamin-2 mutant expression also resulted in impaired albumin and cholera toxin subunit B uptake and reduced transendothelial albumin transport. Thus, Src-mediated phosphorylation of dynamin-2 is an essential requirement for scission of caveolae and the resultant transendothelial transport of albumin.     

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.     

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.     

A distinct class of endosome mediates clathrin-independent endocytosis to the Golgi complex

Mammalian cells endocytose a variety of proteins and lipids without utilising clathrin-coated pits. Detailed molecular mechanisms for clathrin-independent endocytosis are unclear. Several markers for this process, including glycosphingolipid-binding bacterial toxin subunits such as cholera toxin B subunit (CTxB), and glycosyl-phosphatidyl-inositol (GPI)-anchored proteins, are found in detergent-resistant membrane fractions (DRMs), or 'lipid rafts'. The Golgi complex constitutes one principal intracellular destination for these markers. Uptake of both CTxB and GPI-anchored proteins may involve caveolae, small invaginations in the plasma membrane (PM). However, the identity of intermediate organelles involved in PM to Golgi trafficking, as well as the function of caveolins, defining protein components of caveolae, are unclear. This paper shows that molecules which partition into DRMs and are endocytosed in a clathrin-independent fashion, accumulate in a discrete population of endosomes en route to the Golgi complex. These endosomes are devoid of markers for classical early and recycling endosomes, but do contain caveolin-1. Caveolin-1-positive endosomes are sites for the sorting of caveolin-1 away from Golgi-bound cargoes, although caveolin-1 itself is unlikely to have a direct function in PM to Golgi transport.     

Localization of ras antigenicity in rat hepatocyte plasma membrane and rough endoplasmic reticulum fractions

We have examined the antigenicity of plasma membrane (PM) and rough microsomal (RM) fractions from rat liver using anti-ras monoclonal antibodies 142-24EO5 and Y13-259 and immunochemistry as well as electron microscope immunocytochemistry. Proteins immunoprecipitated with monoclonal antibody 142-24E05 were separated using single-dimensional gradient-gel electrophoresis. The separated proteins were then blotted onto nitrocellulose sheets and incubated with [alpha-32P]GTP. Radioautograms of blots indicated the presence of specific 21.5- and 22-kDa labeled proteins in the PM fraction. A 23.5-kDa [alpha-32P] GTP-binding protein was detected in immunoprecipitates of both PM and RM fractions. Monoclonal antibody Y13-259 reacted only with the 21.5-kDa [alpha-32P] GTP-binding protein in the plasma membrane fraction. When anti-ras monoclonal antibody 142-24E05 and the immunogold technique were applied to membrane fractions using a preembedding immunocytochemical method, specific labeling was observed in association with both vesicular structures and membrane sheets in the PM fraction but only with electron-dense vesicular structures in the RM fraction. Thus ras antigenicity is associated with hepatocyte plasma membranes and ras-like antigenicity is probably associated with vesicular (secretory/endocytic) elements in both plasma membrane and rough microsomal preparations.     

Cholesterol depletion inhibits fatty acid uptake without affecting CD36 or caveolin-1 distribution in adipocytes

Caveolin-1 and CD36 are plasma membrane fatty acid binding proteins that participate in adipocyte fatty acid uptake and metabolism. Both are associated with cholesterol-enriched caveolae/lipid rafts in the plasma membrane that are important for long chain fatty acid uptake. Depletion of plasma membrane cholesterol reversibly inhibited oleate uptake by adipocytes without altering the amount or the cell surface distribution of either caveolin-1 or CD36. Cholesterol levels thus regulate fatty acid uptake by adipocytes via a pathway that does not involve altered cell surface localization of caveolin-1 or CD36.     

