Presenilin 1 and presenilin 2 have differential effects on the stability and maturation of nicastrin in Mammalian brain

The presenilins and nicastrin form high molecular mass, multimeric protein complexes involved in the intramembranous proteolysis of several proteins. Post-translational glycosylation and trafficking of nicastrin is necessary for the activity of these complexes. We report here that although there are differences in the post-translational processing of nicastrin in neurons and glia, both of the presenilins are required for the physiological post-translational modification and for the correct subcellular distribution of nicastrin. Absence of presenilin 1 (PS1) is associated with dramatic reductions in the level of mature glycosylated nicastrin and with redistribution of nicastrin away from the cell surface. In contrast, absence of presenilin 2 (PS2) is associated with only modest reductions in the levels of immature nicastrin. It is notable that these differential effects parallel the differential effects of null mutations in PS1 and PS2 on APP and Notch processing. Our data therefore suggest that the differential interactions of PS1 and PS2 with nicastrin reflect different functions for the PS1 and PS2 complexes.     

Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane

The mechanisms involved in angiotensin II type 1 receptor (AT1-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT1-R is not concentrated in caveolae but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT1-R in caveolae, AT1-R.caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT1-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT1-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT1-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT1-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT1-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT1-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT1-R through the exocytic pathway, but this does not result in AT1-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT1-R.     

Positive and negative regulation of the gamma-secretase activity by nicastrin in a murine model

Nicastrin is a component of the gamma-secretase complex that has been shown to adhere to presenilin-1 (PS1), Notch, and APP. Here we demonstrate that Nicastrin-deficient mice showed a phenotype that is indistinguishable from PS1/PS2 double knock-out mice, whereas heterozygotes were healthy and viable. Fibroblasts derived from Nicastrin-deficient embryos were unable to generate amyloid beta-peptide and failed to release the intracellular domain of APP- or Notch1-Gal4-VP16 fusion proteins. Additionally, C- and N-terminal fragments of PS1 and the C-terminal fragments of PS2 were not detectable in Nicastrin-null fibroblasts, whereas full-length PS1 accumulated in null fibroblasts, indicating that Nicastrin is required for the endoproteolytic processing of presenilins. Interestingly, cells derived from Nicastrin heterozygotes produced relatively higher levels of amyloid beta-peptide whether the source was endogenous mouse or transfected human APP. These data demonstrate that Nicastrin is essential for the gamma-secretase cleavage of APP and Notch in mammalian cells and that Nicastrin has both positive and negative functions in the regulation of gamma-secretase activity.     

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.     

Identification and characterization of presenilin-independent Notch signaling.

Presenilin (PS) proteins control the proteolytic cleavage that precedes nuclear access of the Notch intracellular domain. Here we observe that a partial activation of the HES1 promoter can be detected in PS1/PS2 (PS1/2) double null cells using Notch1 Delta E constructs or following Delta 1 stimulation, despite an apparent abolition of the production and nuclear accumulation of the Notch intracellular domain. PS1/2-independent Notch activation is sensitive to Numblike, a physiological inhibitor of Notch. PS1/2-independent Notch signaling is also inhibited by an active gamma-secretase inhibitor in the low micromolar range and is not inhibited by an inactive analogue, similar to PS-dependent Notch signaling. However, experiments using a Notch1-Gal4-VP16 fusion protein indicate that the PS1/2-independent activity does not release Gal4-VP16 and is therefore unlikely to proceed via an intramembranous cleavage. These data reveal that a novel PS1/2-independent mechanism plays a partial role in Notch signal transduction.     

