Growth factor receptors, lipid rafts and caveolae: an evolving story

Growth factor receptors have been shown to be localized to lipid rafts and caveolae. Consistent with a role for these cholesterol-enriched membrane domains in growth factor receptor function, the binding and kinase activities of growth factor receptors are susceptible to regulation by changes in cholesterol content. Furthermore, knockouts of caveolin-1, the structural protein of caveolae, have confirmed that this protein, and by implication caveolae, modulate the ability of growth factor receptors to signal. This article reviews the findings pertinent to the relationship between growth factor receptors, lipid rafts and caveolae and presents a model for understanding the disparate observations regarding the role of membrane microdomains in the regulation of growth factor receptor function.     

Purification and characterization of smooth muscle cell caveolae

Plasmalemmal caveolae are a membrane specialization that mediates transcytosis across endothelial cells and the uptake of small molecules and ions by both epithelial and connective tissue cells. Recent findings suggest that caveolae may, in addition, be involved in signal transduction. To better understand the molecular composition of this membrane specialization, we have developed a biochemical method for purifying caveolae from chicken smooth muscle cells. Biochemical and morphological markers indicate that we can obtain approximately 1.5 mg of protein in the caveolae fraction from approximately 100 g of chicken gizzard. Gel electrophoresis shows that there are more than 30 proteins enriched in caveolae relative to the plasma membrane. Among these proteins are: caveolin, a structural molecule of the caveolae coat; multiple, glycosylphosphatidylinositol-anchored membrane proteins; both G alpha and G beta subunits of heterotrimeric GTP-binding protein; and the Ras-related GTP-binding protein, Rap1A/B. The method we have developed will facilitate future studies on the structure and function of caveolae.     

Reggie-1 and reggie-2 localize in non-caveolar rafts in epithelial cells: cellular localization is not dependent on the expression of caveolin proteins

Reggie-1 and reggie-2 are highly conserved and widely expressed proteins associated with membrane rafts. The molecular function of reggies remains to be clarified, but recent data indicate that they are involved in various cellular processes such as insulin signaling, phagocytosis and actin remodeling. However, there is discrepancy in the literature if reggies are associated with caveolae or non-caveolar rafts. Reggies are expressed and raft associated also in many cells which do not contain caveolae, such as neurons and lymphocytes. However, it is not clear if the function or localization of reggies are dependent on the presence of caveolae and expression of caveolin-1 protein. In this study, we directly addressed this question in epithelial cells. We could show that ectopic expression of caveolin-1 does not result in any change in the cellular localization of reggie-1, which is present at the plasma membrane also in the absence of caveolin-1. On the other hand, caveolin-2, which localizes in caveolae, is dependent on caveolin-1 expression in order to be localized at the plasma membrane. Although reggie-1 and reggie-2 strongly interact with each other, we did not detect a direct interaction between caveolin-1 and reggies by means of a yeast two-hybrid assay, nor could reggies be co-immunoprecipitated with caveolin-1. Furthermore, endogenous reggie-1 and -2 were found not to colocalize with caveolin-1 in epithelial cells. Thus, our data indicate that reggies are localized in microdomains different from caveolae, and the function of reggies is different from and independent of caveolin-1.     

Cellular spelunking: exploring adipocyte caveolae

It has been known for decades that the adipocyte cell surface is particularly rich in small invaginations we now know to be caveolae. These structures are common to many cell types but are not ubiquitous. They have generated considerable curiosity, as manifested by the numerous publications on the topic that describe various, sometimes contradictory, caveolae functions. Here, we review the field from an "adipocentric" point of view and suggest that caveolae may have a function of particular use for the fat cell, namely the modulation of fatty acid flux across the plasma membrane. Other functions for adipocyte caveolae that have been postulated include participation in signal transduction and membrane trafficking pathways, and it will require further experimental scrutiny to resolve controversies surrounding these possible activities.     

