Caveolar membranes contain one or more members of small (18-22 kDa), highly conserved proteins of the caveolin family of which caveolin-1 is the major structural protein of caveolae in cells other than striated muscle. It is present in two isoforms that may represent different gene transcripts . The longer (a) isoform is the major species in most cells. A second family member (caveolin-2) plays a role at the cell surface that is secondary, because caveolae are not detected in caveolin-1 -/- cells, despite the expression of caveolin-2 . Caveolin-3 is the major caveolin of striated muscle cells . A central domain in each caveolin (represented by residues 110-132 in caveolin-1) is highly hydrophobic. Both N-and C-termini are cytoplasmic . This suggests that the protein penetrates (but does not traverse) the plasma membrane bilayer.
In fixed membranes from rat adipocytes or human fibroblasts, myc-tagged cav-eolin-1 was present in the necks but not the bulbs of caveolae . In contrast, in quick-frozen unfixed membranes from 3T3-L1 mouse fibroblastic cells, caveolin-1 was identified as a belt around the caveolar bulb, but was absent from the neck that joins it to the plasma membrane . In both cell lines, the depletion of membrane FC with an extracellular acceptor (cyclodextrin) led to disassembly of cav-eolin multimers and flattening.
The caveolin(82-101) domain has been implicated in self-recognition of caveolin monomers, leading to assembly of a supportive structural basketwork. Based on an estimate of the predicted a-helical content of the caveolin-1 peptide including residues 79-96, it was proposed that filaments made up of caveolin heptamers formed a basketwork surrounding the caveola . However, short peptides can readily assume many different conformations, and secondary structure predictions for the 79-96 aa region in the context of the full primary sequence suggest a more complex structural situation. Other evidence discussed below suggests the presence of significant tertiary structure within the N-terminal half of caveolin-1. High-resolution three-dimensional structural analysis of caveolin will almost certainly be needed to distinguish these alternatives.
The composition of multi-protein complexes containing caveolin in living cells remains a contentious issue. Several dozens of proteins covering a wide range of functions at the cell surface have been identified at one time or another in purified membranes containing caveolae. It has been difficult to prove the existence of many of these in caveolae in living cells. Several factors have contributed to this situation. In earlier research, the range of factors that could modify native complexes in plasma membrane fractions, including detergents and cross-linking antibodies, was not fully appreciated. Major differences between cell lines in the protein composition of caveolae also were not recognized. Proteins in caveolae in primary cells may be found in caveolin-free lipid rafts or nonraft membrane domains in transformed and continuous cell lines, many of which express few, if any, caveolae [46-48]. Growth conditions can have major effects on the structure of caveolae, and on the distribution of caveolin between caveolae and other microdomains . Expression of caveolin in cell lines that are normally caveolin-defi-cient (e. g., FRT cells) may lead to the appearance of only a few cell-surface caveolae; most of the caveolin expressed accumulates on intracellular membrane vesicles . The sum of these factors means that the distribution of membrane proteins between caveolae and other cell-surface domains, and the influence of FC, is highly dependent on the cell or tissue used, technical details of fractionation, and the conditions of analysis.
This being said, certain classes of signaling proteins in tissues and primary cells have been repeatedly shown, by using several different techniques, to be reliably caveola-associated. The following criteria have been used in this chapter to assess if a multi-protein complex including caveolin represents a bona fide caveolar complex: co-localization with caveolin at the cell surface by confocal microscopy; recovery in caveolar vesicles in the absence of detergents; co-precipitation with caveolin antibodies from caveolae; and competitive displacement by caveolin peptides.
In addition to caveolin, caveolae contain other structural proteins which are insufficient by themselves to support membrane invagination, but are possibly important for the biological properties of these domains. Flotillins are a conserved protein family first identified as co-precipitates with caveolin in cells that had been extracted with Triton X-100 and octyl glucoside. Most recent research identifies these proteins mainly in association with GPI-anchored proteins in caveolin-free lipid rafts . Annexins are Ca2+-binding proteins. Annexin-2 forms a stable cyto-plasmic chaperone complex with caveolin-1 in mammalian cells in culture and in zebrafish in vivo (see Chapter 8). However, the inclusion of these proteins in cav-eolae at the cell surface has not yet been conclusively shown.
It is a characteristic of many differentiated peripheral cells that mitosis is strongly dependent on the activity of protein growth factors. These serve as ligands for transmembrane receptors. The protein kinase activity of these receptors initiates signaling cascades that terminate in the activation of nuclear transcription factors. The anchorage-independent growth of virally-transformed and cancer cells reflects their independence from exogenous growth factors, consistent with the frequent reduction of caveolin in cancer cells, and the role proposed for caveolae in growth control and cell attachment [49,52]. Hyperplasia reported in multiple tissues in caveolin-1 -/- mice [6,7] similarly reflects the impairment of normal growth regulation.