A critical role of cavin (polymerase I and transcript release factor) in caveolae formation and organization

Cavin (PTRF) has been shown to be a highly abundant protein component of caveolae, but its functional role there is unknown. Here, we confirm that cavin co-localizes with caveolin-1 in adipocytes by confocal microscopy and co-distributes with caveolin-1 in lipid raft fractions by sucrose gradient flotation. However, cavin does not directly associate with caveolin-1 as solubilization of caveolae disrupts their interaction. Cholesterol depletion with beta-cyclodextrin causes a significant down-regulation of cavin from plasma membrane lipid raft fractions. Overexpression of cavin in HEK293-Cav-1 cells and knockdown of cavin in 3T3-L1 adipocytes enhances and diminishes caveolin-1 levels, respectively, indicating an important role for cavin in maintaining the level of caveolin-1. A truncated form of cavin, eGFP-cavin-1-322, which lacks 74 amino acids from the C-terminal, reveals a microtubular network localization by confocal microscopy. Disruption of cytoskeletal elements with latrunculin B or nocodazole diminishes cavin expression without affecting the caveolin-1 amount. We propose that the presence of cavin on the inside surface of caveolae stabilizes these structures, probably through interaction with the cytoskeleton, and cavin therefore plays an important role in caveolae formation and organization.     

Identification of caveolin-1 as a fatty acid binding protein

In an attempt to identify high affinity, fatty acid binding proteins present in 3T3-L1 adipocytes plasma membranes, we labeled proteins in purified plasma membranes with the photoreactive fatty acid analogue, 11-m-diazirinophenoxy[11-3H]undecanoate. A single membrane protein of 22 kDa was covalently labeled after photolysis. This protein fractionated with caveolin-1 containing caveolae and was immunoprecipitated by an anti-caveolin-1 monoclonal antibody. Furthermore, 2D-PAGE analysis revealed that both the alpha and beta isoforms of caveolin-1 could be labeled by the photoreactive fatty acid upon photolysis, indicating that both bind fatty acids. The saturable binding of the photoreactive fatty acid suggests caveolin-1 has a lipid binding site that may either operate during intracellular lipid traffic or regulate caveolin-1 function.     

Influence of trans-membrane potential and of hydrophobic interactions on dye accumulation in mitochondria of living cells

The lipophilic cationic fluorescent dye azopentylmethylindocarbocyanine (APMC) specifically stains the mitochondria in living cells. The dye contains a photosensitive diazirine ring and is suitable for photoaffinity labelling of mitochondrial proteins. By a combination of photoaffinity labelling of cell cultures of mouse fibroblasts (LM) with APMC, lysis of the labelled cells, subsequent micro-gel electrophoresis and detection of the fluorescence of the labelled proteins in the gel lanes with a sensitive microfluorimeter, we determined the number, apparent molecular masses, and relative intensity of the labelled proteins. In LM cells, three proteins with apparent molecular masses of 31, 40, and 74 kDa were labelled with high intensity, and proteins of 28, 29, 44, 48, 49, 66, and 105 kDa with low intensity. Two effects mainly determine the binding of lipophilic dye cations to mitochondrial proteins in living cells: (1) interaction of the trans-membrane potential of the inner mitochondrial membrane with the dye cations; and (2) hydrophobic interactions between the strongly lipophilic proteins of the inner membrane and the lipophilic dye molecules. Preincubation of the cell cultures with drugs that dissipate the trans-membrane potential, such as valinomycin, 2,4-dinitrophenol (DNP) and 3-chlorcarbonylcyanidephenylhydrazon (CCCP), strongly reduces or even prevents APMC labelling of mitochondrial proteins. The influence of hydrophobic interactions was investigated by competitive staining experiments using dyes with very different lipophilic properties. The lipophilicity of the dyes was characterized by their R _m values in reversed phase thin-layer chromatography. Prestaining with an excess of strongly lipophilic cationic acridine and phenanthridine dyes, such as pentyl acridinium orange chloride (PAO), nonyl acridinium orange chloride (NAO) and tetramethylpropidium chloride (MP), respectively, completely prevents protein labelling with APMC. Obviously, the dyes occupy the same mitochondrial binding sites as APMC. At equal concentrations the intensity of the 40-kDa signal is strongly reduced, whereas the 31-kDa and 74-kDa signals are unaffected. Using phenanthridine dyes with lower lipophilicity, namely propidium chloride (P), M, and N reduces the peak of the 40-kDa protein in APMC labelling, indicating that the 40-kDa protein preferentially binds lipophilic dye cations.     