Relationship between cholesterol trafficking and signaling in rafts and caveolae

Caveolae and lipid rafts are two distinct populations of free cholesterol, sphingolipid (FC/SPH)-rich cell surface microdomains. They differ in stability, shape, and the presence or absence of caveolin (present in caveolae) or GPI-anchored proteins (enriched in lipid rafts). In primary cells, caveolae and rafts support the assembly of different signaling complexes, though signal transduction from both is strongly dependent on the presence of FC. It was initially thought that FC promoted the formation of inactive reservoirs of signaling proteins. Recent data supports the concept of a more dynamic role for FC in caveolae and probably, also lipid rafts. It is more likely that the FC content of these domains is actively modulated as protein complexes are formed and, following signal transduction, disassembled. In transformed cell lines with few caveolae, little caveolin and a preponderance of rafts, complexes normally assembled on caveolae may function in rafts, albeit with altered kinetics. However, caveolae and lipid rafts appear not to be interconvertible. The presence of non-caveolar pools of caveolin in recycling endosomes (RE), the trans-Golgi network (TGN) and in mobile chaperone complexes is now recognized. A role in the uptake of microorganisms by cells ascribed to caveolae now seems more likely to be mediated by cell surface rafts.     

Relationship between cholesterol trafficking and signaling in rafts and caveolae

Caveolae and lipid rafts are two distinct populations of free cholesterol, sphingolipid (FC/SPH)-rich cell surface microdomains. They differ in stability, shape, and the presence or absence of caveolin (present in caveolae) or GPI-anchored proteins (enriched in lipid rafts). In primary cells, caveolae and rafts support the assembly of different signaling complexes, though signal transduction from both is strongly dependent on the presence of FC. It was initially thought that FC promoted the formation of inactive reservoirs of signaling proteins. Recent data supports the concept of a more dynamic role for FC in caveolae and probably, also lipid rafts. It is more likely that the FC content of these domains is actively modulated as protein complexes are formed and, following signal transduction, disassembled. In transformed cell lines with few caveolae, little caveolin and a preponderance of rafts, complexes normally assembled on caveolae may function in rafts, albeit with altered kinetics. However, caveolae and lipid rafts appear not to be interconvertible. The presence of non-caveolar pools of caveolin in recycling endosomes (RE), the trans-Golgi network (TGN) and in mobile chaperone complexes is now recognized. A role in the uptake of microorganisms by cells ascribed to caveolae now seems more likely to be mediated by cell surface rafts.     

Triacylglycerol is synthesized in a specific subclass of caveolae in primary adipocytes

A principal metabolic function of adipocytes is to synthesize triacylglycerol (TG) from exogenous fatty acids. The level of fatty acids has to be tightly controlled in the adipocyte, as they can act as detergents that rapidly dissolve the plasma membrane, causing cell lysis if allowed to accumulate. Fatty acids therefore have to be efficiently converted to TG and stored in the central lipid droplet. We report that in intact primary adipocytes exogenous oleic acid was taken up and directly converted to TG in the plasma membrane, in a novel subclass of caveolae that specifically contains the protein perilipin. Isolated caveolae catalyzed de novo TG synthesis from oleic acid and glycerol 3-phosphate. Electron microscopy revealed the presence of caveolin and perilipin in caveolae and in lipid-laden bulbs in the plasma membrane, and fluorescence microscopy demonstrated colocalization of fatty acids/TG with caveolin and perilipin at the plasma membrane. A second caveolae fraction was isolated, which lacked perilipin and the triacylglycerol synthesizing enzymes. Both caveolae fractions contained caveolin-1 and the insulin receptor. The findings demonstrate that specific subclasses of caveolae carry out specific functions in cell metabolism. In particular, triacylglycerol is synthesized at the site of fatty acid entry in one of these caveolae classes.     

