Membrane Caveolae Research Articles

Characterization of the Molecular Architecture of Human Caveolin-3 and Interaction with the Skeletal Muscle Ryanodine Receptor

Caveolin-3 facilitates both caveolae formation and a range of cell signaling pathways, including Ca(2+) homeostasis. Caveolin-3 forms a disc-shaped nonamer that binds the Ca(2+)-release channel, RyR1. Multiple caveolin-3 nonamers bind to a single RyR1 homotetramer. First three-dimensional structural insights into caveolin-3 assembly, interactions with RyR1 suggest a novel role in muscle contraction and/or for channel localization within the membrane. Caveolin-3 (cav-3), an integral membrane protein, is a building block of caveolae as well as a regulator of a number of physiological processes by facilitating the formation of multiprotein signaling complexes. We report that the expression of cav-3 in insect (Sf9) cells induces caveola formation, comparable in size with those observed in native tissue. We have also purified the recombinant cav-3 determining that it forms an oligomer of ∼220 kDa. We present the first three-dimensional structure for cav-3 (using transmission electron microscopy and single particle analysis methods) and show that nine cav-3 monomers assemble to form a complex that is toroidal in shape, ~16.5 nm in diameter and ~ 5.5 nm in height. Labeling experiments and reconstitution of the purified cav-3 into liposomes have allowed a proposal for the orientation of the protein with respect to the membrane. We have identified multiple caveolin-binding motifs within the ryanodine receptor (RyR1) sequence employing a bioinformatic analysis. We have then shown experimentally that there is a direct interaction between recombinant cav-3 nonamers and purified RyR1 homotetramers that would imply that at least one of the predicted cav-3-binding sites is exposed within the fully assembled RyR1 structure. The cav-3 three-dimensional model provides new insights as to how a cav-3 oligomer can bind multiple partners in close proximity to form signaling complexes. Furthermore, a direct interaction with RyR1 suggests a possible role for cav-3 as a modifier of muscle excitation-contraction coupling and/or for localization of the receptor to regions of the sarcoplasmic reticulum.

Very important pharmacogene summary for VDR

The vitamin D receptor (VDR) binds the active form of vitamin D (1,25-dihydroxyvitamin D3). It belongs to the family of trans-acting transcriptional regulatory factors and shows sequence similarity to the steroid and thyroid hormone receptors. The gene was cloned by Baker and colleagues in 1988 and maps to chromosome 12q13.11 [1]. It consists of nine exons with at least six isoforms of exon 1, spans 63.5 kb, and encodes a 427 amino acid protein [2]. Alternative splicing results in multiple transcript variants encoding the VDR protein of different lengths [3,4]. The interaction of 1,25-dihydroxyvitamin D3 with VDR modulates many biological activities of the neural, immune, and endocrine systems, including calcium and phosphorous homeostasis, apoptosis, and cell differentiation (reviewed by Hewison and colleagues [5-7]). Its pleiotropic property is reflected by the findings that VDR is expressed in at least 37 tissues, which can roughly be grouped into seven biological systems (calcium homeostasis, immune, pancreatic β cells, muscle, cardiovascular, brain, and lung) [8-10]. The most characterized mechanism is the binding of vitamin D3 to nuclear VDR, which activates the receptor to form a heterodimer with the retinoid-X receptor and interacts with a specific DNA sequence on the gene promoter regions called the ‘vitamin D response element’ (VDRE). Transcription repressors occupying the VDRE are then replaced by transcription activators to initiate transcription of targeted genes. Microarray analyses using different human cell lines have identified over 100 genes with VDREs in the promoter regions, all potential targets of the 1,25-dihydroxyvitamin D3–VDR complex [11-13]. In human oral squamous carcinoma SCC25 cells, genes with characterized VDREs and of which demonstrated greater than 10-fold increase after treatment with 1,25-dihydroxy-vitamin D3 include 24-hydroxylase (CYP24), 17β-hydroxy-steroid dehydrogenase (HSD17B2), CD14 (CD14), type XIII collagen (COL13A1), and 5-lipoxygenase (ALOX5) [13]. The effect of 1,25-dihydroxyvitamin D3 by binding to VDR in the nucleus is often described as the slow-acting genomic effect; the effect will take days or hours to be noticed. Alternatively, 1,25-dihydroxyvitamin D3 can bind to VDR located in the caveolae in the plasma membrane to exert rapid responses through production of second messengers [14]. Pathways that have been shown to be activated by the 1,25-dihydroxyvitamin D3–VDR[memb] complex include mitogen-activated protein kinase-dependent [15], cAMP-dependent [16], phospholipase A2-dependent [17], phospholipase C-dependent [18], phosphatidylinositol 3-kinase-dependent [19], and protein kinase C-dependent [20] pathways. Examples of such rapid response include rapid Ca2+ absorption in the duodenum [21,22], opening of Cl− channels and secretion in osteoblasts, insulin secretion from pancreatic β-cells [23,24], and vascular smooth muscle cell migration [19].

