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Vol. 54, Issue 3, 431-467, September 2002
Department of Molecular Pharmacology, Albert Einstein College of Medicine; and the Division of Hormone-Dependent Tumor Biology, Albert Einstein Cancer Center, New York, New York
Abstract
I. Introduction
II. Caveolae
A. Definition and Morphology
B. Tissue Specificity
C. Composition and Biochemical Properties
III. The Caveolin Gene Family
A. The Discovery of Caveolin and Its Relationship to Caveolae
B. The Other Caveolins (Caveolin-2 and -3)
C. Structural Properties of the Caveolins
1. Caveolin Membrane Topology.
2. Caveolin Oligomerization.
3. Structural Relationships between the Caveolins.
IV. Caveolar Biogenesis
A. The Role of Cholesterol
B. The Role of the Caveolins
V. Functional Significance of Caveolae/Caveolins
A. Vesicular Transport
1. Transcytosis.
2. Endocytosis.
3. Mechanisms of Endocytosis/Transcytosis.
4. Potocytosis.
B. Cellular Cholesterol Homeostasis
1. The Effect of Cholesterol on Caveolin-1.
2. Intracellular Transport of de Novo Synthesized cholesterol.
3. Cholesterol Efflux from Cells.
C. Signal Transduction Mechanisms
1. Caveolae As Signalosomes: Compartmentalized Signaling.
2. The Caveolins As Modulators of Signaling.
3. Caveolin-2 and -3 As Signaling Modulators.
4. Signaling Spotlight: Modulation of Endothelial Nitric-Oxide Synthase Function.
5. Signaling Spotlight: the Dynamic Relationship of G-Protein-Coupled Receptors and Caveolae.
D. Oncogenes and Tumorigenesis
1. Caveolae/Caveolins As Targets of Oncogenes.
2. The Caveolins As Tumor Suppressors.
3. Relevance to Human Cancers.
E. Specialized Functions: Caveolin-3 and Muscle Cells
F. Emerging Functions: Caveolins and Lipid Droplets
VI. Animal Models in the Study of Caveolae and Caveolins
A. Studies of Caveolin-1-Deficient Mice
1. Caveolin-1 and Caveolae Biogenesis.
2. Interactions of Caveolin-1 with the Other Caveolins.
3. Caveolin-1 and Cellular Proliferation.
4. Caveolin-1 and Endocytosis.
5. Caveolin-1 in the Lung.
6. The Vascular Physiology of Caveolin-1-Deficient Mice.
7. Caveolin-1 and Lipid Homeostasis.
B. Studies of Caveolin-2-Deficient Mice
1. Relationship with Caveolin-1.
2. The Surprising Role of Caveolin-2 in the Lung.
C. Studies of Caveolin-3-Deficient Mice
1. Caveolin-3 and Muscle Disease.
2. Caveolin-3 and Transverse Tubules.
3. Caveolin-3 and the Dystrophin-Glycoprotein Complex.
D. Dominant-Negative Caveolin Mutations in Human Disease
VII. Conclusions and Future Directions
A. Caveolae and Caveolins
B. Modifiers of Raft Function
Acknowledgments
References
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Among the membrane compartments of a cell, vesicles known as
"caveolae" have long defied functional characterization. However, since the identification of a family of proteins termed
"caveolins", that form and reside in caveolae, a better
understanding has emerged. It is now clear that caveolae do not merely
play a singular role in the cell, but are pleiotropic in
nature
serving to modulate many cellular functions. The purpose of
this review is to explicate what is known about caveolins/caveolae and
highlight growing areas of caveolar research.