Caveolae in fibroblast-like synoviocytes: static structures associated with vimentin-based intermediate filaments

The fibroblast-like synoviocyte is a CD13-positive cell-type containing numerous caveolae, both single and interconnected clusters. In unstimulated cells, all single caveolae at the cell surface and the majority of those localized deeper into the cytoplasm were freely accessible from the medium, as judged from electron microscopy of synoviocytes exposed to the membrane impermeable marker Ruthenium Red. Caveolar internalization could be induced by a CD13 antibody or by cholera toxin B subunit (CTB). Thus, in experiments using sequential labeling with Alexa 488- and 594-conjugated CTB, about 50% of CTB-positive caveolae were internalized by 5 min of chase, and these remained inaccessible from the cell surface for periods up to 24 h. No colocalization with an endosomal marker, EEA1, or Lysotracker was observed, indicating that internalized caveolae clusters represent a static compartment. Vimentin was identified as the most abundant protein in detergent resistant membranes (DRM’s), and by immunogold electron microscopy caveolae were seen in intimate contact with intermediate-size filaments. These observations indicate that vimentin-based filaments are responsible for the spatio-temporal fixation of caveolae clusters. RECK, a glycosylphosphatidylinositol-anchored protein acting as a negative regulator of cell surface metalloproteinases, was also localized to the caveolae clusters. We propose that these clusters function as static reservoirs of specialized lipid raft domains where proteins involved in cell–cell interactions, such as CD13, can be sequestered by binding to RECK in a regulatory manner.     

Docosahexaenoic acid affects endothelial nitric oxide synthase in caveolae

n-3 Polyunsaturated fatty acids are assumed to play an important role in the prevention and treatment of atherosclerosis. Endothelial nitric-oxide synthase (eNOS) is responsible for cardiovascular homeostasis involving in regulation of vascular function, and the subcellular localization is critical for its activation. Here we determined the effect of docosahexaenoic acid (DHA, 22:6 n-3) on distribution of eNOS and its activity. DHA treatment markedly altered lipid environment of caveolae microdomains, which was coincided with selective displacement of caveolin-1 and eNOS from caveolae. Akt was not detected in caveolae fractions and CaM was distributed in both of caveolin-1-enriched membranes and non-caveolar fractions, whose distribution was unaffected by DHA. These data demonstrated for the first time that DHA altered caveolae microenvironment not only by modifying membrane lipid composition, but also by changing distribution of major structural proteins. DHA-induced alterations in caveolae lipid/protein environment may be an important mechanism in the development of pathogenesis of atherosclerosis.     

Role of caveolin and caveolae in insulin signaling and diabetes

Caveolae are specialized membrane microdomains present within the plasma membrane of the vast majority of cell types. They have a unique composition in that they are highly enriched in cholesterol, sphingolipids, and their coat proteins the caveolins (-1, -2, and -3). In recent years it has been recognized that caveolae act as signaling platforms, serving as a concentrating point for numerous signaling molecules, as well as regulating flux through many distinct signaling cascades. Although caveolae are found in a variety of cell types, they are most abundant in adipose tissue. This fact has led to the intense study of the function of these organelles in adipocytes. It has now become apparent that effective insulin signaling in the adipocyte may be strictly dependent on localization of at least two insulin-responsive elements to caveolae (insulin receptor and GLUT4), as well as on a direct functional interaction between caveolin-1 and the insulin receptor. We present a critical discussion of these recent findings.     

A comparative study of the membrane structure in different types of muscle fibers in the frog

The muscle membrane of slow and fast fibers in cruralis and iliofibularis muscles and of intermediate fibers in submaxillaris muscle of the frog is studied in freeze-fracture replicas. A comparison of membrane folds, number, size and distribution of caveolae and of intramembrane particles (IMP) is given. In slow muscle fibers, the membrane folds are systematically present at the level of the I zone with a transversal continuity, whereas in fast and intermediate types the membrane folds are small and are randomly distributed. In slow muscle the caveolae are more numerous at the I zone than in the part corresponding to the center of the sarcomere. In fast muscle, small groups of caveolae form linear patterns, and in intermediate fibers the distribution is random. The number of caveolae in slow muscle fibers is two times more than in fast and intermediate fibers. The mean area of caveolae opening is largest in fast and smallest in slow muscle fibers. The number of IMP is significantly different in the three types of fibers, being highest in slow and lowest in intermediate fibers. The different pattern of folds in slow fibers may correspond to the different contractile properties of this fiber type. The presence of double the number of caveolae in slow fibers correlated to the less elaborate T system in this fiber type shows the possibility that slow fibers may be the result of an arrest during development for the performance of a different function. The difference in IMP density in the three muscle fiber types may be interpreted as the difference in their electrical properties.     

