Caveolae as plasma membrane sensors, protectors and organizers.

Caveolae are submicroscopic, plasma membrane pits that are abundant in many mammalian cell types. The past few years have seen a quantum leap in our understanding of the formation, dynamics and functions of these enigmatic structures. Caveolae have now emerged as vital plasma membrane sensors that can respond to plasma membrane stresses and remodel the extracellular environment. Caveolae at the plasma membrane can be removed by endocytosis to regulate their surface density or can be disassembled and their structural components degraded. Coat proteins, called cavins, work together with caveolins to regulate the formation of caveolae but also have the potential to dynamically transmit signals that originate in caveolae to various cellular destinations. The importance of caveolae as protective elements in the plasma membrane, and as membrane organizers and sensors, is highlighted by links between caveolae dysfunction and human diseases, including muscular dystrophies and cancer.

Glutamine Uptake via SNAT6 and Caveolin Regulates Glutamine-Glutamate Cycle.

SLC38A6 (SNAT6) is the only known member of the SLC38 family that is expressed exclusively in the excitatory neurons of the brain. It has been described as an orphan transporter with an unknown substrate profile, therefore very little is known about SNAT6. In this study, we addressed the substrate specificity, mechanisms for internalization of SNAT6, and the regulatory role of SNAT6 with specific insights into the glutamate-glutamine cycle. We used tritium-labeled amino acids in order to demonstrate that SNAT6 is functioning as a glutamine and glutamate transporter. SNAT6 revealed seven predicted transmembrane segments in a homology model and was localized to caveolin rich sites at the plasma membrane. SNAT6 has high degree of specificity for glutamine and glutamate. Presence of these substrates enables formation of SNAT6-caveolin complexes that aids in sodium dependent trafficking of SNAT6 off the plasma membrane. To further understand its mode of action, several potential interacting partners of SNAT6 were identified using bioinformatics. Among them where CTP synthase 2 (CTPs2), phosphate activated glutaminase (Pag), and glutamate metabotropic receptor 2 (Grm2). Co-expression analysis, immunolabeling with co-localization analysis and proximity ligation assays of these three proteins with SNAT6 were performed to investigate possible interactions. SNAT6 can cycle between cytoplasm and plasma membrane depending on availability of substrates and interact with Pag, synaptophysin, CTPs2, and Grm2. Our data suggest a potential role of SNAT6 in glutamine uptake at the pre-synaptic terminal of excitatory neurons. We propose here a mechanistic model of SNAT6 trafficking that once internalized influences the glutamate-glutamine cycle in presence of its potential interacting partners.

A junctophilin-caveolin interaction enables efficient coupling between ryanodine receptors and BKCa channels in the Ca2+ microdomain of vascular smooth muscle.

Functional coupling between large-conductance Ca2+-activated K+ (BKCa) channels in the plasma membrane (PM) and ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) is an essential mechanism for regulating mechanical force in most smooth muscle (SM) tissues. Spontaneous Ca2+ release through RyRs (Ca2+ sparks) and subsequent BKCa channel activation occur within the PM-SR junctional sites. We report here that a molecular interaction of caveolin-1 (Cav1), a caveola-forming protein, with junctophilin-2 (JP2), a bridging protein between PM and SR, positions BKCa channels near RyRs in SM cells (SMCs) and thereby contributes to the formation of a molecular complex essential for Ca2+ microdomain function. Approximately half of all Ca2+ sparks occurred within a close distance (<400 nm) from fluorescently labeled JP2 or Cav1 particles, when they were moderately expressed in primary SMCs from mouse mesenteric artery. The removal of caveolae by genetic Cav1 ablation or methyl-ß-cyclodextrin treatments significantly reduced coupling efficiency between Ca2+ sparks and BKCa channel activity in SMCs, an effect also observed after JP2 knockdown in SMCs. A 20-amino acid-long region in JP2 appeared to be essential for the observed JP2-Cav1 interaction, and we also observed an interaction between JP2 and the BKCa channel. It can be concluded that the JP2-Cav1 interaction provides a structural and functional basis for the Ca2+ microdomain at PM-SR junctions and mediates cross-talk between RyRs and BKCa channels, converts local Ca2+ sparks into membrane hyperpolarization, and contributes to stabilizing resting tone in SMCs.

Development of an Excipient-Free Peptide Dry Powder Inhalation for the Treatment of Pulmonary Fibrosis.