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I. Introduction |
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Well before the era of molecular biology, electron microscopists
of the 1950s were describing the ultrastructural components of the
cell. Among these were 50- to 100-nm invaginations of the plasma
membrane referred to as either caveolae intracellulare, due
to their cave-like appearance, or plasmalemmal vesicles
(Palade, 1953
; Yamada, 1955). It would be approximately forty years
before the molecular nature of caveolae could be explored following the identification of its signature protein, caveolin. Since that time, the
field of caveolin/caveolae research has blossomed, with caveolae being
implicated and demonstrated to be important in a variety of cellular
functions including endocytic processes, cholesterol and lipid
homeostasis, signal transduction, and tumor suppression. Although
caveolae and the caveolins are continuously implicated in an increasing
array of cellular processes, it is clear that their physiological roles
are vastly different depending on the cell type and organ system
examined. For example, their endocytic and vasoregulatory functions
likely predominate in the vasculature, whereas they play an important
role in the structural integrity of the musculature. In this regard,
insights into caveolar function will not only be interesting from the
standpoint of cell biology but will be rewarding in understanding
mammalian physiology with applications to human disease.
In accordance with increasing knowledge and understanding, the subject
of caveolae and the caveolins has been the focus of numerous review
articles, with most confined to certain aspects of their function
(Parton, 1996; Anderson, 1998; Okamoto et al., 1998; Kurzchalia and
Parton, 1999; Smart et al., 1999; Razani et al., 2000b; Schnitzer
2001). Recently, the field has been invigorated by the characterization
of caveolin/caveolae-deficient mouse models, thus for the first time
enabling investigators to analyze the cellular functions of caveolae
with respect to mammalian physiology. As a consequence, it is the
purpose of this review to provide a broad overview of the field with
detailed discussions on its salient features and to provide a link
between the understanding of caveolae at the cellular level and their
emerging roles at the organismal level.
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II. Caveolae |
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A. Definition and Morphology
Originally caveolae were given the exclusive electron microscopic
description of membrane invaginated "smooth" vesicles of 50 to 100 nm in size (as opposed to the more electron-dense and larger
"coated" vesiclesi.e., clathrin-coated pits). However, with
further investigation the definition of caveolae has expanded to also
include vesicles detached from the plasma membrane proper, associated
in groups as grape-like clusters or rosettes, and even in fused form as
elongated tubules or trans-cellular channels (Fig.
1A). Intriguingly, these
"nontraditional" forms of caveolae are more often found in certain
tissues. For example, grape-like clusters of caveolae are highly
abundant in developing skeletal muscle cells, with rosettes in
adipocytes, and detached vesicles/tubules in endothelial cells
(Simionescu et al., 1975; Scherer et al., 1994; Parton et al., 1997).
Figure 1B shows an electron micrograph of an adipocyte with the several
rosette structures and an endothelial cell with traditional caveolae
and detached plasmalemmal vesicles. Therefore, as more is learned about
these structures, caveolae can no longer be considered static
in-pocketings of the plasma membrane, but can take on disparate shapes
and forms by conglomerating and/or fusing with one another. The
processes involved in the formation of these various caveolar
morphologies are largely unknown.
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B. Tissue Specificity
At the ultrastructural level, morphologically identifiable
caveolae can be found at the plasma membrane of numerous tissues and
cell types, albeit in vastly differing abundance. A compilation of
morphological data from the past several decades reveals that although
most cell types contain some caveolae, certain cells have an
extraordinary abundance of caveolae, namely adipocytes, endothelial
cells, type I pneumocytes, fibroblasts, smooth muscle cells, and
striated muscle cells (Palade, 1953; Napolitano, 1963; Mobley and
Eisenberg, 1975; Gabella, 1976; Gil, 1983). It has been reported that
in tissues such as the lung up to 70% of the alveolar plasma membrane
area (composed of type I pneumocytes and endothelial cells) could be
occupied by caveolae (Gil, 1983). However, it should be noted that
certain cells are curiously devoid of these invaginations (e.g.,
central nervous system neurons and lymphocytes) (Fra et al.,
1994; Cameron et al., 1997). The reasons for such wide-ranging tissue-
and cell-type-specific caveolae expression are not yet known. However,
as will be discussed below, the answer might lie in the fact that some
of the proposed functions for caveolae would suit certain cell types
better than others.
