Caveolar Structures Research Articles

The Role of Caveolae and Membrane Conformation in Load-Induced Ventricular Conduction Slowing

Mechanical load has been shown to alter cardiac electrophysiology, but the underlying mechanisms are still not completely understood. Conduction slowing has been observed during increased ventricular load in the isolated heart and has been associated with changes in intercellular coupling and membrane capacitance. The goal of this work was to investigate the effect of increased ventricular pressure on cardiomyocyte membrane conformation and caveolar structure, and the role of these changes in conduction slowing associated with load. Murine hearts were isolated, retrogradely perfused, and pressure loaded through a cannula inserted into the left ventricular cavity. In the pressure loaded wild-type heart, electron microscopy revealed a tautening of excess membrane folds, fewer sub-sarcolemmal caveolae, and an apparent opening of the neck of sarcolemmal caveolae compared to the unloaded state. Optical mapping revealed a significant 15-18% slowing of conduction velocity with load in both the directions of fastest and slowest conduction (CVmax and CVmin, respectively). Hearts isolated from caveolin-3 deficient mice have no caveolae on the cardiomyocyte membranes, and control hearts treated with methyl-β-cyclodextrin (MβCD) have a diminished number of caveolar structures, as assessed by electron microscopy. These hearts lacked changes in sub-sarcolemmal caveolae and caveolar structure with pressure load, and conduction slowing was attenuated. Cardiomyocyte caveolar localization and conformation change with increased ventricular load, and this alteration appears to play an important role in load-induced conduction slowing, possibly by contributing to changes in effective membrane capacitance.

Close Association of Aquaporin-2 Internalization with Caveolin-1

Aquaporin 2 (AQP2) is a membrane water channel protein that traffics between the intracellular membrane compartment and the plasma membrane in a vasopressin-dependent manner in the renal collecting duct cell to control the amount of water reabsorption. We examined the relation between AQP2 internalization from the plasma membrane and caveolin-1, which is a major protein in membrane microdomain caveolae, in Mardin-Darby canine kidney cells expressing human AQP2 (MDCK-hAQP2 cells). Double-immunofluorescence microscopy showed that AQP2 is colocalized with caveolin-1 in the apical plasma membrane by stimulating the intracellular signaling cascade of vasopressin with forskolin. After washing forskolin, both AQP2 and caveolin-1 were internalized to early endosomes and then separately went back to their individual compartments, which are subapical compartments and the apical membrane, respectively.Double-immunogold electron microscopy in ultrathin cryosections confirmed the colocalization of AQP2 with caveolin-1 at caveolar structures on the apical plasma membrane of forskolin-treated cells and the colocalization within the same intracellular vesicles after washing forskolin. A co-immunoprecipitation experiment showed the close interaction between AQP2 and caveolin-1 in forskolin-treated cells and in cells after washing forskolin. These results suggest that a caveolin-1-dependent and possibly caveolar-dependent pathway is a candidate for AQP2 internalization in MDCK cells.

Estrogen- and xenoestrogen-induced ERK signaling in pituitary tumor cells involves estrogen receptor-α interactions with G protein-αi and caveolin I

Multiple physiologic estrogens (estradiol, estriol, and estrone), as well as xenoestrogenic compounds (including alkylphenols and bisphenol A), can act via nongenomic signaling initiated by liganding of the plasma membrane estrogen receptor-α (mERα). We examined heterotrimeric G protein involvement leading to extracellular-regulated kinase (ERK) activation in GH3/B6/F10 rat anterior pituitary tumor cells that express abundant mERα, and smaller amounts of mERβ and GPR30. A combination of microarrays, immunoblots, and quantitative immunoassays demonstrated the expression of members of all α, β, and γ G protein classes in these cells. Use of selective inhibitors showed that the Gαi subtype was the primary initiator of downstream ERK signaling. Using antibodies against the GTP-bound form of Gα protein subtypes i and s, we showed that xenoestrogens (bisphenol A, nonylphenol) activated Gαi at 15–30s; all alkylphenols examined subsequently suppressed activation by 5min. GTP-activation of Gαi for all estrogens was enhanced by irreversible cumulative binding to GTPγS. In contrast, Gαs was neither activated nor deactivated by these treatments with estrogens. ERα and Gαi co-localized outside nuclei and could be immuno-captured together. Interactions of ERα with Gαi and caveolin I were demonstrated by epitope proximity ligation assays. An ERα/β antagonist (ICI182780) and a selective disruptor of caveolar structures (nystatin) blocked estrogen-induced ERK activation. Conclusions: Xenoestrogens, like physiologic estrogens, can evoke downstream kinase signaling involving selective interactions of ERα with Gαi and caveolin I, but with some different characteristics, which could explain their disruptive actions.