Mitochondria‐localized caveolin in adaptation to cellular stress and injury

We show here that the apposition of plasma membrane caveolae and mitochondria (first noted in electron micrographs >50 yr ago) and caveolae-mitochondria interaction regulates adaptation to cellular stress by modulating the structure and function of mitochondria. In C57Bl/6 mice engineered to overexpress caveolin specifically in cardiac myocytes (Cav-3 OE), localization of caveolin to mitochondria increases membrane rigidity (4.2%; P<0.05), tolerance to calcium, and respiratory function (72% increase in state 3 and 23% increase in complex IV activity; P<0.05), while reducing stress-induced generation of reactive oxygen species (by 20% in cellular superoxide and 41 and 28% in mitochondrial superoxide under states 4 and 3, respectively; P<0.05) in Cav-3 OE vs. TGneg. By contrast, mitochondrial function is abnormal in caveolin-knockout mice and Caenorhabditis elegans with null mutations in caveolin (60% increase free radical in Cav-2 C. elegans mutants; P<0.05). In human colon cancer cells, mitochondria with increased caveolin have a 30% decrease in apoptotic stress (P<0.05), but cells with disrupted mitochondria-caveolin interaction have a 30% increase in stress response (P<0.05). Targeted gene transfer of caveolin to mitochondria in C57Bl/6 mice increases cardiac mitochondria tolerance to calcium, enhances respiratory function (increases of 90% state 4, 220% state 3, 88% complex IV activity; P<0.05), and decreases (by 33%) cardiac damage (P<0.05). Physical association and apparently the transfer of caveolin between caveolae and mitochondria is thus a conserved cellular response that confers protection from cellular damage in a variety of tissues and settings.

Bile Acids Down-Regulate Caveolin-1 in Esophageal Epithelial Cells through Sterol Responsive Element-Binding Protein

Bile acids are synthesized from cholesterol and are major risk factors for Barrett adenocarcinoma (BAC) of the esophagus. Caveolin-1 (Cav1), a scaffold protein of membrane caveolae, is transcriptionally regulated by cholesterol via sterol-responsive element-binding protein-1 (SREBP1). Cav1 protects squamous epithelia by controlling cell growth and stabilizing cell junctions and matrix adhesion. Cav1 is frequently down-regulated in human cancers; however, the molecular mechanisms that lead to this event are unknown. We show that the basal layer of the nonneoplastic human esophageal squamous epithelium expressed Cav1 mainly at intercellular junctions. In contrast, Cav1 was lost in 95% of tissue specimens from BAC patients (n = 100). A strong cytoplasmic expression of Cav1 correlated with poor survival in a small subgroup (n = 5) of BAC patients, and stable expression of an oncogenic Cav1 variant (Cav1-P132L) in the human BAC cell line OE19 promoted proliferation. Cav1 was also detectable in immortalized human squamous epithelial, Barrett esophagus (CPC), and squamous cell carcinoma cells (OE21), but was low in BAC cell lines (OE19, OE33). Mechanistically, bile acids down-regulated Cav1 expression by inhibition of the proteolytic cleavage of 125-kDa pre-SREBP1 from the endoplasmic reticulum/Golgi apparatus and nuclear translocation of active 68-kDa SREBP1. This block in SREBP1's posttranslational processing impaired transcriptional activation of SREBP1 response elements in the proximal human Cav1 promoter. Cav1 was also down-regulated in esophagi from C57BL/6 mice on a diet enriched with 1% (wt/wt) chenodeoxycholic acid. Mice deficient for Cav1 or the nuclear bile acid receptor farnesoid X receptor showed hyperplasia and hyperkeratosis of the basal cell layer of esophageal epithelia, respectively. These data indicate that bile acid-mediated down-regulation of Cav1 marks early changes in the squamous epithelium, which may contribute to onset of Barrett esophagus metaplasia and progression to BAC.