Caveolin moves from caveolae to the Golgi apparatus in response to cholesterol oxidation

Caveolae are a membrane specialization used to internalize molecules by potocytosis. Caveolin, an integral membrane protein, is associated with the striated coat present on the cytoplasmic surface of the caveolae membrane. We now report that oxidation of caveolar cholesterol with cholesterol oxidase rapidly displaces the caveolin from the plasma membrane to intracellular vesicles that colocalize with Golgi apparatus markers. After the enzyme is removed from the medium, caveolin returns to caveolae. When untreated cells are gently homogenized, caveolin on the plasma membrane is accessible to both anti-caveolin IgG and trypsin. After cholesterol oxidase treatment, however, Golgi-associated caveolin is inaccessible to both of these molecules. Brefeldin A, which inhibits ER to Golgi trafficking, blocks the appearance of caveolin in the Golgi apparatus but does not prevent caveolin from leaving the plasma membrane. Indirect immunogold localization experiments show that in the presence of cholesterol oxidase caveolin leaves the plasma membrane and becomes associated with endoplasmic reticulum and Golgi compartments. Surprisingly, the loss of caveolin from the plasma membrane does not affect the number or morphology of the caveolae.     

Segregation of heterotrimeric G proteins in cell surface microdomains. G(q) binds caveolin to concentrate in caveolae, whereas G(i) and G(s) target lipid rafts by default

Select lipid-anchored proteins such as glycosylphosphatidylinositol (GPI)-anchored proteins and nonreceptor tyrosine kinases may preferentially partition into sphingomyelin-rich and cholesterol-rich plasmalemmal microdomains, thereby acquiring resistance to detergent extraction. Two such domains, caveolae and lipid rafts, are morphologically and biochemically distinct, contain many signaling molecules, and may function in compartmentalizing cell surface signaling. Subfractionation and confocal immunofluorescence microscopy reveal that, in lung tissue and in cultured endothelial and epithelial cells, heterotrimeric G proteins (G(i), G(q), G(s), and G(betagamma)) target discrete cell surface microdomains. G(q) specifically concentrates in caveolae, whereas G(i) and G(s) concentrate much more in lipid rafts marked by GPI-anchored proteins (5' nucleotidase and folate receptor). G(q), apparently without G(betagamma) subunits, stably associates with plasmalemmal and cytosolic caveolin. G(i) and G(s) interact with G(betagamma) subunits but not caveolin. G(i) and G(s), unlike G(q), readily move out of caveolae. Thus, caveolin may function as a scaffold to trap, concentrate, and stabilize G(q) preferentially within caveolae over lipid rafts. In N2a cells lacking caveolae and caveolin, G(q), G(i), and G(s) all concentrate in lipid rafts as a complex with G(betagamma). Without effective physiological interaction with caveolin, G proteins tend by default to segregate in lipid rafts. The ramifications of the segregated microdomain distribution and the G(q)-caveolin complex without G(betagamma) for trafficking, signaling, and mechanotransduction are discussed.     

Dynamic modification of sphingomyelin in lipid microdomains controls development of obesity, fatty liver, and type 2 diabetes

Lipid microdomains or caveolae, small invaginations of plasma membrane, have emerged as important elements for lipid uptake and glucose homeostasis. Sphingomyelin (SM) is one of the major phospholipids of the lipid microdomains. In this study, we investigated the physiological function of sphingomyelin synthase 2 (SMS2) using SMS2 knock-out mice, and we found that SMS2 deficiency prevents high fat diet-induced obesity and insulin resistance. Interestingly, in the liver of SMS2 knock-out mice, large and mature lipid droplets were scarcely observed. Treatment with siRNA for SMS2 also decreased the large lipid droplets in HepG2 cells. Additionally, the siRNA of SMS2 decreased the accumulation of triglyceride in liver of leptin-deficient (ob/ob) mice, strongly suggesting that SMS2 is involved in lipid droplet formation. Furthermore, we found that SMS2 exists in lipid microdomains and partially associates with the fatty acid transporter CD36/FAT and with caveolin 1, a scaffolding protein of caveolae. Because CD36/FAT and caveolin 1 exist in lipid microdomains and are coordinately involved in lipid droplet formation, SMS2 is implicated in the modulation of the SM in lipid microdomains, resulting in the regulation of CD36/FAT and caveolae. Here, we established new cell lines, in which we can completely distinguish SMS2 activity from SMS1 activity, and we demonstrated that SMS2 could convert ceramide produced in the outer leaflet of the plasma membrane into SM. Our findings demonstrate the novel and dynamic regulation of lipid microdomains via conformational changes in lipids on the plasma membrane by SMS2, which is responsible for obesity and type 2 diabetes.     