C. Composition and Biochemical Properties
Traditionally, the lipid bilayer of the plasma membrane has been
viewed as a two-dimensional "fluid mosaic" (Singer and Nicolson, 1972). In this so-called "liquid-disordered" or
"liquid-crystalline" state, the plasma membrane is mostly composed
of loosely packed phospholipids capable of rapid lateral diffusion. It
has been known for some time, however, that membranes also exist in
"liquid-ordered" states, where bilayer assembly is more rigid with
confined movement of lipids. Based on a variety of observations, it is
now becoming clear that liquid-ordered and -disordered states can
coexist on the same plasma membrane and that liquid-ordered domains can
remain insular and maintain their relative rigidity among the
neighboring phospholipid bilayer. Such domains form via a coalescence
of cholesterol and sphingolipids (glycosphingolipids and sphingomyelin)
and thus have been termed lipid rafts (Brown and London,
1998; Simons and Toomre, 2000) (Fig. 2A).
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Given their shared biochemical properties, caveolae have traditionally
been considered a specialized form of lipid raft (i.e., an
invaginated/vesicular form) (Fig. 2B) (Brown and London, 1998; Simons
and Toomre, 2000). This generalization is probably not entirely
accurate as it is now known that certain proteins preferentially partition into lipid rafts or caveolae but not both (Liu et al., 1997);
the reader is referred to a more detailed review of lipid rafts (Brown
and London, 1998; Simons and Toomre, 2000). The unusual lipid
composition of lipid rafts/caveolae imparts particular properties to
these microdomains, namely a highly reduced density compared with their
phospholipid counterparts and resistance to solubilization by mild
nonionic detergents such as Triton X-100 at 4°C. These properties
form the basis for the biochemical identification, purification, and
characterization of lipid rafts/caveolae (Brown and Rose, 1992; Lisanti
et al., 1994b). For example, one of the simplest and most commonly used
purification techniques (sucrose gradient ultracentrifugation) utilizes
the detergent resistance and buoyancy of these microdomains to separate
them from all other cellular constituents (Lisanti et al., 1994b).
Although caveolae and lipid rafts share certain biochemical properties, the localization of the caveolin proteins to caveolae distinguishes these membrane domains. The caveolins serve as selective markers for caveolae (Fig. 2B) and thus allow for the specific analysis of caveolae function.
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III. The Caveolin Gene Family |
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A. The Discovery of Caveolin and Its Relationship to Caveolae
The discovery of caveolin, the original member of the
three-protein caveolin family, and its relationship to caveolae was converged upon by investigators from different fields with disparate research interests. In an antibody screen for tyrosine-phosphorylated substrates in Rous sarcoma virus-transformed fibroblasts, Glenney and
Zokas (1989) isolated four predominant proteins. Tyrosine phosphorylation of one of these proteins, a 22-kDa molecule, was strongly correlated with the transformation potential of Rous sarcoma
virus-transformed cells suggesting a potential role in cellular
oncogenesis (Glenney, 1989). Interestingly, antibodies raised against
this protein showed punctate staining on the plasma membrane, with a
tendency for staining at cellular margins or linear membrane arrays
(Glenney and Zokas, 1989; Rothberg et al., 1992). This nonrandom plasma
membrane distribution was curiously similar to that observed for the
flask-shaped caveolae (i.e., concentrations along the leading edge and
the linear stress fibers of cells) and led Rothberg and colleagues to a
series of landmark experiments linking this 22-kDa protein and caveolae
(Rothberg et al., 1992). The answer was arrived at ultrastructurally
when immunogold electron microscopy indicated nearly complete
association between the 22-kDa protein and caveolae. More importantly,
this protein also seemed to be a major structural component of
caveolae. Using rapid-freeze deep-etch techniques, caveolae were shown
to be composed of a series of concentric striated rings that stained with antibodies directed against the 22-kDa protein. Moreover, treatment of cells with cholesterol binding agents, such as nystatin and filipin, had profound effects on caveolar morphology, leading to
flattening of the vesicles and dissociation of the 22-kDa protein-rich striations. Because this protein was so intimately associated with the
structural components of caveolae, it was named caveolin, the first bona fide marker of caveolae microdomains (Rothberg et al.,
1992).