Spatial segregation of transport and signalling functions between human endothelial caveolae and lipid raft proteomes

Lipid rafts and caveolae are biochemically similar, specialized domains of the PM (plasma membrane) that cluster specific proteins. However, they are morphologically distinct, implying different, possibly complementary functions. Two-dimensional gel electrophoresis preceding identification of proteins by MS was used to compare the relative abundance of proteins in DRMs (detergent-resistant membranes) isolated from HUVEC (human umbilical-vein endothelial cells), and caveolae immunopurified from DRM fractions. Various signalling and transport proteins were identified and additional cell-surface biotinylation revealed the majority to be exposed, demonstrating their presence at the PM. In resting endothelial cells, the scaffold of immunoisolated caveolae consists of only few resident proteins, related to structure [CAV1 (caveolin-1), vimentin] and transport (V-ATPase), as well as the GPI (glycosylphosphatidylinositol)-linked, surface-exposed protein CD59. Further quantitative characterization by immunoblotting and confocal microscopy of well-known [eNOS (endothelial nitric oxide synthase) and CAV1], less known [SNAP-23 (23 kDa synaptosome-associated protein) and BASP1 (brain acid soluble protein 1)] and novel [C8ORF2 (chromosome 8 open reading frame 2)] proteins showed different subcellular distributions with none of these proteins being exclusive to either caveolae or DRM. However, the DRM-associated fraction of the novel protein C8ORF2 (approximately 5% of total protein) associated with immunoseparated caveolae, in contrast with the raft protein SNAP-23. The segregation of caveolae from lipid rafts was visually confirmed in proliferating cells, where CAV1 was spatially separated from eNOS, SNAP-23 and BASP1. These results provide direct evidence for the previously suggested segregation of transport and signalling functions between specialized domains of the endothelial plasma membrane.     

Tumour necrosis factor alpha-induced adipose-related protein (TIARP): co-localization with caveolin-1

We previously identified TIARP (TNF(alpha)-induced adipose-related protein, where TNF(alpha) stands for tumour necrosis factor alpha), a novel plasma-membrane protein that is induced during 3T3-L1 preadipocytes differentiation by TNF(alpha). Whereas the biological function of TIARP is currently unknown, its protein sequence is reminiscent of transporter protein and/or NAD(P)/NAD(P)H-dependent oxidoreductase activities. We hypothesized that TIARP could be associated with the 3T3-L1 adipocyte plasma-membrane caveolae domains that contain many proteins involved in cellular trafficking and signalling processes. Studies by confocal microscopy showed that TIARP and caveolin-1, a major protein of caveolae, co-localized as patches at the plasma membrane. Immunoblot analysis of cell extracts indicated that TIARP was completely detergent-extractible from membranes, whereas caveolin-1 was present as both detergent-extractible and -insoluble pools. Since TIARP is compartmentalized with caveolin-1 within caveolae domains, we suggest this protein to be part of a signalling complex in association with caveolin-1 and regulatory proteins.     