Caveolin‐1 regulates proliferation and osteogenic differentiation of human mesenchymal stem cells

Caveolin‐1 is a scaffolding protein of cholesterol‐rich caveolae lipid rafts in the plasma membrane. In addition to regulating cholesterol transport, caveolin‐1 has the ability to bind a diverse array of cell signaling molecules and regulate cell signal transduction in caveolae. Currently, there is little known about the role of caveolin‐1 in stem cells. It has been reported that the caveolin‐1 null mouse has an expanded population of cells expressing stem cell markers in the gut, mammary gland, and brain, suggestive of a role for caveolin‐1 in stem cell regulation. The caveolin‐1 null mouse also has increased bone mass and an increased bone formation rate, and its bone marrow‐derived mesenchymal stem cells (MSCs) have enhanced osteogenic potential. However, the role of caveolin‐1 in human MSC osteogenic differentiation remains unexplored. In this study, we have characterized the expression of caveolin‐1 in human bone marrow derived MSCs. We show that caveolin‐1 protein is enriched in density gradient‐fractionated MSC plasma membrane, consisting of ～100 nm diameter membrane‐bound vesicles, and is distributed in a punctate pattern by immunofluoresence localization. Expression of caveolin‐1 increases in MSCs induced to undergo osteogenic differentiation, and siRNA‐mediated knockdown of caveolin‐1 expression enhances MSC proliferation and osteogenic differentiation. Taken together, these findings suggest that caveolin‐1 normally acts to regulate the differentiation and renewal of MSCs, and increased caveolin‐1 expression during MSC osteogenesis likely acts as a negative feedback to stabilize the cell phenotype. J. Cell. Biochem. 113: 3773–3787, 2012. © 2012 Wiley Periodicals, Inc.     

Bipolar assembly of caveolae in retinal pigment epithelium

Caveolae and their associated structural proteins, the caveolins, are specialized plasmalemmal microdomains involved in endocytosis and compartmentalization of cell signaling. We examined the expression and distribution of caveolae and caveolins in retinal pigment epithelium (RPE), which plays key roles in retinal support, visual cycle, and acts as the main barrier between blood and retina. Electron microscopic observation of rat RPE, in situ primary cultures of rat and human RPE and a rat RPE cell line (RPE-J) demonstrated in all cases the presence of caveolae in both apical and basolateral domains of the plasma membrane. Caveolae were rare in RPE in situ but were frequent in primary RPE cultures and in RPE-J cells, which correlated with increased levels in the expression of caveolin-1 and -2. The bipolar distribution of caveolae in RPE is striking, as all other epithelial cells examined to date (liver, kidney, thyroid, and intestinal) assemble caveolae only at the basolateral side. This might be related to the nonpolar distribution of both caveolin-1 and 2 in RPE because caveolin-2 is basolateral and caveolin-1 nonpolar in other epithelial cells. The bipolar localization of plasmalemmal caveolae in RPE cells may reflect specialized roles in signaling and trafficking important for visual function. caveolin; raft microdomains; membrane traffic; normal rat kidney     

Presenilins and gamma-secretase inhibitors affect intracellular trafficking and cell surface localization of the gamma-secretase complex components

