Caveolae are free cholesterol/sphingolipid (FC/SPH)-rich microdomains of the cell surface that are assembly sites for many transmembrane signaling complexes. Other proteins associated with caveolae include the transporters of some small ligands (glucose, inorganic ions) and catalysts of cell FC homeostasis. The highest levels of caveolae are found in differentiated primary cells, including pulmonary type-1 cells, adipocytes, endothelial cells, smooth muscle cells, and fibroblasts. In blood lymphocytes and many transformed and cancer cell lines, caveolae are sparse or may be completely absent.
While there is no broadly accepted nomenclature, many investigators now recognize two classes of FC/SPH-rich microdomains, caveolae and lipid rafts (Fig. 5.1) [1-3]. Caveolae are invaginated, and contain unique structural proteins (caveolins) that play an essential role in maintaining membrane curvature (see Chapter 2). The mean diameter of caveolar pits is ~60-80 A. A second class of proteins (dynamins) is present in the necks of caveolae , but these are also present in other structures, such as clathrin-coated pits. The central role of caveolins-[particularly caveolin-1, the largest (22 kDa) caveolin-family protein] in maintaining the structure of caveolae is shown by their disappearance from the cell surface if the expression of caveolin is knocked down in wild-type cells . This is confirmed by the absence of caveolae from caveolin-1 -/- cells [6,7]. The structural significance of FC in caveolae is shown by the flattening that first occurs when the plasma membrane is FC-depleted [8,9]. The caveolin skeleton remains at the cell surface. Caveolae reappear when FC is replaced in the plasma membrane. Additional loss of FC may be followed by disassembly of caveolin multimers . Whilst the transfer of caveolin between the cell surface and intracellular pools can be demonstrated using inhibitors of microtubule assembly, it is unclear whether this normally occurs.
Under physiological conditions the caveolin skeleton at the surface of living cells appears to be relatively long-lived. Individual caveolae can be identified at the cell surface over periods of hours or days . In contrast, FC and signaling proteins
move in and out of caveolae much more rapidly (in some cases over a few minutes) in response to signal transduction, mitosis, and cell migration [12,13]. Caveolae are defined here as invaginated cell-surface microdomains stabilized with caveolin whose association with more labile complexes of lipids and other proteins responds to physiological stimuli. Almost all research investigations into the relationship between caveolin and FC have focused on caveolin-1.
Lipid rafts have a similar diameter to caveolae, but are planar and lack caveolin (see Fig. 5.1). Most studies of lipid rafts have been carried out in cells that lack appreciable levels of caveolin or caveolae; however, even in cells rich in caveolae, some caveolin-free lipid rafts are probably present. Glycosylphosphatidylinositol (GPI)-anchored proteins are enriched in lipid rafts, and reduced or absent in caveolae [1,2,14]. The lifetime of lipid rafts appears to be orders of magnitude less than that of caveolae. Single molecules of GPI-anchored protein move between rafts with a t1/2 of seconds or minutes (see Chapter 3), though rafts incorporating multi-protein signaling complexes may be more stable. FC/SPH-rich microdomains with physical properties similar to those of lipid rafts form spontaneously in synthetic lipid bilayers to an extent dependent on the levels of FC and SPH, and reflecting the separation of a FC-rich liquid-ordered (Lo) phase [15-17]. In addition to GPI-anchored proteins, many acylated proteins are raft-associated. It seems likely that acylation is an important promoter of the association of signaling proteins in FC/SPH-rich raft membrane domains.
Much less is known of the organization of lipids in caveolae, but almost certainly there are significant differences from that in lipid rafts. In particular, it is not clear that the lipids of caveolae represent a Lo phase. This need not imply that there is no exchange of lipids between caveolae and rafts, but it could involve equilibration with non-raft membrane microdomains (see Fig. 5.1). The properties of caveolae and lipid rafts are reviewed in Chapters 2, 3, and 7. Here, the focus is on the regulatory role of FC, and in particular its interaction with signaling proteins and caveolin.