Caveolar vesicles generate DNA damage and perpetuate cellular aging

Caveolar vesicles generate DNA damage and perpetuate cellular aging

Regulation of cellular senescence by the essential caveolar component PTRF/Cavin-1

Polymerase I and transcript release factor (PTRF, also known as Cavin-1) is an essential component in the biogenesis and function of caveolae. Here, we show that PTRF expression is increased in senescent human fibroblasts. Importantly, overexpression of PTRF induced features characteristic of cellular senescence, whereas reduced PTRF expression extended the cellular replicative lifespan. Interestingly, we found that PTRF localized primarily to the nuclei of young and quiescent WI-38 human fibroblasts, but translocated to the cytosol and plasma membrane during cellular senescence. Furthermore, electron microscopic analysis demonstrated an increased number of caveolar structures in senescent and PTRF-transfected WI-38 cells. Our data suggest that the role of PTRF in cellular senescence is dependent on its targeting to caveolae and its interaction with caveolin-1, which appeared to be regulated by the phosphorylation of PTRF. Taken together, our findings identify PTRF as a novel regulator of cellular senescence that acts through the p53/p21 and caveolar pathways.

MLC1 trafficking and membrane expression in astrocytes: Role of caveolin-1 and phosphorylation

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare congenital leukodystrophy caused by mutations in the MLC1 gene that encodes a membrane protein of unknown function. In the brain MLC1 protein is mainly expressed in astrocyte end-feet, localizes in lipid rafts and associates with the dystrophin glycoprotein complex (DGC). Using pull-down and co-fractionation assays in cultured human and rat astrocytes, we show here that MLC1 intracellular domains pull-down the DGC proteins syntrophin, dystrobrevin, Kir4.1 and caveolin-1, the structural protein of caveolae, thereby supporting a role for DGC and caveolar structures in MLC1 function. By immunostaining and subcellular fractionation of cultured rat or human astrocytes treated with agents modulating caveolin-mediated trafficking, we demonstrate that MLC1 is also expressed in intracellular vesicles and endoplasmic reticulum and undergoes caveolae/raft-mediated endocytosis. Inhibition of endocytosis, cholesterol lowering and protein kinases A- and C-mediated MLC1 phosphorylation favour the expression of membrane-associated MLC1. Because pathological mutations prevent MLC1 membrane expression, the identification of substances regulating MLC1 intracellular trafficking is potentially relevant for the therapy of MLC.

Interaction of the immunoglobulin-like cell adhesion molecule hepaCAM with caveolin-1

Subsequent to our identification of the novel immunoglobulin-like cell adhesion molecule hepaCAM, we demonstrated that hepaCAM is capable of modulating cell growth and cell–extracellular matrix interactions. In this study, we examined the localization of hepaCAM in lipid rafts/caveolae as well as the interaction of hepaCAM with the caveolar structural protein caveolin-1 (Cav-1). Our results revealed that a portion of hepaCAM resided in detergent-resistant membranes and co-partitioned with Cav-1 to low buoyant density fractions characteristic of lipid rafts/caveolae. In addition, co-localization and coimmunoprecipitation assays confirmed the association of hepaCAM with Cav-1. Deletion analysis of hepaCAM showed that the extracellular first immunoglobulin domain of hepaCAM was required for binding Cav-1. Furthermore, when co-expressed, Cav-1 induced the expression of hepaCAM as well as distributed hepaCAM to intracellular Cav-1-positive caveolar structures. Taken together, our findings indicate that hepaCAM is partially localized in the lipid rafts/caveolae and interacts with Cav-1 through its first immunoglobulin domain.