Endothelin induces rapid, dynamin-mediated budding of endothelial caveolae rich in ET-B

Clathrin-independent trafficking pathways for internalizing G protein-coupled receptors (GPCRs) remain undefined. Clathrin-mediated endocytosis of receptors including ligand-engaged GPCRs can be very rapid and comprehensive (<10 min). Caveolae-mediated endocytosis of ligands and antibodies has been reported to be much slower in cell culture (≫10 min). Little is known about the role of physiological ligands and specific GPCRs in regulating caveolae trafficking. Here, we find that one receptor for endothelin, ET-B but not ET-A, resides on endothelial cell surfaces in both tissue and cell culture primarily concentrated within caveolae. Reconstituted cell-free budding assays show that endothelins (ETs) induce the fission of caveolae from endothelial plasma membranes purified from rat lungs. Electron microcopy of lung tissue sections and tissue subcellular fractionation both show that endothelin administered intravascularly in rats also induces a significant loss of caveolae at the luminal surface of lung vascular endothelium. Endothelial cells in culture show that ET stimulates very rapid internalization of caveolae and cargo including caveolin, caveolae-targeting antibody, and itself. The ET-B inhibitor BQ788, but not the ET-A inhibitor BQ123, blocks the ET-induced budding of caveolae. Both the pharmacological inhibitor Dynasore and the genetic dominant negative K44A mutant of dynamin prevent this induced budding and internalization of caveolae. Also shRNA lentivirus knockdown of caveolin-1 expression prevents rapid internalization of ET and ET-B. It appears that endothelin can engage ET-B already highly concentrated in caveolae of endothelial cells to induce very rapid caveolae fission and endocytosis. This transport requires active dynamin function. Caveolae trafficking may occur more rapidly than previously documented when it is stimulated by a specific ligand to signaling receptors already located in caveolae before ligand engagement.     

Oligomers of the ATPase EHD2 confine caveolae to the plasma membrane through association with actin

Caveolae are specialized domains present in the plasma membrane (PM) of most mammalian cell types. They function in signalling, membrane regulation, and endocytosis. We found that the Eps-15 homology domain-containing protein 2 (EHD2, an ATPase) associated with the static population of PM caveolae. Recruitment to the PM involved ATP binding, interaction with anionic lipids, and oligomerization into large complexes (60-75S) via interaction of the EH domains with intrinsic NPF/KPF motifs. Hydrolysis of ATP was essential for binding of EHD2 complexes to caveolae. EHD2 was found to undergo dynamic exchange at caveolae, a process that depended on a functional ATPase cycle. Depletion of EHD2 by siRNA or expression of a dominant-negative mutant dramatically increased the fraction of mobile caveolar vesicles coming from the PM. Overexpression of EHD2, in turn, caused confinement of cholera toxin B in caveolae. The confining role of EHD2 relied on its capacity to link caveolae to actin filaments. Thus, EHD2 likely plays a key role in adjusting the balance between PM functions of stationary caveolae and the role of caveolae as vesicular carriers.     

Ecto-ATPases and 5′-nucleotidases in the caveolae of smooth muscle. Enzyme-histochemical evidence may indicate a role for caveolae in neurotransmission.

We have demonstrated the localization of ecto-Ca-ATPase and 5′-nucleotidase activity in the caveolae of smooth muscle cells of guinea pig vas deferens and the ileum longitudinal muscle strips with a cerium-precipitation enzyme-cytochemical method. The activities seemed to be strongest in the caveolae. Since the simultaneous presence of the 5′-nucleotidase activity supports the hypothesis that this ecto-Ca-ATPase activity does not have a pump function, but, together with 5′-nucleotidase, may play a role in neurotransmission, these specific membrane invaginations, the caveolae, have a functional relationship with transverse tubules of striated muscle.     