The caveolin scaffolding domain peptide (CSP) is being developed for the therapeutic intervention of a lethal lung disease, idiopathic pulmonary fibrosis. While direct respiratory delivery of CSP7 (a 7-mer fragment of CSP) is considered an effective route, proper formulation and processing of the peptide are required. First, air-jet milling technology was performed in order to micronize the neat peptide powder. Next, the fine particles were subjected to a stability study with physical and chemical characterizations. In addition, the in vivo efficacy of processed CSP7 powder was evaluated in an animal model of lung fibrosis. The results revealed that, with jet milling, the particle size of CSP7 was reduced to a mass median aerodynamic diameter of 1.58 ± 0.1 µm and 93.3 ± 3.3% fine particle fraction, optimal for deep lung delivery. A statistically significant reduction of collagen was observed in diseased lung tissues of mice that received CSP7 powder for inhalation. The particles remained chemically and physically stable after micronization and during storage. This work demonstrated that jet milling is effective in the manufacturing of a stable, excipient-free CSP7 inhalation powder for the treatment of pulmonary fibrosis.

Secondary structure of caveolins: a mini review.

Caveolae are 50-100 nm invaginations found within the plasma membrane of cells. Caveolae are involved in many processes that are essential for homeostasis, most notably endocytosis, mechano-protection, and signal transduction. Within these invaginations, the most important proteins are caveolins, which in addition to participating in the aforementioned processes are structural proteins responsible for caveolae biogenesis. When caveolin is misregulated or mutated, many disease states can arise which include muscular dystrophy, cancers, and heart disease. Unlike most integral membrane proteins, caveolin does not have a transmembrane orientation; instead, it is postulated to adopt an unusual topography where both the N- and C-termini lie on the cytoplasmic side of the membrane, and the hydrophobic span adopts an intramembrane loop conformation. While knowledge concerning the biology of caveolin has progressed apace, fundamental structural information has proven more difficult to obtain. In this mini-review, we curate as well as critically assess the structural data that have been obtained on caveolins to date in order to build a robust and compelling model of the caveolin secondary structure.

Spelunking for lipids in caveolae.

Phosphatidylserine (PtdSer) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) have been implicated in the maintenance of caveolae, but direct evidence that these lipids are required for normal caveolar structure and dynamics in living cells has been lacking. A new study by Fairn and colleagues uses sophisticated tools to perturb specific lipids in living cells to assess the consequences for caveolae. This study demonstrates disparate roles for these lipids in the stability and mobility of caveolae and points the way for future work to understand how these lipids contribute to the biology of caveolae.

Caveolins sequester FA on the cytoplasmic leaflet of the plasma membrane, augment triglyceride formation, and protect cells from lipotoxicity.

Ectopic expression of caveolin-1 in HEK293 cells enhances FA sequestration in membranes as measured by a pH-sensitive fluorescent dye (1). We hypothesized that sequestration of FA is due to the enrichment of caveolin in the cytosolic leaflet and its ability to facilitate the formation of lipid rafts to buffer high FA levels. Here we show that ec-topic expression of caveolin-3 also results in enhanced FA sequestration. To further discriminate the effect that caveolins have on transmembrane FA movement and distribution, we labeled the outer membrane leaflet with fluorescein-phosphatidylethanolamine (FPE), whose emission is quenched by the presence of FA anions. Real-time measurements made with FPE and control experiments with positively charged fatty amines support our hypothesis that caveolins promote localization of FA anions through interactions with basic amino acid residues (lysines and arginines) present at the C termini of caveolins-1 and -3.

Inhibition of Bufo arenarum oocyte maturation induced by cholesterol depletion by methyl-beta-cyclodextrin. Role of low-density caveolae-like membranes.

The invaginated structure of caveolae seems to provide an optimal environment for hormone binding leading to oocyte meiotic maturation. We conducted a quantitative analysis of lipids and proteins of detergent-free low-density membranes isolated from Bufo arenarum oocytes and we modulated cellular cholesterol to further understand how these domains perform their regulatory functions in the amphibian system. Light membranes derive from the plasma membrane as suggested by the enrichment in the activity of 5'nucleotidase. Lipid analysis by chromatography techniques revealed that this fraction is enriched in phosphatidylserine and cholesterol and that it evidences an important level of sphingomyelin. The finding of a single 21 kDa caveolin in light membranes indicates the presence of caveolae-like structures in B. arenarum oocytes. In support of this finding, c-Src is significantly associated to this fraction. Cholesterol content of oocytes treated with methyl-beta-cyclodextrin (MbetaCD) decreased when compared to control oocytes. Drug treatment inhibited meiotic maturation in a dose-dependent manner and affected the localization of caveolin and c-Src among membrane fractions. Repletion of cholesterol showed a recovery of the ability of MbetaCD-treated oocytes to mature, particularly at the 25 mM concentration in which reversibility was close to the control level. Results highlight the importance of caveolae-like microdomains for maturation signaling in Bufo oocytes.