Cav‐3 is associated with and regulates the actin cytoskeleton in BV2 cells

Caveolins are canonically localized to plasma membrane (PM) caveolae. BV2 cells express all three caveolin isoforms (Cav‐1, ‐2, ‐3). Cav‐1 is localized to the PM but Cav‐3 is concentrated at cell processes and perinuclear. When BV2 cells are grown in DMEM 10% FBS (D10%), a rounded morphology with limited Cav‐3 expression is exhibited. In serum free media (HYC), expression of Cav‐3 increases and can be reversed by D10% incubation. Sucrose density fractionation shows Cav‐3 in heavy membrane fractions with limited protein in buoyant lipid raft fractions. Cell compartmentation shows partitioning of Cav‐1 specifically in the membrane, while Cav‐3 partitions to the cytoskeleton. Analysis with actin or microtubule disrupting agents reveals a close connection between Cav‐1 and Cav‐3 with the cytoskeleton. In HYC cultures treated with nocodazole or D10% with taxol, Cav‐1 was increased in HYC and decreased in D10%. In HYC cultures treated with cytochalasin D or D10% with jasplakinoide (JASP), Cav‐3 expression was decreased in HYC and increased in D10%. HYC cultures treated with JASP redistributed Cav‐3 to PM. G‐actin was sequestered with latruculin B resulting in reduced cell proliferation in HYC or enhanced proliferation in D10%. Cav‐3, not Cav‐1, was observed associated with mitotic spindles. Thus, we propose a novel mechanistic role for Cav‐3 in the regulation of microglia morphology and mitosis.

WNT6 is a novel target gene of caveolin-1 promoting chemoresistance to epirubicin in human gastric cancer cells

Resistance to chemotherapy is a major obstacle for curative treatment of human gastric cancer (GC). However, the underlying molecular mechanisms are largely unknown. Wingless-type MMTV integration site family members (WNTs) are secreted glycoproteins involved in embryogenesis and, on inappropriate expression in the adult, in cancer. Here, we show expression of WNT6 in GC patient specimens, human GC cell lines and in a mouse model of GC. In human GC cells, WNT6 expression was enhanced by caveolin-1 (Cav1), a scaffold protein of plasma membrane caveolae. WNT6 knock-down and overexpression experiments demonstrated that WNT6 increased the resistance to apoptotic cell death induced by the anthracycline chemotherapeutics epirubicin (Epi) and doxorubicin (Dox). Epi increased the activity of the human WNT6 promoter through Cav1-dependent binding of β-catenin to the proximal WNT6 promoter. Epi increased both WNT6/Wnt6 and Cav1 expression in human GC cells and within the tumor area of a murine model of GC (CEA424-SV40 TAg). In GC patients, WNT6 expression was positively associated with the tumor stage and the nodal status, and inversely correlated with the response to ECF (Epi, cisplatin, 5-fluorouracil) chemotherapy. These results showed that WNT6 and Cav1 are upregulated by chemotherapeutics and enhance the resistance of GC cells to anthracycline drugs. Understanding the molecular mechanisms driving WNT6/Cav1-induced drug resistance will provide benefits in developing new therapies for GC.