Caveolar endocytosis and virus entry

The endocytic function of caveolae has been controversial for a long time. However, a real-time-imaging analysis of Simian virus 40 (SV40) 's entry in cells has indicated the existence of caveolar endocytosis during virus entry. The caveolae engulfed SV40 virions begin budding from plasma membrane depending on dynamin. SV40 enclosed in caveolae vesicles move to the caveosome, then to the endoplasmic reticulum. In addition, it was demonstrated that human coronavirus-229E enters the cell through caveolae. This review examines the involvement of caveolae in endocytosis used by the viral entry system.     

Depletion of plasma membrane cholesterol dampens hydrostatic pressure and shear stress-induced mechanotransduction pathways in osteoblast cultures

The preferential association of cholesterol and sphingolipids within plasma membranes forms organized compartments termed lipid rafts. Addition of caveolin proteins to this lipid milieu induces the formation of specialized invaginated plasma membrane structures called caveolae. Both lipid rafts and caveolae are purported to function in vesicular transport and cell signaling. We and others have shown that disassembly of rafts and caveolae through depletion of plasma membrane cholesterol mitigates mechanotransduction processes in endothelial cells. Because osteoblasts are subjected to fluid-mechanical forces, we hypothesize that cholesterol-rich plasma membrane microdomains also serve the mechanotransduction process in this cell type. Cultured human fetal osteoblasts were subjected to either sustained hydrostatic pressure or laminar shear stress using a pressure column or parallel-plate apparatus, respectively. We found that sustained hydrostatic pressure induced protein tyrosine phosphorylation, activation of extracellular signal-regulated kinase (ERK)1/2, and enhanced expression of c-fos in both time- and magnitude-dependent manners. Similar responses were observed in cells subjected to laminar shear stress. Both sustained hydrostatic pressure- and shear stress-induced signaling were significantly reduced in osteoblasts pre-exposed to either filipin or methyl--cyclodextrin. These mechanotransduction responses were restored on reconstitution of lipid rafts and caveolae, which suggests that cholesterol-rich plasma membrane microdomains participate in the mechanotransduction process in osteoblasts. In addition, mechanical force-induced phosphoproteins were localized within caveolin-containing membranes. These data support the concept that lipid rafts and caveolae serve a general function as cell surface mechanotransduction sites within the plasma membrane. lipid rafts; caveolae; extracellular signal-regulated kinase     

Potocytosis

Potocytosis represents a mechanism by which small and large molecules as well as macromolecular complexes are sequestered and transported by caveolae. Caveolae are flask-shaped plasma membrane specializations characterized by a filamentous coat consisting of caveolins that decorates the inside surface of each caveola membrane. They have endocytotic functions that differ from the clathrin-coated pit pathway. Ligands bound to receptors that are internalized by caveolae can be delivered to four different locations in the cell bypassing the lysosome and at least four different caveolae membrane traffic patterns during potocytosis can be distinguished. Hence, cells have two endocytic machines and each is designed to accomplish different tasks. This review provides a brief summary of the discovery of caveolae and of potocytosis, and focuses on recent discoveries of the unique endocytic capabilities of caveolae in a variety of different cells.     

Theoretical model for the formation of caveolae and similar membrane invaginations

We study a physical model for the formation of bud-like invaginations on fluid lipid membranes under tension, and apply this model to caveolae formation. We demonstrate that budding can be driven by membrane-bound proteins, provided that they exert asymmetric forces on the membrane that give rise to bending moments. In particular, caveolae formation does not necessarily require forces to be applied by the cytoskeleton. Our theoretical model is able to explain several features observed experimentally in caveolae, where proteins in the caveolin family are known to play a crucial role in the formation of caveolae buds. These include 1), the formation of caveolae buds with sizes in the 100-nm range and 2), that certain N- and C-termini deletion mutants result in vesicles that are an order-of-magnitude larger. Finally, we discuss the possible origin of the morphological striations that are observed on the surfaces of the caveolae.