A caveolin dominant negative mutant associates with lipid bodies and induces intracellular cholesterol imbalance.

Recent studies have indicated a role for caveolin in regulating cholesterol-dependent signaling events. In the present study we have analyzed the role of caveolins in intracellular cholesterol cycling using a dominant negative caveolin mutant. The mutant caveolin protein, cav-3(DGV), specifically associates with the membrane surrounding large lipid droplets. These structures contain neutral lipids, and are accessed by caveolin 1-3 upon overexpression. Fluorescence, electron, and video microscopy observations are consistent with formation of the membrane-enclosed lipid rich structures by maturation of subdomains of the ER. The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis. The amphiphile U18666A acts synergistically with cav(DGV) to increase intracellular accumulation of FC. Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets. We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts.

Caveolin-2 is targeted to lipid droplets, a new "membrane domain" in the cell.

Caveolin-1 and -2 constitute a framework of caveolae in nonmuscle cells. In the present study, we showed that caveolin-2, especially its beta isoform, is targeted to the surface of lipid droplets (LD) by immunofluorescence and immunoelectron microscopy, and by subcellular fractionation. Brefeldin A treatment induced further accumulation of caveolin-2 along with caveolin-1 in LD. Analysis of mouse caveolin-2 deletion mutants revealed that the central hydrophobic domain (residues 87-119) and the NH(2)-terminal (residues 70-86) and COOH-terminal (residues 120-150) hydrophilic domains are all necessary for the localization in LD. The NH(2)- and COOH-terminal domains appeared to be related to membrane binding and exit from ER, respectively, implying that caveolin-2 is synthesized and transported to LD as a membrane protein. In conjunction with recent findings that LD contain unesterified cholesterol and raft proteins, the result implies that the LD surface may function as a membrane domain. It also suggests that LD is related to trafficking of lipid molecules mediated by caveolins.

CD26 mediates dissociation of Tollip and IRAK-1 from caveolin-1 and induces upregulation of CD86 on antigen-presenting cells.

CD26 is a T-cell costimulatory molecule with dipeptidyl peptidase IV enzyme activity in its extracellular region. We have previously reported that the addition of recombinant soluble CD26 resulted in enhanced proliferation of human T lymphocytes induced by the recall antigen tetanus toxoid (TT) via upregulation of CD86 on monocytes and that caveolin-1 was a binding protein of CD26, and the CD26-caveolin-1 interaction resulted in caveolin-1 phosphorylation (p-cav-1) as well as TT-mediated T-cell proliferation. However, the mechanism involved in this immune enhancement has not yet been elucidated. In the present work, we perform experiments to identify the molecular mechanisms by which p-cav-1 leads directly to the upregulation of CD86. Through proteomic analysis, we identify Tollip (Toll-interacting protein) and IRAK-1 (interleukin-1 receptor-associated serine/threonine kinase 1) as caveolin-1-interacting proteins in monocytes. We also demonstrate that following stimulation by exogenous CD26, Tollip and IRAK-1 dissociate from caveolin-1, and IRAK-1 is then phosphorylated in the cytosol, leading to the upregulation of CD86 via activation of NF-kappaB. Binding of CD26 to caveolin-1 therefore regulates signaling pathways in antigen-presenting cells to induce antigen-specific T-cell proliferation.

Lipid droplets: proteins floating on a pool of fat.

Proteins on the surface of lipid storage droplets are crucial to droplet structure and function, but are poorly understood. Recent reports reveal a function for perilipins, major lipid droplet proteins in adipocytes, and show that caveolin proteins can accumulate on lipid droplets.

Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells.