The subsequent cloning of the caveolin gene led to yet another surprise
about the possible functions of the protein (Glenney, 1992; Glenney and
Soppet, 1992). In an attempt to identify the cellular machinery
involved in the differential sorting of vesicles to the apical or
basolateral surface of polarized epithelial cells, Simons and
colleagues had cloned
VIP-211 (vesicular
integral protein of 21 kDa), an integral membrane protein component of
trans-Golgi-derived transport vesicles (Kurzchalia et al.,
1992). As it turned out, the caveolin sequence was identical to that of
VIP-21, thereby showing that the same protein could possibly serve as a
structural component of plasma membrane caveolae, as well as have roles
in oncogenesis and vesicular traffickingall at the same time (Glenney
1992). This series of discoveries marked the beginning of the past
decade's investigations into the intimate role between caveolae and
caveolin and their intricate functions, topics that we will in turn
discuss below.
B. The Other Caveolins (Caveolin-2 and -3)
Since the initial characterization of caveolin, two additional
caveolin gene family members have been discovered (caveolin-2 and -3);
thus, the original caveolin protein is now known as caveolin-1 (Cav-1)
(Way and Parton, 1995; Scherer et al., 1996; Tang et al., 1996).
Caveolin-2 (Cav-2) was cloned in an effort to identify other novel
resident proteins of adipocyte caveolae (where there is an abundance of
caveolae and caveolin-1); caveolin-3 (Cav-3) was found by classic cDNA
library screening for Cav-1 homologous genes. In addition to the
full-length proteins (
-isoform), both caveolin-1 and -2 have other
smaller sized isoforms. The 
-isoform of Cav-1 is derived from an
alternative translational start site that creates a protein truncated
by 32 amino acids (Scherer et al., 1995). The - and 
-isoforms of
Cav-2 have not been analyzed in detail.
An alignment of the human caveolin sequences and an overview of the three caveolin proteins is provided in Fig. 3 and Table 1. There is a relatively high degree of identity between Cav-1 and -3, whereas Cav-2 is by far the most divergent of the three proteins. The highest degree of identity among all three caveolins in the majority of species examined is a stretch of amino acids "FEDVIAEP" and is now referred to as the "caveolin signature motif". The structural/functional significance of this motif remains unknown. Other important domains in the caveolin sequences are the membrane-spanning, oligomerization, and scaffolding domains, all of which will be discussed below (Fig. 3).
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All three proteins are present at their highest levels in terminally
differentiated cells. As all caveolins are classically known as markers
of caveolae, it is not surprising that tissues expressing caveolin-1,
-2, and/or -3 are also abundant in caveolae. Interestingly, the
expression pattern of caveolin-1 and -2 are largely distinct from that
of caveolin-3. Adipocytes, endothelial cells, pneumocytes, and
fibroblasts have the highest levels of caveolin-1 and -2, whereas
caveolin-3 expression is limited to muscle cell types (i.e., cardiac,
skeletal, and smooth muscle cells) (Table 1) (Scherer et al., 1994,
1996; Tang et al., 1996). Certain cell types (namely smooth muscle)
have expression of all three proteins possibly due to the hybrid
fibroblastic/muscle-like nature of this cell. The expression profiles
of these proteins in mammalian tissues is intriguing in light of the
following observations: 1) despite the divergence of the Cav-2
sequence, it is selectively detected in Cav-1 expressing tissues; and
2) the highly non-overlapping expression patterns of Cav-1 and -3 indicates that the two proteins might have disparate functions in vivo
in spite of their high degree of identity.