1α,25(OH)2D3‐dependent modulation of Akt in proliferating and differentiating C2C12 skeletal muscle cells

We previously reported that 1α,25-dihydroxy-vitamin D(3) [1α,25(OH)(2)D(3)] induces non-transcriptional rapid responses through activation of Src and MAPKs in the skeletal muscle cell line C2C12. In the present study we investigated the modulation of Akt by the secosteroid hormone in C2C12 cells at proliferative stage (myoblasts) and at early differentiation stage. In proliferating cells, 1α,25(OH)(2)D(3) activates Akt by phosphorylation in Ser473 in a time-dependent manner (5-60 min). When these cells were pretreated with methyl-beta-cyclodextrin to disrupt caveolae microdomains, hormone-induced activation of Akt was suppressed. Similar results were obtained by siRNA silencing of caveolin-1 expression, further indicating that hormone effects on cell membrane caveolae are required for downstream signaling. PI3K and p38 MAPK, but not ERK1/2, participate in 1α,25(OH)(2)D(3) activation of Akt in myoblasts. The involvement of p38 MAPK in Akt phosphorylation by the hormone probably occurs through MAPK-activated protein kinase 2 (MK2), which is activated by the steroid. In addition, the participation of Src in Akt phosphorylation by 1α,25(OH)(2)D(3) was demonstrated using the inhibitor PP2 and antisense oligodeoxynucleotides that suppress Src expression. We also observed that PI3K participates in hormone-induced proliferation. During the early phase of C2C12 cell differentiation 1α,25(OH)(2)D(3) also increases Akt phosphorylation and activates Src. Of relevance, Src and PI3K are involved in Akt activation and in MHC and myogenin increased expression by 1α,25(OH)(2)D(3). Altogether, these data suggest that 1α,25(OH)(2)D(3) upregulates Akt through Src, PI(3)K, and p38 MAPK to stimulate myogenesis in C2C12 cells.

Proteomic Identification of S-Nitrosylated Golgi Proteins: New Insights into Endothelial Cell Regulation by eNOS-Derived NO

BackgroundEndothelial nitric oxide synthase (eNOS) is primarily localized on the Golgi apparatus and plasma membrane caveolae in endothelial cells. Previously, we demonstrated that protein S-nitrosylation occurs preferentially where eNOS is localized. Thus, in endothelial cells, Golgi proteins are likely to be targets for S-nitrosylation. The aim of this study was to identify S-nitrosylated Golgi proteins and attribute their S-nitrosylation to eNOS-derived nitric oxide in endothelial cells.MethodsGolgi membranes were isolated from rat livers. S-nitrosylated Golgi proteins were determined by a modified biotin-switch assay coupled with mass spectrometry that allows the identification of the S-nitrosylated cysteine residue. The biotin switch assay followed by Western blot or immunoprecipitation using an S-nitrosocysteine antibody was also employed to validate S-nitrosylated proteins in endothelial cell lysates.ResultsSeventy-eight potential S-nitrosylated proteins and their target cysteine residues for S-nitrosylation were identified; 9 of them were Golgi-resident or Golgi/endoplasmic reticulum (ER)-associated proteins. Among these 9 proteins, S-nitrosylation of EMMPRIN and Golgi phosphoprotein 3 (GOLPH3) was verified in endothelial cells. Furthermore, S-nitrosylation of these proteins was found at the basal levels and increased in response to eNOS stimulation by the calcium ionophore A23187. Immunofluorescence microscopy and immunoprecipitation showed that EMMPRIN and GOLPH3 are co-localized with eNOS at the Golgi apparatus in endothelial cells. S-nitrosylation of EMMPRIN was notably increased in the aorta of cirrhotic rats.ConclusionOur data suggest that the selective S-nitrosylation of EMMPRIN and GOLPH3 at the Golgi apparatus in endothelial cells results from the physical proximity to eNOS-derived nitric oxide.