Cholesterol Substitution Increases the Structural Heterogeneity of Caveolae

Caveolin-1 binds cholesterol and caveola formation involves caveolin-1 oligomerization and cholesterol association. The role of cholesterol in caveolae has so far been addressed by methods that compromise membrane integrity and abolish caveolar invaginations. To study the importance of sterol specificity for the structure and function of caveolae, we replaced cholesterol in mammalian cells with its immediate precursor desmosterol by inhibiting 24-dehydrocholesterol reductase. Desmosterol could substitute for cholesterol in maintaining cell growth, membrane integrity, and preserving caveolar invaginations. However, in desmosterol cells the affinity of caveolin-1 for sterol and the stability of caveolin oligomers were decreased. Moreover, caveolar invaginations became more heterogeneous in dimensions and in the number of caveolin-1 molecules per caveola. Despite the altered caveolar structure, caveolar ligand uptake was only moderately inhibited. We found that in desmosterol cells, Src kinase phosphorylated Cav1 at Tyr(14) more avidly than in cholesterol cells. Taken the role of Cav1 Tyr(14) phosphorylation in caveolar endocytosis, this may help to preserve caveolar uptake in desmosterol cells. We conclude that a sterol C24 double bond interferes with caveolin-sterol interaction and perturbs caveolar morphology but facilitates Cav1 Src phosphorylation and allows caveolar endocytosis. More generally, substitution of cholesterol by a structurally closely related sterol provides a method to selectively modify membrane protein-sterol affinity, structure and function of cholesterol-dependent domains without compromising membrane integrity.

Epithelial Growth Factor-induced Phosphorylation of Caveolin 1 at Tyrosine 14 Stimulates Caveolae Formation in Epithelial Cells

Caveolae are flask-shaped endocytic structures composed primarily of caveolin-1 (Cav1) and caveolin-2 (Cav2) proteins. Interestingly, a cytoplasmic accumulation of Cav1 protein does not always result in a large number of assembled caveolae organelles, suggesting a regulatory mechanism that controls caveolae assembly. In this study we report that stimulation of epithelial cells with epithelial growth factor (EGF) results in a profound increase in the number of caveolar structures at the plasma membrane. Human pancreatic tumor cells (PANC-1) and normal rat kidney cells (NRK), as a control, were treated with 30 ng/ml EGF for 0, 5, and 20 min before fixation and viewing by electron microscopy. Cells fixed without EGF treatment exhibited modest numbers of plasma membrane-associated caveolae. Cells treated with EGF for 5 or 20 min showed an 8-10-fold increase in caveolar structures, some forming long, pronounced caveolar "towers" at the cell-cell borders. It is known that Cav1 is Src-phosphorylated on tyrosine 14 in response to EGF treatment, although the significance of this modification is unknown. We postulated that phosphorylation could provide the stimulus for caveolae assembly. To this end, we transfected cells with mutant forms of Cav1 that could not be phosphorylated (Cav1Y14F) and tested if this altered protein reduced the number of EGF-induced caveolae. We observed that EGF-stimulated PANC-1 cells expressing the mutant Cav1Y14F protein exhibited a 90-95% reduction in caveolae number compared with cells expressing wild type Cav1. This study provides novel insights into how cells regulate caveolae formation and implicates EGF-based signaling cascades in the phosphorylation of Cav1 as a stimulus for caveolae assembly.

