The class B, type I scavenger receptor (SR-BI) mediates the selective uptake of high density lipoprotein (HDL) cholesteryl esters and the efflux of free cholesterol. SR-BI is predominantly associated with caveolae in Chinese hamster ovary cells. The caveola protein, caveolin-1, binds to cholesterol and is involved in intracellular cholesterol trafficking. We previously demonstrated a correlative increase in caveolin-1 expression and the selective uptake of HDL cholesteryl esters in phorbol ester-induced differentiated THP-1 cells. The goal of the present study was to determine if the expression of caveolin-1 is the causative factor in increasing selective cholesteryl ester uptake in macrophages. To test this, we established RAW and J-774 cell lines that stably expressed caveolin-1. Transfection with caveolin-1 cDNA did not alter the amount of 125I-labeled HDL that associated with the cells, although selective uptake of HDL [3H]cholesteryl ether was decreased by approximately 50%. The amount of [3H]cholesterol effluxed to HDL was not affected by caveolin-1. To directly address whether caveolin-1 inhibits SR-BI-dependent selective cholesteryl ester uptake, we overexpressed caveolin-1 by adenoviral vector gene transfer in Chinese hamster ovary cells stably transfected with SR-BI. Caveolin-1 inhibited the selective uptake of HDL [3H]cholesteryl ether by 50-60% of control values without altering the extent of cell associated HDL. We next used blocking antibodies to CD36 and SR-BI to demonstrate that the increase in selective [3H]cholesteryl ether uptake previously seen in differentiated THP-1 cells was independent of SR-BI. Finally, we used beta-cyclodextrin and caveolin overexpression to demonstrate that caveolae depleted of cholesterol facilitate SR-BI-dependent selective cholesteryl ester uptake and caveolae containing excess cholesterol inhibit uptake. We conclude that caveolin-1 is a novel negative regulator of SR-BI-dependent selective cholesteryl ester uptake. (+info)
Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo.
(50/744)
The role of endothelial cell caveolae in the uptake and transport of macromolecules from the blood-space to the tissue-space remains controversial. To address this issue directly, we employed caveolin-1 gene knock-out mice that lack caveolin-1 protein expression and caveolae organelles. Here, we show that endothelial cell caveolae are required for the efficient uptake and transport of a known caveolar ligand, i.e. albumin, in vivo. Caveolin-1-null mice were perfused with 5-nm gold-conjugated albumin, and its uptake was followed by transmission electron microscopy. Our results indicate that gold-conjugated albumin is not endocytosed by Cav-1-deficient lung endothelial cells and remains in the blood vessel lumen; in contrast, gold-conjugated albumin was concentrated and internalized by lung endothelial cell caveolae in wild-type mice, as expected. To quantitate this defect in uptake, we next studied the endocytosis of radioiodinated albumin using aortic ring segments from wild-type and Cav-1-null mice. Interestingly, little or no uptake of radioiodinated albumin was observed in the aortic segments from Cav-1-deficient mice, whereas aortic segments from wild-type mice showed robust uptake that was time- and temperature-dependent and competed by unlabeled albumin. We conclude that endothelial cell caveolae are required for the efficient uptake and transport of albumin from the blood to the interstitium. (+info)
N-terminal protein acylation confers localization to cholesterol, sphingolipid-enriched membranes but not to lipid rafts/caveolae.