Caveolin targeting to late endosome/lysosomal membranes is induced by perturbations of lysosomal pH and cholesterol content

Caveolin-1 is an integral membrane protein of plasma membrane caveolae. Here we report that caveolin-1 collects at the cytosolic surface of lysosomal membranes when cells are serum starved. This is due to an elevation of the intralysosomal pH, since ionophores and proton pump inhibitors that dissipate the lysosomal pH gradient also trapped caveolin-1 on late endosome/lysosomes. Accumulation is both saturable and reversible. At least a portion of the caveolin-1 goes to the plasma membrane upon reversal. Several studies suggest that caveolin-1 is involved in cholesterol transport within the cell. Strikingly, we find that blocking cholesterol export from lysosomes with progesterone or U18666A or treating cells with low concentrations of cyclodextrin also caused caveolin-1 to accumulate on late endosome/lysosomal membranes. Under these conditions, however, live-cell imaging shows cavicles actively docking with lysosomes, suggesting that these structures might be involved in delivering caveolin-1. Targeting of caveolin-1 to late endosome/lysosomes is not observed normally, and the degradation rate of caveolin-1 is not altered by any of these conditions, indicating that caveolin-1 accumulation is not a consequence of blocked degradation. We conclude that caveolin-1 normally traffics to and from the cytoplasmic surface of lysosomes during intracellular cholesterol trafficking.

Additive Phenotype of MG53 and Dysferlin Deficiencies in Membrane Repair Function of Skeletal Muscle Fibers

Dysferlin and MG53 have been implicated as members of the sarcolemma repair machinery. Skeletal muscle fibers isolated from mice lacking expression of one or the other protein display defective membrane repair process following acute injuries. Our previous studies show that MG53 can nucleate the assembly of the membrane repair machinery by facilitating translocation of intracellular vesicles to membrane injury sites. Dysferlin appears to participate in Ca2+-dependent fusion of these vesicles for formation of a repair patch, but dysferlin itself cannot translocate to the injury site in the absence of MG53. To test the complementary function of MG53 and dysferlin in cell membrane repair, we generated a double knockout mouse lacking both MG53 and dysferlin. While serum creatine kinase (CK) levels of dysferlin-/-, mg53-/-, and wild-type mice were not significantly different from each other, CK levels of the mg53-/-dysferlin-/- mice were significantly elevated at resting condition. This suggests a more severe membrane repair defect. Plasma membrane targeted UV-irradiation of isolated skeletal muscle fibers in the presence of a membrane impermeant dye (FM1-43) was used to directly assay membrane repair. This experimental procedure was modified from previously established protocols for a quantitative assessment of the muscle membrane repair capacity. Compared with dysferlin-/- and mg53-/- fibers, significant elevated entry of FM1-43 dye was observed in mg53-/-dysferlin-/- muscle following UV-irradiation. These results suggest that the involvement of these two proteins in the membrane repair mechanism is more complicated than that as two points along a linear pathway. The additive effects of MG53 and dysferlin in muscle membrane repair suggest the possibility that these two proteins may either sense different membrane injury signals, or that they may act at different compartments of the sarcolemma membrane (caveolae or transverse-tubule network).

PTRF Anchors MG53 to Cell Injury Site for Initiation of Membrane Repair

Abstract Plasma membrane repair is an essential process for maintenance of homeostasis at the cellular and tissue levels while compromised repair capacity contributes to degenerative human diseases. Our recent studies show that MG53 is essential for muscle membrane repair, and defects in MG53 function are linked to muscular dystrophy and cardiac dysfunction. Here we report PTRF (polymerase I and transcript release factor), a gene known to regulate caveolae membrane structure, is an indispensible component of the membrane repair machinery. PTRF acts as a docking protein for MG53 during membrane repair potentially by binding exposed membrane cholesterol at the injury site. Cells lacking expression of endogenous PTRF show defective trafficking of MG53 to membrane injury sites. A mutation in PTRF associated with human disease results in aberrant nuclear localization of PTRF and disrupts MG53 function in membrane resealing. While RNAisilencing of PTRF leads to defective muscle membrane repair, overexpression of PTRF can rescue membrane repair defects in dystrophic muscle. Our data suggests that membranedelimited interaction between MG53 and PTRF contributes to initiation of cell membrane repair, which can be an attractive target for treatment or prevention of tissue injury in human diseases.

