Caveolae and Caveolin1

Our understanding of plasma membrane microdomains in general, and of caveolae and lipid rafts in particular, has grown tremendously during the past few years. One important paradigm shift occurred with the clear demonstration that two very similar domains exist on the plasma membrane called caveolae and lipid rafts [1,2]. Caveolae in most cells and tissues are defined by the presence of a 22-kDa protein called caveolin-1, whereas lipid rafts are defined by the presence of glycosyl-phosphatidylinositol (GPI)-anchored proteins such as CD55. Caveolae and lipid rafts are similar in that both types of domain are enriched in cholesterol, sphingomyelin, and signaling proteins [3]. However, caveolae contain significantly more cholesterol than lipid rafts, and it is this high relative concentration of cholesterol that is thought to be responsible for the invaginated morphology of caveolae in some cell types [4,5]. In contrast, caveolae that are depleted of cholesterol can lose their invaginated morphology and flatten within the plane of the plasma membrane [6]. Thus, the contents of caveolin and cholesterol in cells appear to be critical elements in the formation, number and morphology of caveolae. The localization of caveolin-1 in caveolae is critical in the organization of caveolae, and this is a combined result of the effect of caveolin on the cholesterol content, organization of the membrane lipid components, and the effect of caveolin as it forms oligomers and is able to bind to and scaffold other proteins.

Few studies have been conducted in which the levels and distribution of caveolin-1 and the absolute content of cholesterol within caveolae have been analyzed. Variation has been observed in the content of caveolin in caveolin-expressing cells and tissues that differ by more than two orders of magnitude. Quite clearly, a "caveolin-containing" cell that has a relative 100-fold enrichment of caveolin compared to another "caveolin- containing" cell is quite likely to have a dramatically different organization of caveolae, a different proportion of caveolae within the plasma membrane, and a different distribution of caveolin-organized domains within other intracellular membranes causing variation in caveolin's role in the regulation of trafficking and signaling pathways. However, few studies have taken into account the heterogeneity and functional differences between distinct cell types. In a parallel argument, cells in which caveolin expression has been lost or ablated in culture will respond differently, reflecting the alteration of steady-state equilibria between caveolae domains, lipid rafts, and phospholipids regions of the plasma membrane and intracellular membranes. Thus, some signaling or trafficking pathways normally regulated by caveolin may show little impact of caveolin disruption due to compensatory activation of secondary pathways localized to lipid rafts or even phospholipids domains. A number of ligands that initiate signaling in caveolae can be internalized by clathrin-mediated endocytosis, although in cav-eolin-expressing cells the rapid caveolae-mediated endocytic pathway dominates under normal physiologic conditions. This also holds true for membrane components. Overexpression of caveolin-1 changed the uptake of specific glycosphingo-lipids and shifted GM1 from the clathrin endocytic pathway to a caveolae-endocytic pathway [7]. The abolition of GPI synthesis to cause cell depletion of GPI-anchored proteins resulted in an increase in caveolae at the cell surface, indicating that some type of inverse relationship existed between the amount of lipid rafts and caveolae expressed on the cell surface [8]. The manipulation of total cell cholesterol content with cyclodextrin has been shown to have profound effects on caveolae organization and localization of caveolae-associated proteins, including caveolin-1. The effects of cyclodextrin require careful measurement of caveolae cholesterol content before and after treatment to determine whether such effects either disrupt cav-eolae or deplete the entire plasma membrane surface of cholesterol [9]. Physiological manipulations, such as the addition of lipoproteins which bind to receptors and alter cholesterol distribution in caveolae, result in changes in caveolae cholesterol and caveolin content, and change caveolae signaling and trafficking as a consequence [10,11]. These studies document the importance of careful quantitative analysis of the level of expression of caveolin protein and the mass of cholesterol in the cell as a whole, and within the caveolae compartment specifically, to demonstrate the physiological relevance of altered caveolae cholesterol levels to the change that occurs in a specific pathway or activity. Otherwise, the change could be due to alterations in cholesterol levels in noncaveolae membrane domains which cause a change in a pathway associated with these domains.

Because of the steady-state interactions between caveolae and other membrane domains, the merging of endocytic pathways and capacity for redundancy in endocytosis, and redundancy in signal transduction that has been identified since the first null mouse without an obvious phenotype was produced, care must be taken in interpreting the results of gross manipulations which alter protein and lipid content of cells and caveolae. In particular, studies in cells in which caveolin is absent, or in which the expression has been ablated, must be carefully evaluated. Several studies have been published from which conclusions have been drawn using negative data about the role (or lack of role) of caveolae and caveolin in a particular pathway, without having incorporated controls to ensure that the model system being employed is physiologically relevant or evaluation of changes in the activity of compensatory, overlapping, or redundant pathways.