The intramembranous cleavage of Alzheimer beta-amyloid precursor protein and the signaling receptor Notch is mediated by the presenilin (PS, PS1/PS2)-gamma-secretase complex, the components of which also include nicastrin, APH-1, and PEN-2. In addition to its essential role in gamma-secretase activity, we and others have reported that PS1 plays a role in intracellular trafficking of select membrane proteins including nicastrin. Here we examined the fate of PEN-2 in the absence of PS expression or gamma-secretase activity. We found that PEN-2 is retained in the endoplasmic reticulum and has a much shorter half-life in PS-deficient cells than in wild type cells, suggesting that PSs are required for maintaining the stability and proper subcellular trafficking of PEN-2. However, the function of PS in PEN-2 trafficking is distinct from its contribution to gamma-secretase activity because inhibition of gamma-secretase activity by gamma-secretase inhibitors did not affect the PEN-2 level or its egress from the endoplasmic reticulum. Instead, membrane-permeable gamma-secretase inhibitors, but not a membrane-impermeable derivative, markedly increased the cell surface levels of PS1 and PEN-2 without affecting that of nicastrin. In support of its role in PEN-2 trafficking, PS1 was also required for the gamma-secretase inhibitor-induced plasma membrane accumulation of PEN-2. We further showed that gamma-secretase inhibitors specifically accelerated the Golgi to the cell surface transport of PS1 and PEN-2. Taken together, we demonstrate an essential role for PSs in intracellular trafficking of the gamma-secretase components, and that selective gamma-secretase inhibitors differentially affect the trafficking of the gamma-secretase components, which may contribute to an inactivation of gamma-secretase.     

Raft association and lipid droplet targeting of flotillins are independent of caveolin

Lipid rafts are liquid ordered platforms that dynamically compartmentalize membranes. Caveolins and flotillins constitute a group of proteins that are enriched in these domains. Caveolin-1 has been shown to be an essential component of caveolae. Flotillins were also discovered as an integral component of caveolae and have since been suggested to interact with caveolins. However, flotillins are also expressed in non-caveolae-containing cells such as lymphocytes and neuronal cells. Hence, a discrepancy exists in the literature regarding the caveolin dependence of flotillin expression and their subcellular localization. To address this controversy, we used mouse embryonic fibroblasts (MEFs) from caveolin-1 knockout (Cav-1(-/-)) and wild-type mice to study flotillin expression and localization. Here we show that both membrane association and lipid raft partitioning of flotillins are not perturbed in Cav-1(-/-) MEFs, whereas membrane targeting and raft partitioning of caveolin-2, another caveolin family protein, is severely impaired. Moreover, we demonstrate that flotillin-1, but not flotillin-2, associates with lipid droplets upon oleic acid treatment and that this association is completely independent of caveolin. Taken together, our results show that flotillins are localized in lipid rafts independent of caveolin-1 and that translocation of flotillin-1 to lipid droplets is a caveolin-independent process.     

Overexpression of Caveolin‐1 Is Sufficient to Phenocopy the Behavior of a Disease‐Associated Mutant

Mutations and alterations in caveolin‐1 expression levels have been linked to a number of human diseases. How misregulation of caveolin‐1 contributes to disease is not fully understood, but has been proposed to involve the intracellular accumulation of mutant forms of the protein. To better understand the molecular basis for trafficking defects that trap caveolin‐1 intracellularly, we compared the properties of a GFP‐tagged version of caveolin‐1 P132L, a mutant form of caveolin‐1 previously linked to breast cancer, with wild‐type caveolin‐1. Unexpectedly, wild‐type caveolin‐1‐GFP also accumulated intracellularly, leading us to examine the mechanisms underlying the abnormal localization of the wild type and mutant protein in more detail. We show that both the nature of the tag and cellular context impact the subcellular distribution of caveolin‐1, demonstrate that even the wild‐type form of caveolin‐1 can function as a dominant negative under some conditions, and identify specific conformation changes associated with incorrectly targeted forms of the protein. In addition, we find intracellular caveolin‐1 is phosphorylated on Tyr14, but phosphorylation is not required for mistrafficking of the protein. These findings identify novel properties of mistargeted forms of caveolin‐1 and raise the possibility that common trafficking defects underlie diseases associated with overexpression and mutations in caveolin‐1.     