CDP-diacylglycerol phospholipid synthesis in detergent-soluble, non-raft, membrane microdomains of the endoplasmic reticulum

Phosphatidylinositol (PI) is essential for numerous cell functions and is generated by consecutive reactions catalyzed by CDP-diacylglycerol synthase (CDS) and PI synthase. In this study, we investigated the membrane organization of CDP-diacylglycerol synthesis. Separation of mildly disrupted A431 cell membranes on sucrose density gradients revealed cofractionation of CDS and PI synthase activities with cholesterol-poor, endoplasmic reticulum (ER) membranes and partial overlap with plasma membrane caveolae. Cofractionation of CDS activity with caveolae was also observed when low-buoyant density caveolin-enriched membranes were prepared using a carbonate-based method. However, immunoisolation studies determined that CDS activity localized to ER membrane fragments containing calnexin and type III inositol (1,4,5)-trisphosphate receptors but not to caveolae. Membrane fragmentation in neutral pH buffer established that CDP-diacylglycerol and PI syntheses were restricted to a subfraction of the calnexin-positive ER. In contrast to lipid rafts enriched for caveolin, cholesterol, and GM1 glycosphingolipids, the CDS-containing ER membranes were detergent soluble. In cell imaging studies, CDS and calnexin colocalized in microdomain-sized patches of the ER and also unexpectedly at the plasma membrane. These results demonstrate that key components of the PI pathway localize to nonraft, phospholipid-synthesizing microdomains of the ER that are also enriched for calnexin.

Stop the Flow: A Paradigm for Cell Signaling Mediated by Reactive Oxygen Species in the Pulmonary Endothelium

The lung endothelium is exposed to mechanical stimuli through shear stress arising from blood flow and responds to altered shear by activation of NADPH (NOX2) to generate reactive oxygen species (ROS). This review describes the pathway for NOX2 activation and the downstream ROS-mediated signaling events on the basis of studies of isolated lungs and flow-adapted endothelial cells in vitro that are subjected to acute flow cessation (ischemia). Altered mechanical stress is detected by a cell-associated complex involving caveolae and other membrane proteins that results in endothelial cell membrane depolarization and then the activation of specific kinases that lead to the assembly of NOX2 components. ROS generated by this enzyme amplify the mechanosignal within the endothelial cell to regulate activation and/or synthesis of proteins that participate in cell growth, proliferation, differentiation, apoptosis, and vascular remodeling. These responses indicate an important role for NOX2-derived ROS associated with mechanotransduction in promoting vascular homeostasis.

1α,25(OH)2-Vitamin D and a Nongenomic Vitamin D Analogue Inhibit Ultraviolet Radiation–Induced Skin Carcinogenesis

Exposure to ultraviolet radiation (UVR) can lead to a range of deleterious responses in the skin. An important form of damage is the DNA photolesion cyclobutane pyrimidine dimer (CPD). CPDs can be highly mutagenic if not repaired prior to cell division and can lead to UV-induced immunosuppression, making them potentially carcinogenic. UVR exposure also produces vitamin D, a prehormone. Different shapes of the steroid hormone 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] can produce biological responses through binding either to its cognate nuclear receptor (VDR) to regulate gene transcription or to the VDR associated with plasma membrane caveolae to produce, via signal transduction, nongenomic physiologic responses. Here, we show that both 1,25(OH)₂D₃ and 1α,25(OH)₂-lumisterol (JN), a conformationally restricted analogue that can generate only nongenomic responses, are effective inhibitors of UV damage in an immunocompetent mouse (Skh:hr1) model susceptible to UV-induced tumors. Both 1,25(OH)₂D₃ and JN significantly reduced UVR-induced CPD, apoptotic sunburn cells, and immunosuppression. Furthermore, these compounds inhibited skin tumor development, both papillomas and squamous cell carcinomas, in these mice. The observed reduction of these UV-induced effects by 1,25(OH)₂D₃ and JN suggests a role for these compounds in prevention against skin carcinogenesis. To the best of our knowledge, this is the first comprehensive report of an in vivo long-term biological response generated by chronic dosing with a nongenomic-selective vitamin D steroid.