The relationship between glycosylphosphatidyl inositol (GPI)-linked proteins and caveolins remains controversial. Here, we derived fibroblasts from Cav-1 null mouse embryos to study the behavior of GPI-linked proteins in the absence of caveolins. These cells lack morphological caveolae, do not express caveolin-1, and show a approximately 95% down-regulation in caveolin-2 expression; these cells also do not express caveolin-3, a muscle-specific caveolin family member. As such, these caveolin-deficient cells represent an ideal tool to study the role of caveolins in GPI-linked protein sorting. We show that in Cav-1 null cells GPI-linked proteins are preferentially retained in an intracellular compartment that we identify as the Golgi complex. This intracellular pool of GPI-linked proteins is not degraded and remains associated with intracellular lipid rafts as judged by its Triton insolubility. In contrast, GPI-linked proteins are transported to the plasma membrane in wild-type cells, as expected. Furthermore, recombinant expression of caveolin-1 or caveolin-3, but not caveolin-2, in Cav-1 null cells complements this phenotype and restores the cell surface expression of GPI-linked proteins. This is perhaps surprising, as GPI-linked proteins are confined to the exoplasmic leaflet of the membrane, while caveolins are cytoplasmically oriented membrane proteins. As caveolin-1 normally undergoes palmitoylation on three cysteine residues (133, 143, and 156), we speculated that palmitoylation might mechanistically couple caveolin-1 to GPI-linked proteins. In support of this hypothesis, we show that palmitoylation of caveolin-1 on residues 143 and 156, but not residue 133, is required to restore cell surface expression of GPI-linked proteins in this complementation assay. We also show that another lipid raft-associated protein, c-Src, is retained intracellularly in Cav-1 null cells. Thus, Golgi-associated caveolins and caveola-like vesicles could represent part of the transport machinery that is necessary for efficiently moving lipid rafts and their associated proteins from the trans-Golgi to the plasma membrane. In further support of these findings, GPI-linked proteins were also retained intracellularly in tissue samples derived from Cav-1 null mice (i.e., lung endothelial and renal epithelial cells) and Cav-3 null mice (skeletal muscle fibers).

Caveolin-1 maintains activated Akt in prostate cancer cells through scaffolding domain binding site interactions with and inhibition of serine/threonine protein phosphatases PP1 and PP2A.

Previously it has been reported that caveolin-1 (cav-1) has antiapoptotic activities in prostate cancer cells and functions downstream of androgenic stimulation. In this study, we demonstrate that cav-1 overexpression significantly reduced thapsigargin (Tg)-stimulated apoptosis. Examination of the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling cascade revealed higher activities of PDK1 and Akt but not PI3-K in cav-1-stimulated cells compared to control cells. We subsequently found that cav-1 interacts with and inhibits serine/threonine protein phosphatases PP1 and PP2A through scaffolding domain binding site interactions. Deletion of the cav-1 scaffolding domain significantly reduces phosphorylated Akt and cell viability compared with wild-type cav-1. Analysis of potential substrates for PP1 and PP2A revealed that cav-1-mediated inhibition of PP1 and PP2A leads to increased PDK1, Akt, and ERK1/2 activities. We demonstrate that increased Akt activities are largely responsible for cav-1-mediated cell survival using dominant-negative Akt mutants and specific inhibitors to MEK1/MEK and show that cav-1 increases the half-life of phosphorylated PDK1 and Akt after inhibition of PI3-K by LY294002. We further demonstrate that cav-1-stimulated Akt activities lead to increased phosphorylation of multiple Akt substrates, including GSK3, FKHR, and MDM2. In addition, overexpression of cav-1 significantly increases translocation of phosphorylated androgen receptor to nucleus. Our studies therefore reveal a novel mechanism of Akt activation in prostate cancer and potentially other malignancies.

Detergent and detergent-free methods to define lipid rafts and caveolae.

Lipid rafts and their related membrane vesicular structures, caveolae, are cholesterol- and sphingolipid-rich microdomains of the plasma membrane that have attracted considerable interest because of their ability to concentrate numerous signaling proteins. Efforts to define the proteins that reside in lipid rafts and caveolae as well as investigations into the functional role of these microdomains in signaling, endocytosis, and other cellular processes have led to the hypothesis that they compartmentalize or prearrange molecules involved in regulating these pathways. This chapter describes biochemical approaches for defining lipid rafts and caveolae. Included are detergent- and nondetergent-based fractionations on sucrose-density gradients that isolate buoyant lipid rafts and caveolae as well as caveolin antibody-based immunoisolation of detergent-insoluble membranes that selectively isolates caveolae and not lipid rafts. Also, a general method to disrupt lipid rafts and caveolae using beta-cyclodextrin that is useful for probing the role of these microdomains in cellular processes is described. The advantages and disadvantages of the respective approaches are discussed. Taken together, these methods are useful for defining the role of lipid rafts and caveolae in cell signaling.

The caveolin triad: caveolae biogenesis, cholesterol trafficking, and signal transduction.