Caveolin Protein Structure, Domains, and Membrane Interactions

Three caveolins have been identified in mammalian cells, termed caveolin-1, -2, and -3. Caveolin-1 and -2 are co-expressed in a wide range of tissues, exhibiting high expression in lung, vascular tissue, fibroblasts and adipocytes, whereas cav-eolin-3 shows limited expression and is the dominant caveolin in striated muscle. Caveolin-1, a 21- to 24-kDa protein, is a major integral membrane component of caveola membranes (Fig. 8.1). Caveolin-1 contains an additional N-terminal 27 amino acids (aa) that are not found in caveolin-3, and is also 16 amino acids longer than caveolin-2. Thus, all three caveolins resolve at distinct molecular weights by sodium dodecylsulfate-polyacryamide gel electrophoresis (SDS-PAGE). The predicted secondary structure of caveolin contains an N-terminal cytoplasmic region, a membrane-associated domain, and a cytosolic C-terminal tail. All three caveolins exhibit a similar structure, with the N- and C-terminal ends of the protein in the cytoplasm flanking a ~20 aa hydrophobic domain that forms a hairpin in the membrane. Several regions of caveolin-1 have been identified that mediate interactions

Fig. 8.1 Caveolin protein structure and domains. The distinct functional domains in caveolin-1 (Cav-1) are shown graphically and labeled as described in the text. Both caveolin-1 and caveolin-2 have alternative splice sites and exist as full-length alpha and truncated beta isomers. Domains showing potential conservation based on primary sequence homology are aligned for caveolin-3 (below Cav-1) and caveolin-2 (above Cav-1). Caveolin-3 shows high sequence homology in the hairpin, oligomerization, scaffolding and C-

terminal cytoplasmic domains, including conservation of the position of three cysteines that are acylated in caveolin-1, as discussed in the text. Caveolin-2 shows homology in the hairpin domain and the N-terminal half of the oligomerization domain, but differs significantly in the primary sequence of the scaffolding domain and the C-terminal cytoplasmic domain. All three caveolins contain unique sequences in the N-terminal region that aligns with the first 50 amino acids of caveolin-1.

Fig. 8.1 Caveolin protein structure and domains. The distinct functional domains in caveolin-1 (Cav-1) are shown graphically and labeled as described in the text. Both caveolin-1 and caveolin-2 have alternative splice sites and exist as full-length alpha and truncated beta isomers. Domains showing potential conservation based on primary sequence homology are aligned for caveolin-3 (below Cav-1) and caveolin-2 (above Cav-1). Caveolin-3 shows high sequence homology in the hairpin, oligomerization, scaffolding and C-

terminal cytoplasmic domains, including conservation of the position of three cysteines that are acylated in caveolin-1, as discussed in the text. Caveolin-2 shows homology in the hairpin domain and the N-terminal half of the oligomerization domain, but differs significantly in the primary sequence of the scaffolding domain and the C-terminal cytoplasmic domain. All three caveolins contain unique sequences in the N-terminal region that aligns with the first 50 amino acids of caveolin-1.

with itself and other proteins. The N-terminal membrane proximal region (residues 61-101) of caveolin-1 is sufficient to mediate the formation of caveolin homo-oligomers, and a portion of this controls exit from the Golgi [12]. A part of this region (residues 82-101) has been termed the "scaffolding domain", and mediates the putative interaction of caveolin-1 with signaling molecules such as small GTP-binding proteins, Src family tyrosine kinases, and endothelial nitric oxide synthase (eNOS) [13,14]. Other domains within caveolin have also been reported to interact with and serve as scaffolds to bind signaling molecules to caveolae [15].

Caveolin-1 has three cysteines, caveolin-2 has five cysteines, and caveolin-3 has eight cysteines. Caveolin-1 and -3 are both highly homologous at the protein level, with conservation of the location of the three cysteines C-terminal to the hairpin domain that we have shown are functionally acylated in caveolin-1 (discussed below). Caveolin-2 shows much less homology, and none of its cysteines aligns with the cysteines in caveolin-1. It is not known whether caveolin-2 or caveolin-3 are acylated proteins. The potential role of acylation in the control of membrane interactions of caveolin-1 are discussed below, but the acylation and potential functions associated with acylation in caveolin-2 or caveolin-3 remain to be elucidated.

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