C. Structural Properties of the Caveolins
In several ways, the caveolins are highly unusual proteins, whose overall structure and membrane topology has thus far remained recalcitrant to a complete molecular analysis. Based on a compilation of work conducted mainly on Cav-1 as the archetypal caveolin, two main insights have emerged: 1) Cav-1 is a nonconventional membrane-spanning protein; and 2) it exists primarily as a higher ordered oligomeric complex of ~14 to 16 monomers.
1. Caveolin Membrane Topology.
Early work on caveolin-1
showed that it is resistant to extraction with sodium carbonate, a
property indicative of its integral association with the plasma
membrane (Kurzchalia et al., 1992; Sargiacomo et al., 1993). Based on
various topological analyses, it is now well accepted that the N and C
termini of Cav-1 remain cytoplasmic. For example, i)
antibodies recognizing the N and C termini of Cav-1 can bind the
protein only when cells are permeabilized with detergents (Dupree et
al., 1993); ii) membrane-associated Cav-1 remains sensitive
to proteolysis, a condition indicative of cytoplasmically directed N
and C termini (Monier et al., 1995); and iii) there are
known phosphorylation and palmitoylation sites in the N-and C-terminal
domains of Cav-1, respectively (Dietzen et al., 1995; Li et al.,
1996b). By extension, it is reasonable to assume that Cav-1 is a
double-pass membrane-spanning protein. However, cell-surface
biotinylation of cells shows no labeling of caveolin-1, indicating the
inaccessibility of its domains to the extracellular milieu (Sargiacomo
et al., 1995). Analysis of the Cav-1 protein sequence indicates that it
contains a single hydrophobic region (residues 102-134). As a 32-amino
acid stretch is not long enough to enable a complete hairpin loop
through the lipid bilayer, a topological view of Cav-1 that took into
account its tight association with the plasma membrane was a conundrum.
). This process is
dependent on the 32-residue hydrophobic domain of Cav-1, which acts as
a "signal peptide" (note: Cav-1 does not contain a classical
N-terminal signal sequence). Interestingly, the N terminus of the
protein is required for its hairpin loop configuration, as fusion of
the N-terminal domain from a secreted protein (growth hormone) causes
Cav-1 to take on a membrane-spanning topology (Monier et al., 1995).
A holistic view that incorporates all of these observations led
investigators to propose an incomplete hairpin structure for Cav-1,
where the 32-amino acid hydrophobic domain acted as the putative
"membrane-spanning domain" of the protein. Through exhaustive mutational/deletional analyses, recent evidence has elaborated on and
complicated this initial model. Fusion proteins containing only the N-
or C-terminal domains of Cav-1 (i.e., lacking the transmembrane domain)
are also able to bind tightly to membranes (Luetterforst et al., 1999;
Schlegel et al., 1999a; Schlegel and Lisanti, 2000). Further deletion
analysis shows that residues 82-101 of the N terminus and residues
135-150 of the C terminus are the minimal regions mediating membrane
attachment (Schlegel and Lisanti, 2000). These regions are now known as
the N-terminal membrane attachment domain (N-MAD) and the C-terminal
membrane attachment domain (C-MAD). C-MAD is a membrane attachment
domain that directs the trans-Golgi localization of Cav-1,
whereas N-MAD specifically directs Cav-1 to caveolae membranes. In
addition, Cav-1 is palmitoylated on three cysteine residues on the C
terminus (133, 143, and 156) (Dietzen et al., 1995). Although these
lipid modifications are not necessary for the membrane binding or
caveolar localization of Cav-1 (Schlegel and Lisanti, 2000), they act
to stabilize the overall structure of the protein at the membrane (see
below) (Monier et al., 1996). The present view of caveolin-1's structure at the plasma membrane is shown in Fig.
4.
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2. Caveolin Oligomerization.