Belt-like localisation of caveolin in deep caveolae and its re-distribution after cholesterol depletion

Caveolae are specialised vesicular microdomains of the plasma membrane. Using freeze-fracture immunogold labelling and stereoscopic imaging, the distribution of labelled caveolin 1 in caveolae of 3T3-L1 mouse fibroblast cells was shown. Immunogold-labelled caveolin structures surrounded the basolateral region of deeply invaginated caveolae like a belt whereas in the apical region distal to the plasma membrane, the caveolin labelling was nearly absent. Shallow caveolar membranes showed a dispersed caveolin labelling. After membrane cholesterol reduction by methyl-beta-cyclodextrin treatment, a dynamic re-distribution of labelled caveolin 1 and a flattening of caveolar structures was found. The highly curved caveolar membrane got totally flat, and the initial belt-like caveolin labelling disintegrated to a ring-like structure and later to a dispersed order. Intramembrane particle-free domains were still observable after cholesterol depletion and caveolin re-distribution. These results indicate that cholesterol interacting with caveolin structures at the basolateral part of caveolae is necessary for the maintenance of the deeply invaginated caveolar membranes.

The Cytosolic Phospholipase A2 Pathway, a Safeguard of β2-Adrenergic Cardiac Effects in Rat

We have recently demonstrated that in human heart, beta2-adrenergic receptors (beta2-ARs) are biochemically coupled not only to the classical adenylyl cyclase (AC) pathway but also to the cytosolic phospholipase A2 (cPLA2) pathway (Pavoine, C., Behforouz, N., Gauthier, C., Le Gouvello, S., Roudot-Thoraval, F., Martin, C. R., Pawlak, A., Feral, C., Defer, N., Houel, R., Magne, S., Amadou, A., Loisance, D., Duvaldestin, P., and Pecker, F. (2003) Mol. Pharmacol. 64, 1117-1125). In this study, using Fura-2-loaded cardiomyocytes isolated from adult rats, we showed that stimulation of beta2-ARs triggered an increase in the amplitude of electrically stimulated [Ca2+]i transients and contractions. This effect was abolished with the PKA inhibitor, H89, but greatly enhanced upon addition of the selective cPLA2 inhibitor, AACOCF3. The beta2-AR/cPLA2 inhibitory pathway involved G(i) and MSK1. Potentiation of beta2-AR/AC/PKA-induced Ca2+ responses by AACOCF3 did not rely on the enhancement of AC activity but was associated with eNOS phosphorylation (Ser1177) and L-NAME-sensitive NO production. This was correlated with PKA-dependent phosphorylation of PLB (Ser16). The constraint exerted by the beta2-AR/cPLA2 pathway on the beta2-AR/AC/PKA-induced Ca2+ responses required integrity of caveolar structures and was impaired by Filipin III treatment. Immunoblot analyses demonstrated zinterol-induced translocation of cPLA and its cosedimentation with MSK1, eNOS, PLB, and sarcoplasmic reticulum Ca2+ pump (SERCA) 2a in a low density caveolin-3-enriched membrane fraction. This inferred the gathering of beta2-AR signaling effectors around caveolae/sarcoplasmic reticulum (SR) functional platforms. Taken together, these data highlight cPLA as a cardiac beta2-AR signaling pathway that limits beta2-AR/AC/PKA-induced Ca2+ responses in adult rat cardiomyocytes through the impairment of eNOS activation and PLB phosphorylation.

Caveolin-1 Facilitates Mechanosensitive Protein Kinase B (Akt) Signaling In Vitro and In Vivo

Mechanotransduction represents an integral part of vascular homeostasis and contributes to vascular lesion formation. Previously, we demonstrated a mechanosensitive activation of phosphoinositide 3-kinase (PI3-K)/protein kinase B (Akt) resulting in p27Kip1 transcriptional downregulation and cell cycle entry of vascular smooth muscle cells (VSMC). In this study, we further elucidated the signaling from outside-in toward PI3-K/Akt in vitro and in an in vivo model of elevated tensile force. When VSMC were subjected to cyclic stretch (0.5 Hz at 125% resting length), PI3-K, Akt, and Src kinases were found activated. Disrupting caveolar structures with beta-cyclodextrin or transfection of VSMC with caveolin-1 antisense oligonucleotides (ODN) prevented PI3-K and Akt activation and cell cycle entry. Furthermore, PI3-K and Akt were resistant to activation when Src kinases were inhibited pharmacologically or by overexpression of a kinase-dead c-Src mutant. alpha(V)beta3 integrins were identified to colocalize with PI3-K/caveolin-1 complexes, and blockade of alpha(V)beta3 integrins prevented Akt activation. The central role of caveolin-1 in mechanotransduction was further examined in an in vivo model of elevated tensile force. Interposition of wild-type (WT) jugular veins into WT carotid arteries resulted in a rapid Akt activation within the veins that was almost abolished when veins of caveolin-1 knockout (KO) mice were used. Furthermore, late neointima formation within the KO veins was significantly reduced. Our study provides evidence that PI3-K/Akt is critically involved in mechanotransduction of VSMC in vitro and within the vasculature in vivo. Furthermore, caveolin-1 is essential for the integrin-mediated activation of PI3-K/Akt.