Agonist-modulated targeting of the EDG-1 receptor to plasmalemmal caveolae. eNOS activation by sphingosine 1-phosphate and the role of caveolin-1 in sphingolipid signal transduction

Plasmalemmal caveolae are membrane microdomains that are specifically enriched in sphingolipids and contain a wide array of signaling proteins, including the endothelial isoform of nitric-oxide synthase (eNOS). EDG-1 is a G protein-coupled receptor for sphingosine 1-phosphate (S1P) that is expressed in endothelial cells and has been implicated in diverse vascular signal transduction pathways. We analyzed the subcellular distribution of EDG-1 in COS-7 cells transiently transfected with cDNA constructs encoding epitope-tagged EDG-1. Subcellular fractionation of cell lysates resolved by ultracentrifugation in discontinuous sucrose gradients revealed that approximately 55% of the EDG-1 protein was recovered in fractions enriched in caveolin-1, a resident protein of caveolae. Co-immunoprecipitation experiments showed that EDG-1 could be specifically precipitated by antibodies directed against caveolin-1 and vice versa. The targeting of EDG-1 to caveolae-enriched fractions was markedly increased (from 51 +/- 11% to 93 +/- 14%) by treatment of transfected cells with S1P (5 microm, 60 min). In co-transfection experiments expressing EDG-1 and eNOS cDNAs in COS-7 cells, we found that S1P treatment significantly and specifically increased nitric-oxide synthase activity, with an EC(50) of 30 nm S1P. Overexpression of transfected caveolin-1 cDNA together with EDG-1 and eNOS markedly diminished S1P-mediated eNOS activation; caveolin overexpression also attenuated agonist-induced phosphorylation of EDG-1 receptor by >90%. These results suggest that the interaction of the EDG-1 receptor with caveolin may serve to inhibit signaling through the S1P pathway, even as the targeting of EDG-1 to caveolae facilitates the interactions of this receptor with ligands and effectors that are also targeted to caveolae. The agonist-modulated targeting of EDG-1 to caveolae and its dynamic inhibitory interactions with caveolin identify new points for regulation of sphingolipid-dependent signaling in the vascular wall.     

Cellular Factor XIIIA Transglutaminase Localizes in Caveolae and Regulates Caveolin-1 Phosphorylation,Homo-oligomerization and c-Src Signaling in Osteoblasts

Transglutaminases (TGs) are a family of widely distributed enzymes that catalyze protein crosslinking by forming a covalent isopeptide bond between the substrate proteins. We have shown that MC3T3-E1 osteoblasts express Factor XIII-A (FXIII-A), and that the extracellular crosslinking activity of FXIII-A is involved in regulating matrix secretion and deposition. In this study, we have investigated the localization and potential role of intracellular FXIII-A. Conventional immunofluorescence microscopy and TIRF microscopy analyses showed that FXIII-A co-localizes with caveolin-1 in specialized membrane structures, caveolae, in differentiating osteoblasts. The caveolae-disrupting agent methyl-β-cyclodextrin abolished FXIII-A staining and co-localization with caveolin-1 from the osteoblast plasma membrane. The presence of FXIII-A in caveolae was confirmed by preparing caveolae-enriched cellular fractions using sucrose density gradient ultracentrifugation followed by western blotting. Despite this association of FXIII-A with caveolae, there was no detectable transglutaminase activity in caveolae, as measured by monodansylcadaverine incorporation. TG inhibitor NC9—which can alter TG enzyme conformation—localized to caveolae and displaced FXIII-A from these structures when added to the osteoblast cultures. The decreased FXIII-A levels in caveolae after NC9 treatment increased c-Src activation, which resulted in caveolin-1 phosphorylation, homo-oligomerization and Akt phosphorylation, suggesting cellular FXIII-A has a role in regulating c-Src signaling in osteoblasts.     

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.     

Rabbit small intestine does not contain an annexin II/caveolin 1 complex as a target for 2-azetidinone cholesterol absorption inhibitors