Using velocity gradient
centrifugation, it was first discovered that caveolin-1 migrates as a
high molecular weight complex of approximately 350 to 400 kDa in vivo
(Monier et al., 1995; Sargiacomo et al., 1995). This complex was
exclusively composed of the Cav-1 protein and was dissociated only
under harsh detergent treatment at elevated temperatures, indicating
the presence of a highly stable caveolin-1 homo-oligomer of
approximately 14 to 16 monomers (Monier et al., 1995; Sargiacomo et
al., 1995; Li et al., 1996c). Furthermore, pulse-chase analysis showed
that the complex was formed relatively rapidly after synthesis of Cav-1 in the ER and prior to completion of Golgi transit (Monier et al.,
1995).
; Song et al.,
1997b) (Fig. 4).
3. Structural Relationships between the Caveolins.
Although it is well established that Cav-1 can form large
homo-oligomeric complexes in vivo, Cav-1 has recently been shown to
undergo higher ordered interactions with caveolin-2 as well. Cav-1
and -2 can interact to form very stable high molecular mass hetero-oligomers, akin to the large homo-oligomeric complexes observed
for Cav-1 (Scherer et al., 1997). Interestingly, in the absence of
Cav-1, Cav-2 is not capable of forming large homo-oligomers and rather
exists in a monomeric/dimeric form and is retained at the level of the
Golgi compartment (Scherer et al., 1996; Mora et al., 1999; Parolini et
al., 1999). Upon Cav-1 expression, the Cav-1 and Cav-2 proteins form a
large complex and are found predominantly localized to plasma membrane
caveolae (Scherer et al., 1996; Mora et al., 1999; Parolini et al.,
1999). Therefore, Cav-2 is dependent on caveolin-1 both structurally
and for appropriate subcellular trafficking (Mora et al., 1999;
Parolini et al., 1999). Based on the primary sequence, the
oligomerization domain of Cav-2 is ~50% identical/75% similar to
that of Cav-1, and it was believed that oligomer formation between the
two proteins is mediated by their respective oligomerization domains.
However, recent deletion mapping studies surprisingly indicate that the
interaction is mediated by the membrane-spanning domains of Cav-1 and
-2 (Das et al., 1999). The tissue-specific expression of Cav-1 and
Cav-2 is highly coregulated (i.e., all tissues with Cav-1 expression are also the primary sites of Cav-2 expression) (Scherer et al., 1996).
Therefore, despite the well established fact that Cav-1 can form
large homo-oligomeric complexes in vivo, it seems more likely that
the Cav-1 protein is constitutively associated with Cav-2 under
physiological conditions.
). However, the
two proteins are not simply redundant in all respects, e.g., the
C-terminal domain of Cav-1 can bind to full-length Cav-1 oligomers
(homotypic interactions) but not full-length Cav-3 oligomers (Song et
al., 1997b), and unlike Cav-1, Cav-3 does not form a complex with Cav-2
(Das et al., 1999).
Therefore, despite their high identity, it appears that Cav-1 and -3 have distinct structural properties that might affect their
localization and function in the cell. For example, in the widely used
myocyte cell line, C2C12, Cav-1 and -3 are both expressed and
colocalized to caveolae in the myoblast stage. Upon myocyte differentiation, however, Cav-1 levels steadily decline (albeit remaining associated with the cell periphery) while Cav-3 levels increase and associate with the developing T-tubule network (Parton et
al., 1997).
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IV. Caveolar Biogenesis |
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A. The Role of Cholesterol
The crucial role of cholesterol in caveolar biogenesis has been
evident for some time. Treatment of cells with cholesterol binding
agents such as nystatin, filipin, or cyclodextrin leads to a complete
ablation of morphologically identifiable caveolae (Rothberg et al.,
1992; Schnitzer et al., 1994; Hailstones et al., 1998). Furthermore,
absolute cellular levels of cholesterol need to rise above a certain
threshold level before caveolae formation can occur (Hailstones et al.,
1998). Although caveolae microdomains are highly enriched in
cholesterol, the selective requirement for cholesterol in caveolar
biogenesis is not straightforward. Recent reports have clearly shown
that manipulation of cellular cholesterol levels also has effects on
the biogenesis of clathrin-coated endocytic vesicles and synaptic
vesicles, membrane entities that differ in many respects from lipid
rafts and caveolae (Rodal et al., 1999; Thiele et al., 2000). If the
formation of many types of post-Golgi-derived vesicles depends on
critical levels of cholesterol, the formation of caveolae must involve
more than just the role played by cholesterol.