Maxi-K channels localize to caveolae in human myometrium: a role for an actin-channel-caveolin complex in the regulation of myometrial smooth muscle K+ current

Multiple cell-signaling pathways converge to modulate large-conductance, voltage- and Ca2+-sensitive K+ channel (maxi-K channel) activity and buffer cell excitability in human myometrial smooth muscle cells (hMSMCs). Recent evidence indicates that maxi-K channel proteins can target to membrane microdomains; however, their association with other proteins within these macromolecular complexes has not been elucidated. Biochemical isolation of detergent-resistant membrane fractions from human myometrium demonstrates the presence of maxi-K channels in lipid raft microdomains, which cofractionate with caveolins. In both nonpregnant and late-pregnant myometrium, maxi-K channels associate and colocalize with caveolar scaffolding proteins caveolin-1 and caveolin-2, but not caveolin-3. Disruption of cultured hMSMC caveolar complexes by cholesterol depletion with cyclodextrin increases an iberiotoxin-sensitive K+ current. Co-immunoprecipitations have indicated that the maxi-K channel also is associated with both alpha- and gamma-actin. Immunocytochemical analysis indicates colocalization of maxi-K channels, actin, and caveolin-1 in primary cultures of hMSMCs. Further experiments using immunoelectron microscopy have shown the proximity of both actin and the maxi-K channel within the same cell surface caveolar structures. Functionally, disruption of the actin cytoskeleton in cultured hMSMCs by cytochalasin D and latrunculin A greatly increased the open-state probability of the channel, while stabilization of actin cytoskeleton with jasplakinolide abolished the effect of latrunculin A. These data indicate that the actin cytoskeleton is involved as part of a caveolar complex in the regulation of myometrial maxi-K channel function.

Nitric oxide modulates caveolin-1 and matrix metalloproteinase-9 expression and distribution at the endothelial cell/tumor cell interface.

We used a two-compartment coculture model comprising human endothelial cells (EC) and non-small cell lung carcinoma (CA) cells to study capillary formation. Elevated NO concentrations, contributed in part by CA cells, lead to inhibited capillary formation (Phillips PG, Birnby LM, Narendran A, and Milonovich WL. Am J Physiol Lung Cell Mol Physiol 281: L278-L290, 2001). Here we demonstrate using gelatin substrate zymography that high NO concentrations, whether produced endogenously or by NO donor spermine-NONOate or peroxynitrite-generating compound SIN-1, significantly inhibit MMP-9 expression and activation. Furthermore, high NO concentrations decrease Cav-1 abundance and alter its cellular distribution in EC. Cav-1 is essential for capillary formation in this model because Cav-1 antisense treatments targeted to EC significantly inhibit capillary formation. Laser confocal microscopy demonstrated extensive colocalization of MMP-9 with Cav-1 in sprouting EC, primarily at the basolateral surfaces of EC in focal structures associated with directed migration. This codistribution was NO concentration dependent, and elevated NO concentrations lead to marked dissociation of these two proteins. We propose that compartmentalization of MMP-9 within caveolar structures does occur, and that this could facilitate directed proteolysis essential for early migratory and invasive processes. Our data suggest elevated NO concentrations could impact on capillary formation via a combination of direct effects on MMP activation and by altering the distribution or abundance of Cav-1. Consequences of Cav-1 alterations may include impaired activation of proteolytic enzymes that utilize caveolar structure for stabilization and/or compartmentalization of MMP-9 as well as other putative members of an ECM proteolytic cascade.