Transmembrane signaling kinases have been localized to caveolae in many different peripheral cell lines, using many different criteria. Receptor proteins for the platelet-derived, epidermal and vascular endothelial growth factors (PDGF, EGF,
VEGF) co-purified with the caveolar membrane fraction, and were co-precipitated with caveolin-1 antibodies. PDGFR co-purified with caveolae in 3T3 cells [53,54], normal human fibroblasts  and human vascular smooth muscle cells . EGFR co-purified with caveolae in primary human fibroblasts  and in a mouse fibroblast (3T3) line , but not laryngeal (Hep-2) or epidermal (A431) cancer cell lines [57,58]. VEGFR co-purified with caveolae and caveolin in both bovine aortic and human umbilical endothelial cells [59-61].
Additional evidence of a structural and functional association of caveolin with growth factors comes from studies of its (Y14)-phosphorylation, mediated by nonreceptor c-Src family kinases, which are themselves substrates for the tyrosine kinase activities of PDGFR, EGFR, and VEGFR (see Chapter 6). These data provide strong support for the existence of functional complexes between caveolin-1, transmembrane kinases, and nonreceptor signaling kinases such as c-Src and Fyn in living cells . Membrane transporter proteins are also frequently recovered in preparations of caveolae. Glucose (Glut-4) transporters were localized to caveolae in 3T3 cells . Potassium (K+(KATP), Na+, and Ca2+ channels were found in this fraction in SMC [64,65].
ATP-binding cassette transporter-A1 (ABCA1) ferries phospholipids and possibly FC across cell membranes, but it was not found with caveolin in detergent-extracted membranes . However, the opposite result was more recently obtained, under detergent-free conditions, using the plasma membrane fraction of rat endothelial cells, after purification with caveolin antibody . These contrasting data illustrate the ambiguity in protein composition data of caveolae from detergent-treated membranes.
Another recent study showed that a second ABC transporter (p-glycoprotein) can regulate the distribution of FC across the membrane bilayer . The localization of p-glycoprotein to caveolae in primary tissues and cells has been shown in several studies. [47,69,70]. In contrast, in 2780AD ovarian carcinoma cells (which lack caveolae completely), p-glycoprotein was recovered with lipid rafts .
Several other proteins able to modify the level of plasma membrane FC have been identified less consistently in caveolae. Scavenger receptor BI (SR-BI) catalyzes the selective uptake of cholesteryl esters and FC from HDL. It also plays a key physiological role in supplying substrate for steroid hormone production by adrenal and gonadal cells. SR-BI was identified in the caveolae of both overexpressing Chinese hamster ovary (CHO) cells and mouse adrenal (Y1) cells on the basis of immunofluorescence co-localization . A second study using SV-40 transformed human fibroblasts (WI38-VA13 cells) found little SR-BI in caveolae; the majority of this protein was in microvilli of the cell surface . The transformed cells contained relatively few caveolae. In adrenal cells in vivo, SR-BI was mainly associated with a novel class of double-membrane channels associated with microvilli containing few, if any, caveolae . The association of SR-BI with caveolae, when present, remains unsettled at this time. In contrast, the beta-subunit of ATP synthase, identified as a high-density lipoprotein (HDL) binding protein, was localized to caveolae, and immunoprecipitated with caveolin . These data, and that relating to ABCA1 , suggest that caveolae may be able to regulate their own FC content.
One particularly well-characterized complex of caveolar lipids and proteins is that of FC with caveolin and endothelial nitric oxide synthase (eNOS) Numerous studies have shown the co-purification of eNOS and caveolin, and its inhibition of this activity by caveolin peptide [76-80] (see also Chapter 11).
Several proteins which control growth and differentiation by other pathways are also present in caveolae. These include receptors for bone morphogenetic proteins  and the cell-surface receptor proteins for estrogen and vitamin D, identical to the transcription factors regulated by these hormones in the nucleus [82,83].
To summarize, the distribution of proteins between different cell-surface domains in living cells is not maintained after detergent extraction. Even in the absence of detergents, the distribution of these proteins in continuous cell lines may be modified from that found in intact tissues and primary cells. Nevertheless, a subset of proteins is reproducibly associated with caveolae in tissues and primary cells. These include transmembrane signaling kinases, eNOS, and some membrane transporters. These caveola-associated proteins are characterized both by caveolin-binding, and a marked dependence on FC. The structural and functional relationship between these properties, and its implications, are considered in the following sections.
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