Lipids of Caveolae
The lipid composition of caveolae has been determined in only a few cases. The results of early studies in this area are difficult to interpret due to the presence of the detergents that are now recognized to modify the native distribution of lipids and proteins in membrane fractions . A second problem has been the combination of caveolae and lipid rafts in a FC/SPH-rich "lipid raft" plasma membrane fraction, in which the contribution of caveolin-containing vesicles was undetermined. An additional variable has been the extent to which a "rim" of circumferential membrane and "neck" joining the caveolar bulb to the cell surface was included with the membrane "bowl" which made up the rest of the caveolar surface . Recently, techniques for detergent-free isolation of caveolae from the cell surface have been described . One detailed recent report is of the composition of caveolae from primary rat adipocytes . These were purified from a total plasma membrane fraction by gradient ultracentrifugation, and then by immuno-precipitation with caveolin antibody.
Caveolae contain significantly higher levels of FC and sphingomyelin than do unfractionated plasma membranes. That of glycerophospholipids is essentially unchanged, so the total number of lipid molecules per unit area is increased (Fig. 5.2). The level of gangliosides is higher in caveolae than in plasma mem-
branes overall, though it represents only a small part (about 1 %) of the total sphin-golipid present. The major ganglioside of adipocytes is GM3. The same has been found for other differentiated peripheral cells rich in caveolae. GM1, though often identified as a specific marker for caveolae by its reaction with cholera toxin, in primary adipocytes was enriched only 2.6-fold in this fraction.
Additional analyses were reported from a line of human epidermal carcinoma (KB) cells stably transfected with mouse caveolin-1 cDNA . The membrane fraction in this study was not immunopurified, and so probably included some caveolin-free lipid rafts. Nevertheless, as in the adipocyte study, the caveola-en-riched fraction of KB cells contained increased levels of FC and sphingomyelin, relative to the rest of the plasma membrane. An unexpected finding was the enrichment there of arachidonyl ethanolamine plasmalogens, though this was also found in the raft fraction from a control, caveolin-free KB cell line.
Another unexpected finding was the rapidity with which FC moved between caveolae and other microdomains or extracellular acceptors in response to signal transduction . FC was transferred readily to either cyclodextrin, a synthetic extracellular FC acceptor, or lipid-poor high-density lipoprotein, prebeta-HDL [22,23]. Dehydroergosterol, a fluorescent sterol marker for FC, was transferred more rapidly from of a caveola-rich fraction of fibroblast-derived l-cells than from other membrane domains . In synthetic membranes, the effect of lowering FC is to decrease membrane stiffness and increase diffusion rates from the bilayer [25,26]. In contrast, FC depletion from caveola-rich membranes, which are linked to the sub-skeleton via the actin-family protein filamin  led to increased membrane stiffness [28,29].
Further evidence for a unique organization of FC in caveolae was presented in a recent study of the water permeability of caveolae from rat lung. This was 5- to 10-fold greater than that of noncaveolar membranes of comparable lipid composition . Aquaporin has been reported in lung caveolae , but aquaporin did not significantly stimulate water flux in caveolae in this study . If confirmed with caveolae from other cells, this observation could help to explain the rapid changes in FC in caveolae to physiological stimuli  compared to the stability of their caveolin skeleton . FC-FC bonds within caveolae appear to be weaker than in other membrane microdomains.
Taken together, these data suggest that the organization of FC in caveolae and lipid rafts is fundamentally different. FC and SPH are enriched in both (relative to other membrane fractions), but within caveolae FC is labile not stabilized and the lipid bilayer is more, rather than less, permeable to water. The key factor may be that caveolin binds FC directly [31-33]. If this is correct, the properties of FC in lipid rafts would be determined mainly by its interaction with other lipids particularly SPH; in caveolae, they would be determined mainly by caveolin [13,33].
In addition to the major lipid classes (FC, sphingolipids, glycerolipids), caveolae also contain relatively high levels (compared to other plasma membrane fractions) of signaling lipids, including phosphatidic acid, diglyceride, ceramide and asialo-gangliosides. Most of these are probably generated in situ since caveolae are enriched in phospholipase D2 , lipid phosphate phosphohydrolase , sphingo-
myelinase and ceramidase [36,37] and sialidase  While the primary role of these lipids appears to be in signaling, some may also act locally to regulate the composition and properties of caveolae. Ceramide generated by extracellular bacterial sphingomyelinase reduced the interaction of FC and caveolin ; however, whether the low levels of bioactive lipids present under physiological conditions would be sufficient for such effects to be significant remains uncertain.
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