Localization of VEGFR-2 and PLD2 in endothelial caveolae is involved in VEGF-induced phosphorylation of MEK and ERK

To clarify the role of caveolae in VEGF/VEGF receptor-2 (VEGFR-2)-mediated signaling cascades, primary cultured human umbilical vein endothelial cells (HUVECs) were fractionated to isolate caveolae-enriched cell membranes. Interestingly, VEGFR-2, phospholipase D2 (PLD2), and Ras were enriched in caveolae-enriched fractions. Moreover, VEGF increased PLD activity in a time- and dose-dependent manner in HUVECs, whereas a ligand specific for VEGFR-1 placental growth factor did not change PLD activity. A PLD inhibitor, 1-butanol, almost completely suppressed VEGF-induced ERK phosphorylation and cellular proliferation, whereas the negative control for 1-butanol, 3-butanol, did not produce significant changes. Addition of phosphatidic acid negated the 1-butanol-induced suppression. Pharmacological analyses using several inhibitors indicated that PKC-delta regulates the VEGF-induced activation of PLD/ERK. Thus PLD2 could be involved in MEK/ERK signaling cascades that are induced by the VEGF/VEGFR-2/PKC-delta pathway in endothelial cells. Pretreatment with the cholesterol depletion agent methyl-beta-cyclodextrin (MbetaCD) almost completely disassembled caveolar structures, whereas the addition of cholesterol to MbetaCD-treated cells restored caveolar structures. Pretreatment with MbetaCD largely abolished phosphorylation of MEK/ERK by VEGF, whereas the addition of cholesterol restored VEGF-induced MEK/ERK phosphorylations. These results indicate that intact caveolae are required for the VEGF/VEGFR-2-mediated MEK/ERK signaling cascade.

A CAV3 microdeletion differentially affects skeletal muscle and myocardium.

Caveolin-3 is the muscle-specific protein product of the caveolin gene family and an integral membrane component of caveolae. Mutations in the gene encoding caveolin-3 (CAV3) underlie four distinct disorders of skeletal muscle: the autosomal dominant form of limb-girdle muscular dystrophy type 1C (LGMD-1C), rippling muscle disease (RMD), sporadic and familial forms of hyperCKemia, and distal myopathy. To characterize a multigenerational Italian family affected by an autosomal dominant myopathic disorder and to assess the expression of caveolin-3, dystrophin, dystrophin-associated glycoproteins, and neuronal nitric oxide synthase in the myocardium of an affected patient. Clinical analysis involved 15 family members. Skeletal muscle expression of sarcolemmal proteins was evaluated by immunohistochemistry and western blot analysis in three affected individuals. Caveolar structures were analyzed through electron microscopy in muscle biopsies and in one heart biopsy. CAV3 genetic analysis showed a heterozygous 3-bp microdeletion (328-330del) in affected individuals, resulting in the loss of a phenylalanine (Phe97del) in the transmembrane domain. In the skeletal muscle, the mutation was associated with severe caveolin-3 deficiency and caveolar disorganization, whereas the expression of the other analyzed muscle proteins was unaltered. Remarkably, caveolin-3 was expressed in myocardium at a level corresponding to about 60% of that of control individuals and was correctly localized at the myocardial cell membranes, with preservation of cardiac myofiber caveolar structures. Clinical analysis revealed the concomitant presence in this family of the following phenotypes: RMD, LGMD, and hyperCKemia. Intrafamilial phenotypic heterogeneity is associated with caveolin-3 Phe97 microdeletion. The molecular network interacting with caveolin-3 in skeletal muscle and heart may differ.

Differential caveolin-1 polarization in endothelial cells during migration in two and three dimensions.