(51/744)
When variably fatty acylated N-terminal amino acid sequences were appended to a green fluorescent reporter protein (GFP), chimeric GFPs were localized to different membranes in a fatty acylation-dependent manner. To explore the mechanism of localization, the properties of acceptor membranes and their interaction with acylated chimeric GFPs were analyzed in COS-7 cells. Myristoylated GFPs containing a palmitoylated or polybasic region colocalized with cholesterol and ganglioside GM(1), but not with caveolin, at the plasma membrane and endosomes. A dipalmitoylated GFP chimera colocalized with cholesterol and GM(1) at the plasma membrane and with caveolin in the Golgi region. Acylated GFP chimeras did not cofractionate with low-density caveolin-rich lipid rafts prepared with Triton X-100 or detergent-free methods. All GFP chimeras, but not full-length p62(c-yes) and caveolin, were readily solubilized from membranes with various detergents. These data suggest that, although N-terminal acylation can bring GFP to cholesterol and sphingolipid-enriched membranes, protein-protein interactions are required to localize a given protein to detergent-resistant membranes or caveolin-rich membranes. In addition to restricting acceptor membrane localization, N-terminal fatty acylation could represent an efficient means to enrich the concentration of signaling proteins in the vicinity of detergent-resistant membranes and facilitate protein-protein interactions mediating transfer to a detergent-resistant lipid raft core. (+info)
Internalization of cholera toxin by different endocytic mechanisms.
(52/744)
The mechanism of cholera toxin (CT) internalization has been investigated using Caco-2 cells transfected with caveolin to induce formation of caveolae, HeLa cells with inducible synthesis of mutant dynamin (K44A) and BHK cells in which antisense mRNA to clathrin heavy chain can be induced. Here we show that endocytosis and the ability of CT to increase the level of cAMP were unaltered in caveolin-transfected cells grown either in a non-polarized or polarized manner. Treatment of Caco-2 cells with filipin reduced CT-uptake by less than 20%, suggesting that caveolae do not play a major role in the uptake. Extraction of cholesterol by methyl-beta-cyclodextrin, which removes caveolae and inhibits uptake from clathrin-coated pits, gave 30-40% reduction of CT-endocytosis. Also, CT-uptake in HeLa K44A cells was reduced by 50-70% after induction of mutant dynamin, which inhibits both caveolae- and clathrin-dependent endocytosis. These cells contain few caveolae, and nystatin and filipin had no effect on CT-uptake, indicating major involvement of clathrin-coated pits in CT-internalization. Similarly, in BHK cells, where clathrin-dependent endocytosis is blocked by induction of antisense clathrin heavy chain, the CT-uptake was reduced by 50% in induced cells. In conclusion, a large fraction of CT can be endocytosed by clathrin-dependent as well as by caveolae- and clathrin-independent endocytosis in different cell types. (+info)
Distinction between signaling mechanisms in lipid rafts vs. caveolae.
(53/744)
The relative importance of lipid rafts vs. specialized rafts termed caveolae to influence signal transduction is not known. Here we show that in cells lacking caveolae, the dually acylated protein, endothelial nitric oxide synthase (eNOS), localizes to cholesterol-rich lipid raft domains of the plasma membrane. In these cells, expression of caveolin-1 (cav-1) stimulates caveolae biogenesis, promotes the interaction of cav-1 with eNOS, and the inhibition of NO release from cells. Interestingly, in cells where cav-1 does not drive caveolae assembly, despite equal levels of cav-1 and eNOS and localization of both proteins to raft domains of the plasmalemma, the physical interaction of eNOS with cav-1 is dramatically less resulting in less inhibition of NO release. Thus, cav-1 concentrated in caveolae, not in rafts, is in closer proximity to eNOS and is necessary for negative regulation of eNOS function, thereby providing the first clear example of spatial regulation of signaling in this organelle that is distinct from raft domains. (+info)
The chemokine receptor CCR2 mediates the binding and internalization of monocyte chemoattractant protein-1 along brain microvessels.