Cross-talk between caveolae and glycosylphosphatidylinositol-rich domains.

Most mammalian cells have in their plasma membrane at least two types of lipid microdomains, non-invaginated lipid rafts and caveolae. Glycosylphosphatidylinositol (GPI)-anchored proteins constitute a class of proteins that are enriched in rafts but not caveolae at steady state. We have analyzed the effects of abolishing GPI biosynthesis on rafts, caveolae, and cholesterol levels. GPI-deficient cells were obtained by screening for resistance to the pore-forming toxin aerolysin, which uses this class of proteins as receptors. Despite the absence of GPI-anchored proteins, mutant cells still contained lipid rafts, indicating that GPI-anchored proteins are not crucial structural elements of these domains. Interestingly, the caveolae-specific membrane proteins, caveolin-1 and 2, were up-regulated in GPI-deficient cells, in contrast to flotillin-1 and GM1, which were expressed at normal levels. Additionally, the number of surface caveolae was increased. This effect was specific since recovery of GPI biosynthesis by gene recomplementation restored caveolin expression and the number of surface caveolae to wild type levels. The inverse correlation between the expression of GPI-anchored proteins and caveolin-1 was confirmed by the observation that overexpression of caveolin-1 in wild type cells led to a decrease in the expression of GPI-anchored proteins. In cells lacking caveolae, the absence of GPI-anchored proteins caused an increase in cholesterol levels, suggesting a possible role of GPI-anchored proteins in cholesterol homeostasis, which in some cells, such as Chinese hamster ovary cells, can be compensated by caveolin up-regulation.     

Lipid rafts/caveolae are essential for insulin-like growth factor-1 receptor signaling during 3T3-L1 preadipocyte differentiation induction

Lipid rafts/caveolae are found to be essential for insulin-like growth factor (IGF)-1 receptor signaling during 3T3-L1 preadipocyte differentiation induction. In 3T3-L1 cells, IGF-1 receptor is located in lipid rafts/caveolae of the plasma membrane and can directly interact with caveolin-1, the major protein component in caveolae. Disruption of lipid rafts/caveolae by depleting cellular cholesterol with cholesterol-binding reagent, beta-methylcyclodextrin or filipin, blocks the IGF-1 receptor signaling in 3T3-L1 preadipocyte. Both hormonal induced adipocyte differentiation and mitotic clonal expansion are inhibited by lipid rafts/caveolae disruption. However, a nonspecific lipid binding reagent, xylazine, does not affect adipocyte differentiation or mitotic clonal expansion. Further studies indicate that lipid rafts/caveolae are required only for IGF-1 receptor downstream signaling and not the activation of receptor itself by ligand. Thus, our results suggest that localization in lipid rafts/caveolae and association with caveolin enable IGF-1 receptor to have a close contact with downstream signal molecules recruited into lipid rafts/caveolae and transmit the signal through these signal molecule complexes.     

Specificity of presenilin‐1‐ and presenilin‐2‐dependent γ‐secretases towards substrate processing

The two presenilin‐1 (PS1) and presenilin‐2 (PS2) homologs are the catalytic core of the γ‐secretase complex, which has a major role in cell fate decision and Alzheimer's disease (AD) progression. Understanding the precise contribution of PS1‐ and PS2‐dependent γ‐secretases to the production of β‐amyloid peptide (Aβ) from amyloid precursor protein (APP) remains an important challenge to design molecules efficiently modulating Aβ release without affecting the processing of other γ‐secretase substrates. To that end, we studied PS1‐ and PS2‐dependent substrate processing in murine cells lacking presenilins (PSs) (PS1KO, PS2KO or PS1‐PS2 double‐KO noted PSdKO) or stably re‐expressing human PS1 or PS2 in an endogenous PS‐null (PSdKO) background. We characterized the processing of APP and Notch on both endogenous and exogenous substrates, and we investigated the effect of pharmacological inhibitors targeting the PSs activity (DAPT and L‐685,458). We found that murine PS1 γ‐secretase plays a predominant role in APP and Notch processing when compared to murine PS2 γ‐secretase. The inhibitors blocked more efficiently murine PS2‐ than murine PS1‐dependent processing. Human PSs, especially human PS1, expression in a PS‐null background efficiently restored APP and Notch processing. Strikingly, and contrary to the results obtained on murine PSs, pharmacological inhibitors appear to preferentially target human PS1‐ than human PS2‐dependent γ‐secretase activity.     