B. The Role of the Caveolins
Overexpression of caveolin-1 in cells lacking endogenous
caveolin/caveolae (lymphocytes and transformed fibroblasts) results in
the de novo production of ~50- to 100-nm membrane invaginations and
vesicles (Fra et al., 1995b; Engelman et al., 1997; Li et al., 1998).
Conversely, the targeted down-regulated caveolin-1 in cells with
endogenous caveolae results in the loss of caveolae (Galbiati et al.,
1998; Liu et al., 2001). These experiments clearly link caveolin
expression with caveolae formation.
Several characteristics of caveolin-1 suggest the way in which it
transforms the morphology of the plasma membrane into flask-shaped caveolae. Cav-1 binds the two primary components of lipid rafts, namely
cholesterol and sphingolipids, both in vitro and in vivo (Fra et al.,
1995a; Murata et al., 1995; Thiele et al., 2000). In fact, cholesterol
has an unusually high affinity toward Cav-1, remaining associated with
caveolin oligomers even in the presence of harsh detergents such as SDS
(Murata et al., 1995). A high local concentration of cholesterol and
caveolin maintained at a critical level could provide the appropriate
lipid-protein microenvironment for membrane invagination (Fig.
5).
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The tendency of caveolins to form high molecular weight oligomeric
complexes likely contributes to caveolae formation. As mentioned
before, caveolin-1 is able to form extremely strong detergent-resistant
homo-oligomeric complexes composed of ~14 to 16 monomers (Monier et
al., 1995; Sargiacomo et al., 1995). If these oligomers are purified
from plasma membrane lysates and detergents are subsequently removed by
dialysis, the oligomers self-associate into very large/sedimentable
complexes of ~20- to 40-nm in diameter (Sargiacomo et al., 1995).
Furthermore, deletion analysis has revealed that the C-terminal domain
of Cav-1 (namely residues 135-178), is able to interact with the
full-length Cav-1 molecule (Song et al., 1997b; Schlegel and Lisanti,
2000). Taken together, this evidence suggests that caveolin oligomers
can form higher order oligomer-oligomer complexes by interactions of
the respective C-terminal domains. This meshwork of caveolin protein complexes most likely elicits the bending of the plasma membrane, a
process similar to that of the clathrin triskelion in coated-pit formation (Fig. 5).
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V. Functional Significance of Caveolae/Caveolins |
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Since their discovery in the 1950s, the function of caveolae has been a matter of speculation. Consequently, numerous roles have been attributed to these microdomains and their caveolar marker protein, the caveolins. Here, we will discuss the most thoroughly studied roles of caveolae and their potential relevance to physiology.
A. Vesicular Transport
1. Transcytosis.
Based purely on the vesicular morphology and
abundance of caveolae in endothelial cells (Palade 1953), caveolae were
hypothesized to function as conduits for the general transport of
proteins through capillary cells (i.e., transcytosis) (Fig.
6) (Simionescu et al., 1975). The primary
approach in these initial reports and numerous follow-up studies
involves the use of labeled "tracer" serum proteins (e.g.,
gold-conjugated albumin) and following their dynamic interaction with
the capillary endothelium via transmission electron microscopy
(Simionescu et al., 1975; Ghitescu and Bendayan, 1992; Schnitzer et
al., 1994; Predescu et al., 1997, 1998). Despite very convincing
time-lapse imaging of caveolae-mediated tracer transport to the
abluminal side of endothelial cells, the functionality of caveolae as
transcytotic vesicles has been contentious. It is not clear whether
this observed mode of transport is specific to caveolae or is simply a
nonspecific component of bulk fluid flow, which would include
paracellular transport. Furthermore, it has been argued that the
observed tracer labeling of invaginated caveolae (i.e., cytoplasmic
caveolae in transit to the abluminal side) could actually be labeling
of a continuation of the plasma membrane only appearing to be
intracellular due to microscopic sectioning artifacts.