Mitochondrial Cholesterol: A Connection Between Caveolin, Metabolism, and Disease

Caveolin (CAV) is an essential component of caveolae, cholesterol-enriched invaginations of the plasma membrane of most mammalian cells. However, CAV is not restricted to plasma membrane caveolae, and pools of CAV are present in myriad intracellular membranes. CAV proteins tightly bind cholesterol and contribute to regulation of cholesterol fluxes and distributions within cells. In this context, we recently showed that CAV1 regulates the poorly understood process controlling mitochondrial cholesterol levels. Cholesterol accumulates in mitochondrial membranes in the absence of CAV1, promoting the organelle's dysfunction with important metabolic consequences for cells and animals. In this article, we suggest a working hypothesis that addresses the role of CAV1 within the homeostatic network that regulates the influx/efflux of mitochondrial cholesterol.

Pacsin 2 is recruited to caveolae and functions in caveolar biogenesis

The pacsin (also termed syndapin) protein family is well characterised structurally. They contain F-BAR domains associated with the generation or maintenance of membrane curvature. The cell biology of these proteins remains less understood. Here, we initially confirm that EHD2, a protein previously shown biochemically to be present in caveolar fractions and to bind to pacsins, is a caveolar protein. We go on to report that GFP-pacsin 2 can be recruited to caveolae, and that endogenous pacsin 2 partially colocalises with caveolin 1 at the plasma membrane. Analysis of the role of pacsin 2 in caveolar biogenesis using small interfering RNA (siRNA) reveals that loss of pacsin 2 function results in loss of morphologically defined caveolae and accumulation of caveolin proteins within the plasma membrane. Overexpression of the F-BAR domain of pacsin 2 (but not the related F-BAR domains of CIP4 and FBP17) disrupts caveolar morphogenesis or trafficking, implying that pacsin 2 interacts with components required for these processes. We propose that pacsin 2 has an important role in the formation of plasma membrane caveolae.

Serotonin 5-HT2A receptor-mediated hypertrophy is negatively regulated by caveolin-3 in cardiomyoblasts and neonatal cardiomyocytes

The serotonin 5-HT2A receptor belongs to the G-protein-coupled receptors (GPCRs) superfamily and mediates the hypertrophic response to serotonin (5-HT) in cardiac myocytes. At present the regulatory mechanisms of 5-HT2A receptor-induced myocyte hypertrophy are not fully understood. The localization and the compartmentation of GPCRs within specialized membrane microdomains are known to modulate their signalling pathway. Therefore, we hypothesized that caveolae microdomains and caveolin-3, the predominant isoform of cardiac caveolae, might be regulators of 5-HT2A receptor signalling. We demonstrate that 5-HT2A receptors interact with caveolin-3 upon 5-HT stimulation and traffic into caveolae membrane microdomains. We provide evidence that caveolin-3 knockdown abolishes the redistribution of 5-HT2A receptors into caveolae and enhances 5-HT2A receptor-induced myocyte hypertrophic markers such as cell size, protein synthesis and ANF gene expression. Importantly, we demonstrate that caveolin-3 and caveolae structures are negative regulators of 5-HT2A receptor-induced nuclear factor of activated T cells (NFAT) transcriptional activation. Taken together, our data demonstrate that caveolin-3 and caveolae microdomains are important regulators of the hypertrophic response induced by 5-HT2A receptors. These findings thus open new insights to target heart hypertrophy under the enhanced serotonin system. This article is part of a Special Issue entitled “Local Signaling in Myocytes”.

Compartmentalizing Proximal FGFR1 Signaling in Placental Artery Endothelial Cell Caveolae.