Endothelial cell (EC) migration is a critical event during multiple physiological and pathological processes. ECs move in the plane of the endothelium to heal superficially injured blood vessels but migrate in three dimensions during angiogenesis. We herein investigate differences in these modes of movement focusing on caveolae and their defining protein caveolin-1. Using a novel approach for morphological analysis of transmigrating cells, we show that ECs exhibit a polarized distribution of caveolin-1 when traversing a filter pore. Strikingly, in these cells caveolin-1 seems to be released from caveolar structures in the cell rear and to relocalize at the cell front in a cytoplasmic form. In contrast, during planar movement caveolin-1 is concentrated at the rear of ECs, colocalizing with caveolae. The phosphorylatable Tyr14 residue of caveolin-1 is required for polarization of the protein during transmigration but does not alter polarization during planar movement. Palmitoylation of caveolin-1 is not essential for redistribution of the protein during either mode of movement. Thus, ECs migrating in three dimensions uniquely exhibit dissociation of caveolin-1 from caveolae and phosphorylation-dependent relocalization to the cell front.

Characterization of a Distinct Plasma Membrane Macrodomain in Differentiated Adipocytes

Caveolae are small invaginations of the cell surface that are abundant in mature adipocytes. A recent study (Kanzaki, M., and Pessin, J. E. (2002) J. Biol. Chem. 277, 25867-25869) described novel caveolin- and actin-containing structures associated with the adipocyte cell surface that contain specific signaling proteins. We have characterized these structures, here termed "caves," using light and electron microscopy and observe that they represent surface-connected wide invaginations of the basal plasma membrane that are sometimes many micrometers in diameter. Rather than simply a caveolar domain, these structures contain all elements of the plasma membrane including clathrin-coated pits, lipid raft markers, and non-raft markers. GLUT4 is recruited to caves in response to insulin stimulation. Caves can occupy a significant proportion of the plasma membrane area and are surrounded by cortical actin. Caveolae density in caves is similar to that on the bulk plasma membrane, but because these structures protrude much deeper into the plane of focus of the light microscope molecules such as caveolin and other plasma membrane proteins appear more concentrated in caves. We conclude that the adipocyte surface membrane contains numerous wide invaginations that do not represent novel caveolar structures but rather large surface caves.

Localization of the human oxytocin receptor in caveolin-1 enriched domains turns the receptor-mediated inhibition of cell growth into a proliferative response.

In this study, we investigated the functional role of the localization of human OTR in caveolin-1 enriched membrane domains. Biochemical fractionation of MDCK cells stably expressing the WT OTR-GFP indicated that only minor quantities of receptor are partitioned in caveolin-1 enriched domains. However, when fused to caveolin-2, the OTR protein proved to be exclusively localized in caveolin-1 enriched fractions, where it bound the agonist with increased affinity and efficiently coupled to Galpha(q/11). Interestingly, the chimeric protein was unable to undergo agonist-induced internalization and remained confined to the plasma membrane even after prolonged agonist exposure (120 min). A striking difference in receptor stimulation was observed when the OT-induced effect on cell proliferation was analysed: stimulation of the human WT OTR inhibited cell growth, whereas the chimeric protein had a proliferative effect. These data indicate that the localization of human OTR in caveolin-1 enriched microdomains radically alters its regulatory effects on cell growth; the fraction of OTR residing in caveolar structures may therefore play a crucial role in regulating cell proliferation.

Association of Smooth Muscle Cell Tissue Factor With Caveolae

There is still no satisfactory explanation for the low catalytic activity of tissue factor (TF)/factor VII(a) complexes towards coagulation factor X, as found on the apical surface side of cell layers. It has been hypothesized that TF exists in a latent form. Layers of cultured human smooth muscle cells, constitutively expressing TF, were immunogold-labeled for TF in situ and processed for electron microscopy. We showed that, besides internalization and accumulation in lysosomal-like structures, TF remained associated with noncoated, flask-shaped microinvaginations of the plasma membrane. These invaginations were identified as caveolae. In regions in which intercellular contacts were interrupted, more TF-positive caveolae were observed. Enzymatically detached smooth muscle cells exhibited a similar enlargement of caveolar structures. Concomitantly, an increase of catalytic activity of apically formed TF/VIIa complexes towards factor X was found on the suspended cells. We speculate that caveolae-associated TF may function as a latent pool of procoagulant activity, which can rapidly be activated at sites in which vessel wall integrity is lost.