(54/744)
Previous results from this laboratory revealed the presence of high-affinity saturable binding sites for monocyte chemoattractant protein-1 (MCP-1) along human brain microvessels (Andjelkovic et al., 1999; Andjelkovic and Pachter, 2000), which suggested that CC chemokine receptor 2 (CCR2), the recognized receptor for this chemokine, was expressed by the brain microvascular endothelium. To test the role of CCR2 directly in mediating MCP-1 interactions with the brain microvasculature, we assessed MCP-1 binding activity in murine brain microvessels isolated from wild-type mice and from CCR2 (-/-) mice engineered to lack this receptor. Results demonstrate that MCP-1 binding is greatly attenuated in microvessels prepared from CCR2 (-/-) mice compared with wild-type controls. Moreover, microvessels from wild-type mice exhibited MCP-1-induced downmodulation in MCP-1 binding and a recovery of binding activity that was not dependent on de novo protein synthesis. Furthermore, MCP-1 was shown to be internalized within wild-type microvessels, but not within microvessels obtained from CCR2 (-/-) mice, additionally demonstrating that CCR2 is obligatory for MCP-1 endocytosis. Last, internalization of MCP-1, but not transferrin, was observed to be inhibited by disruption of caveolae. Internalized MCP-1 also colocalized at some sites with caveolin-1, a major protein of caveolae, implying that this chemokine is endocytosed, in part, via nonclathrin-coated vesicles. These results prompt consideration that MCP-1 signals may be relayed across the blood-brain barrier by highly specialized interactions of this chemokine with its cognate receptor, CCR2, along brain microvascular endothelial cells. (+info)
Caveolin-1 is a negative regulator of caveolae-mediated endocytosis to the endoplasmic reticulum.
(55/744)
Caveolae are flask-shaped invaginations at the plasma membrane that constitute a subclass of detergent-resistant membrane domains enriched in cholesterol and sphingolipids and that express caveolin, a caveolar coat protein. Autocrine motility factor receptor (AMF-R) is stably localized to caveolae, and the cholesterol extracting reagent, methyl-beta-cyclodextrin, inhibits its internalization to the endoplasmic reticulum implicating caveolae in this distinct receptor-mediated endocytic pathway. Curiously, the rate of methyl-beta-cyclodextrin-sensitive endocytosis of AMF-R to the endoplasmic reticulum is increased in ras- and abl-transformed NIH-3T3 cells that express significantly reduced levels of caveolin and few caveolae. Overexpression of the dynamin K44A dominant negative mutant via an adenovirus expression system induces caveolar invaginations sensitive to methyl-beta-cyclodextrin extraction in the transformed cells without increasing caveolin expression. Dynamin K44A expression further inhibits AMF-R-mediated endocytosis to the endoplasmic reticulum in untransformed and transformed NIH-3T3 cells. Adenoviral expression of caveolin-1 also induces caveolae in the transformed NIH-3T3 cells and reduces AMF-R-mediated endocytosis to the endoplasmic reticulum to levels observed in untransformed NIH-3T3 cells. Cholesterol-rich detergent-resistant membrane domains or glycolipid rafts therefore invaginate independently of caveolin-1 expression to form endocytosis-competent caveolar vesicles via rapid dynamin-dependent detachment from the plasma membrane. Caveolin-1 stabilizes the plasma membrane association of caveolae and thereby acts as a negative regulator of the caveolae-mediated endocytosis of AMF-R to the endoplasmic reticulum. (+info)
Cellular cholesterol efflux.
(56/744)
Efflux of free cholesterol (FC) continues even when cellular FC mass is unchanged. This reflects a recirculation of preformed FC between cells and extracellular fluids which has multiple functions in cell biology including receptor recycling and signaling as well as cellular FC homeostasis. Total FC efflux is heterogeneous. Simple diffusion to mature high density lipoprotein (HDL), mainly via albumin as intermediate, initiates FC net transport driven by plasma lecithin:cholesterol acyltransferase activity. A second major efflux component reflects protein-facilitated transport from cell surface domains (caveolae, rafts) driven by FC binding to lipid-poor, pre-beta-migrating HDL (pre-beta-HDL). Facilitated efflux from caveolae, unlike simple diffusion, is highly regulated. Neither ABC1 (the protein defective in Tangier disease) nor other ATP-dependent transporters now appear likely to contribute directly to FC efflux. Their role is limited to the initial formation of a particle precursor to circulating pre-beta-HDL, which recycles without further lipid input from ATP-dependent transporter proteins. Lipid-free apolipoprotein A-I, previously considered a surrogate for pre-beta-HDL, has a reactivity much lower than that of native lipoprotein FC acceptors. (+info)