Fibroblast growth factor-2 (FGF2) signaling via FGF receptor 1 (FGFR1) is critical for fetal placental angiogenesis. We have recently shown that the flask-shaped plasma membrane microdomain caveolae function as a platform for compartmentalizing ERK1/2 and AKT pathways activated by FGF2 and VEGF in placental artery endothelial cells. However, whether the proximal FGFR1 signaling events are also compartmentalized in the caveolae has not been explored. Proximal FGFR1 signaling is unique in that it is mediated by direct binding to and phosphorylating an adaptor protein FGFR substrate 2 (FRS2); this results in Grb2-mediated ERK2/1 activation and Gab1-mediated PI3K/Akt signaling. The objective of this study was to determine if the proximal FGFR1 signaling events are compartmentalized in placental endothelial cell caveolae. In ovine fetoplacental artery endothelial cells (oFPAEC), FGF2 treatment induced FRS2 phosphorylation on Tyr196 in a time- and dose-dependent fashion. Co-immunoprecipitation (Co-IP) experiments demonstrated that FGF2 rapidly and transiently increased the levels of FGFR1 bound FRS2 and the formation of a ternary complex FRS2-Gab1-Grb2 in oFPAEC. Co-IP and double fluorescence labeling experiments showed that FGFR1 and FRS2 was associated with the major caveolar structural protein caveolin-1 and co-purified with caveolin-1 in caveolae membranes. In oFPAEC treated with the cholesterol depletant methyl-β-cyclodextrin (MβCD) to disrupt caveolae, FGF2-induced FRS2 phosphorylation was inhibited and FGF2-induced formation of FRS2-Gab1-Grb2 complex was greatly attenuated. Subsequently, FGF2 was no longer able to activate the ERK/2 and Akt signaling pathways. Thus, integral caveolae compartmentalizes the proximal FGFR1 signaling via FRS2 and the formation of a ternary complex FRS2-Gab1-Grb2 (Supported by RO1 HL74947). (poster)

Alterations in membrane caveolae and BKCa channel activity in skin fibroblasts in Smith–Lemli–Opitz syndrome

The Smith–Lemli–Opitz syndrome (SLOS) is an inherited disorder of cholesterol synthesis caused by mutations in DHCR7 which encodes the final enzyme in the cholesterol synthesis pathway. The immediate precursor to cholesterol synthesis, 7-dehydrocholesterol (7-DHC) accumulates in the plasma and cells of SLOS patients which has led to the idea that the accumulation of abnormal sterols and/or reduction in cholesterol underlies the phenotypic abnormalities of SLOS. We tested the hypothesis that 7-DHC accumulates in membrane caveolae where it disturbs caveolar bilayer structure–function. Membrane caveolae from skin fibroblasts obtained from SLOS patients were isolated and found to accumulate 7-DHC. In caveolar-like model membranes containing 7-DHC, subtle, but complex alterations in intermolecular packing, lipid order and membrane width were observed. In addition, the BKCa K+ channel, which co-migrates with caveolin-1 in a membrane fraction enriched with cholesterol, was impaired in SLOS cells as reflected by reduced single channel conductance and a 50mV rightward shift in the channel activation voltage. In addition, a marked decrease in BKCa protein but not mRNA expression levels was seen suggesting post-translational alterations. Accompanying these changes was a reduction in caveolin-1 protein and mRNA levels, but membrane caveolar structure was not altered. These results are consistent with the hypothesis that 7-DHC accumulation in the caveolar membrane results in defective caveolar signaling. However, additional cellular alterations beyond mere changes associated with abnormal sterols in the membrane likely contribute to the pathogenesis of SLOS.

Essential role of PACSIN2/syndapin-II in caveolae membrane sculpting

Caveolae are flask-shaped invaginations of the plasma membrane that are associated with tumor formation, pathogen entry and muscular dystrophy, through the regulation of lipids, signal transduction and endocytosis. Caveolae are generated by the fusion of caveolin-1-containing vesicles with the plasma membrane, which then participate in endocytosis via dynamin. Proteins containing membrane-sculpting F-BAR (or EFC) domains organize the membrane in clathrin-mediated endocytosis. Here, we show that the F-BAR protein PACSIN2 sculpts the plasma membrane of the caveola. The PACSIN2 F-BAR domain interacts directly with caveolin-1 by unmasking autoinhibition of PACSIN2. Furthermore, the membrane invaginations induced by the PACSIN2 F-BAR domain contained caveolin-1. Knockdown of PACSIN2 resulted in abnormal morphology of caveolin-1-associated plasma membranes, presumably as a result of decreased recruitment of dynamin-2 to caveolin-1. These results indicate that PACSIN2 mediates membrane sculpting by caveolin-1 in caveola morphology and recruits dynamin-2 for caveola fission.