Notch, epidermal growth factor receptor, and beta1-integrin pathways are coordinated in neural stem cells

Notch1 and beta1-integrins are cell surface receptors involved in the recognition of the niche that surrounds stem cells through cell-cell and cell-extracellular matrix interactions, respectively. Notch1 is also involved in the control of cell fate choices in the developing central nervous system (Lewis, J. (1998) Semin. Cell Dev. Biol. 9, 583-589). Here we report that Notch and beta1-integrins are co-expressed and that these proteins cooperate with the epidermal growth factor receptor in neural progenitors. We describe data that suggests that beta1-integrins may affect Notch signaling through 1) physical interaction (sequestration) of the Notch intracellular domain fragment by the cytoplasmic tail of the beta1-integrin and 2) affecting trafficking of the Notch intracellular domain via caveolin-mediated mechanisms. Our findings suggest that caveolin 1-containing lipid rafts play a role in the coordination and coupling of beta1-integrin, Notch1, and tyrosine kinase receptor signaling pathways. We speculate that this will require the presence of the adequate beta1-activating extracellular matrix or growth factors in restricted regions of the central nervous system and namely in neurogenic niches.     

Caveolae Are Highly Immobile Plasma Membrane Microdomains, Which Are not Involved in Constitutive Endocytic Trafficking

To investigate whether caveolae are involved in constitutive endocytic trafficking, we expressed N- and C- terminally green fluorescent protein (GFP)-tagged caveolin- 1 fusion proteins in HeLa, A431, and Madin-Darby canine kidney cells. The fusion proteins were shown by immunogold labeling to be sorted correctly to caveolae. By using confocal microscopy and photobleaching techniques, it was found that although intracellular structures labeled with GFP-tagged caveolin were dynamic, GFP-labeled caveolae were very immobile. However, after incubation with methyl- beta-cyclodextrin, distinct caveolae disappeared and the mobility of GFP-tagged caveolin in the plasma membrane increased. Treatment of cells with cytochalasin D caused lateral movement and aggregation of GFP-labeled caveolae. Therefore, both cholesterol and an intact actin cytoskeleton are required for the integrity of GFP-labeled caveolae. Moreover, stimulation with okadaic acid caused increased mobility and internalization of the labeled caveolae. Although the calculated mobile fraction (for t = infinity) of intracellular, GFP-tagged caveolin- associated structures was 70-90%, GFP-labeled caveolae in unstimulated cells had a mobile fraction of <20%, a value comparable to that previously reported for E-cadherin in junctional complexes. We therefore conclude that caveolae are not involved in constitutive endocytosis but represent a highly stable plasma membrane compartment anchored by the actin cytoskeleton.     

Caveolins and cellular cholesterol balance

Caveolins are major integral membrane components of caveolae. Over the last few years, evidence has accumulated for a close link between caveolin, caveolae, and the regulation of cellular cholesterol levels. However, the exact role of caveolin in this process, the intracellular trafficking routes followed by caveolin/cholesterol complexes, and the relationship of caveolin-cholesterol to other caveolin-mediated processes such as signal transduction have remained unclear. Recent findings from a number of systems suggest that specific signaling pathways require precise regulation of cellular cholesterol. Here we review evidence for caveolin regulation of cholesterol transport and consider how this may relate to signal transduction.