Intestinal cholesterol absorption is specifically inhibited by the 2-azetidinone cholesterol absorption inhibitor ezetimibe. Photoreactive ezetimibe analogues specifically label a 145-kDa protein in the brush border membrane of enterocytes from rabbit small intestine identified as aminopeptidase N (CD13). In zebrafish and mouse small intestinal cytosol, a heterocomplex of M_r 52 kDa between annexin II and caveolin 1 was suggested as a target of ezetimibe. In contrast, in the cytosol and brush border membrane vesicles (BBMV) from rabbit small intestine of control animals or rabbits treated with the nonabsorbable cholesterol absorption inhibitor AVE 5530, both annexin II and caveolin 1 were exclusively present as monomers without any heterocomplex formation. Upon immunoprecipitation with annexin II a 52-kDa band was observed after immunostaining with annexin II antibodies, whereas no staining of a 52-kDa band occurred with anti-caveolin 1 antibodies. Vice versa, a 52-kDa band obtained by immunoprecipitation with caveolin 1 antibodies did not stain with annexin II-antibodies. The intensity of the 52-kDa band was dependent on the amount of antibody and was also observed with anti-actin or anti-APN antibodies suggesting that the 52-kDa band is a biochemical artefact. After incubation of cytosol or BBMV with radioactively labelled ezetimibe analogues, no significant amounts of the ezetimibe analogues could be detected in the immunoprecipitate with caveolin-1 or annexin II antibodies. Photoaffinity labelling of rabbit small intestinal BBMV with ezetimibe analogues did not result in labelling of proteins being immunoreactive with annexin II, caveolin 1 or a 52-kDa heterocomplex. These findings indicate that the rabbit small intestine does not contain an annexin II/caveolin 1 heterocomplex as a target for ezetimibe.     

Cell surface orifices of caveolae and localization of caveolin to the necks of caveolae in adipocytes

Caveolae are noncoated invaginations of the plasma membrane that form in the presence of the protein caveolin. Caveolae are found in most cells, but are especially abundant in adipocytes. By high-resolution electron microscopy of plasma membrane sheets the detailed structure of individual caveolae of primary rat adipocytes was examined. Caveolin-1 and -2 binding was restricted to the membrane proximal region, such as the ducts or necks attaching the caveolar bulb to the membrane. This was confirmed by transfection with myc-tagged caveolin-1 and -2. Essentially the same results were obtained with human fibroblasts. Hence caveolin does not form the caveolar bulb in these cells, but rather the neck and may thus act to retain the caveolar constituents, indicating how caveolin participates in the formation of caveolae. Caveolae, randomly distributed over the plasma membrane, were very heterogeneous, varying in size between 25 and 150 nm. There was about one million caveolae in an adipocyte, which increased the surface area of the plasma membrane by 50%. Half of the caveolae, those larger than 50 nm, had access to the outside of the cell via ducts and 20-nm orifices at the cell surface. The rest of the caveolae, those smaller than 50 nm, were not open to the cell exterior. Cholesterol depletion destroyed both caveolae and the cell surface orifices.     

Cathepsin B localizes to plasma membrane caveolae of differentiating myoblasts and is secreted in an active form at physiological pH

Our in vitro studies support a functional link between the induction of cathepsin B gene expression and the catabolic restructuring associated with myotube formation during myogenesis in vivo. We have tested two predictions that are basic to this hypothesis: (1) that active cathepsin B is localized to plasma membrane caveolae of fusing myoblasts; and (2) that active cathepsin B is secreted from fusing myoblasts at physiological pH. During differentiation, L6 rat myoblasts demonstrated a fusion-related increase in activity associated with the 25/26-kDa, fully processed, active form of cathepsin B. Immunocytochemical studies demonstrated a redistribution of lysosomal cathepsin B protein toward the membrane of fusing myoblasts, and a colocalization of cathepsin B with caveolin-3, the muscle-specific structural protein of membrane caveolae. Sucrose density fractionation and Western blot analysis demonstrated that an active form of cathepsin B localizes to caveolar fractions along with caveolin-3, annexin-VII, beta-dystroglycan and dystrophin. Finally, 'real-time' activity assays and Western blot analysis demonstrated that active cathepsin B is secreted from fusing myoblasts at physiological pH. Collectively, these studies support an association of active cathepsin B with plasma membrane caveolae and the secretion of active cathepsin B from differentiating myoblasts during myoblast fusion.