Caveolins are a family of proteins that coat the cytoplasmic face of caveolae, vesicular invaginations of the plasma membrane. These proteins are central to the organization of the proteins and lipids that reside in caveolae. Caveolins transport cholesterol to and from caveolae, and they regulate the activity of signaling proteins that reside in caveolae. Through studying the genes encoding the caveolae coat proteins, we have learned much about how they perform these multiple functions.

Conformational defects slow Golgi exit, block oligomerization, and reduce raft affinity of caveolin-1 mutant proteins.

Caveolin-1, a structural protein of caveolae, is cleared unusually slowly from the Golgi apparatus during biosynthetic transport. Furthermore, several caveolin-1 mutant proteins accumulate in the Golgi apparatus. We examined this behavior further in this mutant study. Golgi accumulation probably resulted from loss of Golgi exit information, not exposure of cryptic retention signals, because several deletion mutants accumulated in the Golgi apparatus. Alterations throughout the protein caused Golgi accumulation. Thus, most probably acted indirectly, by affecting overall conformation, rather than by disrupting specific Golgi exit motifs. Consistent with this idea, almost all the Golgi-localized mutant proteins failed to oligomerize normally (even with an intact oligomerization domain), and they showed reduced raft affinity in an in vitro detergent-insolubility assay. A few mutant proteins formed unstable oligomers that migrated unusually slowly on blue native gels. Only one mutant protein, which lacked the first half of the N-terminal hydrophilic domain, accumulated in the Golgi apparatus despite normal oligomerization and raft association. These results suggested that transport of caveolin-1 through the Golgi apparatus is unusually difficult. The conformation of caveolin-1 may be optimized to overcome this difficulty, but remain very sensitive to mutation. Disrupting conformation can coordinately affect oligomerization, raft affinity, and Golgi exit of caveolin-1.

Sphingomyelin-enriched microdomains at the Golgi complex.

Sphingomyelin- and cholesterol-enriched microdomains can be isolated as detergent-resistant membranes from total cell extracts (total-DRM). It is generally believed that this total-DRM represents microdomains of the plasma membrane. Here we describe the purification and detailed characterization of microdomains from Golgi membranes. These Golgi-derived detergent-insoluble complexes (GICs) have a low buoyant density and are highly enriched in lipids, containing 25% of total Golgi phospholipids including 67% of Golgi-derived sphingomyelin, and 43% of Golgi-derived cholesterol. In contrast to total-DRM, GICs contain only 10 major proteins, present in nearly stoichiometric amounts, including the alpha- and beta-subunits of heterotrimeric G proteins, flotillin-1, caveolin, and subunits of the vacuolar ATPase. Morphological data show a brefeldin A-sensitive and temperature-sensitive localization to the Golgi complex. Strikingly, the stability of GICs does not depend on its membrane environment, because, after addition of brefeldin A to cells, GICs can be isolated from a fused Golgi-endoplasmic reticulum organelle. This indicates that GIC microdomains are not in a dynamic equilibrium with neighboring membrane proteins and lipids. After disruption of the microdomains by cholesterol extraction with cyclodextrin, a subcomplex of several GIC proteins including the B-subunit of the vacuolar ATPase, flotillin-1, caveolin, and p17 could still be isolated by immunoprecipitation. This indicates that several of the identified GIC proteins localize to the same microdomains and that the microdomain scaffold is not required for protein interactions between these GIC proteins but instead might modulate their affinity.

Identification of a major protein on the cytosolic face of caveolae.

Cav-p60, a specific and ubiquitous caveolar protein, was immunoprecipitated from solubilized rat adipocyte plasma membranes and identified as similar to a GeneBank entry annotated mouse polymerase transcript release factor (PTRF) by MALDI-TOF and MS-MS of major fragments. Cloning and virtual translation of the corresponding rat adipocyte cDNA sequence revealed 98.7% identity with mouse PTRF. In vitro translation of this sequence produced a protein, which was recognized by antibodies to both cav-p60 and PTRF. EM gold labeling studies showed that a rabbit antiserum against murine PTRF immunolabeled caveolae specifically in adipocytes from both mouse and rat. In view of the reported function of the protein, which is exerted in the cell nucleus, its subcellular localization was investigated. We found that the protein could be purified by differential solubilization of a plasma membrane fraction followed by SDS-PAGE, and that the protein was as abundant as caveolin in this fraction. We were unable to detect the protein in cell nuclei by subcellular fractionation or fluorescence microscopy. The results show that in a large number of cell types, PTRF is essentially located to caveolae, and that each caveola harbors many copies of the protein. Consequently, we suggest the name Cavin for this protein.