View larger version (34K):
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Fig. 6.
Caveoale vesicular trafficking: transcytosis,
endocytosis, and potocytosis. Caveolae appear to mediate the selective
uptake and transport of several molecules via different processes
(transcytosis, endocytosis, and potocytosis). In transcytosis, caveolae
transport proteins from the luminal side of the endothelial cell to the
interstitial compartment for subsequent uptake by underlying tissues.
In caveolae-mediated endocytosis (distinct from that of clathrin-coated
pits), caveolae bud off from the plasma membrane and fuse with various
intracellular compartments. Possible transport routes include the
recently characterized caveolae-caveosome-ER pathway. In potocytosis,
caveolae mediate the uptake of small solutes (<1 kDa) by pinching off
but remaining associated with the plasma membrane. The molecular
machinery involved in this caveolar fission/fusion is the same as that
used for numerous other vesicular transport processes with requirements
for dynamin, VAMP, SNAP-25, the SNARE complex, and GTP hydrolysis.
have developed antibodies capable of specifically labeling the
extracellular (luminal) domains of proteins residing in endothelial cell caveolae. In situ labeling experiments showed that these antibodies are capable of completely and rapidly crossing the capillary
wall directly through caveolae; bulk fluid flow (as determined by
nonspecific antibodies) occurred significantly more slowly (Schnitzer,
2001). These observations show that caveolae-mediated transport is a
major and kinetically preferred route for the transcytosis of certain proteins.
2. Endocytosis.
Caveolae have also been implicated in
endocytic processes. Traditionally, endocytosis has been almost
entirely associated with clathrin-coated vesicle transport and the
molecular mechanisms underlying this process have been studied in
detail (for review, see Takei and Haucke, 2001). Numerous receptors and
their cognate ligands are taken up via this pathway leading to
termination or desensitization of signaling cascades, among other
things. However, it has become clear that certain receptors and
extracellular macromolecules are exclusively transported via caveolae
rather than clathrin-coated pits (Fig. 6). The first demonstration of
this selectivity came when investigators found that caveolae can bind
and internalize cholera and tetanus toxins (Montesano et al., 1982).
Biochemical evidence bolstered this argument when it was shown that
treatment of cells with cholesterol binding agents (nystatin or
filipin) abrogated the endocytosis of certain macromolecules (e.g.,
albumin) without affecting the uptake of clathrin-dependent ones
(Schnitzer et al., 1994). Similar results have been seen with several
membrane-bound proteins including alkaline phosphatase (Montesano et
al., 1982; Anderson et al., 1992; Parton et al., 1994). This
selectivity is in large part due to the caveolar localization of the
cognate receptor for these molecules [e.g., the glycosphingolipid GM1 in the case of cholera toxin or the glycosylphosphatidylinositol (GPI)
modification of alkaline phosphatase] (Fig. 6) (see also Table
2 for a more complete list of
caveolae-localized proteins).
|
; Stang et al., 1997; Shin et al., 2000). The relatively small size
of caveolae (~50-100 nm) obviously places limitations on the
endocytosis of such large entities. In the case of bacteria, it appears
that phagocytic cells circumvent such problems by harboring mechanisms
that enable several caveolae to coalesce into significantly larger
endocytic microdomains (Shin et al., 2000).
Exactly where in the cell the endocytosed caveolar vesicle targets is
not clear. Among the possibilities are direct transport to endosomes
with or without further trafficking to other intracellular compartments. For example, in the case of cholera toxin, it is believed
that caveolar endocytosis on the apical membrane is followed by
retrograde transport to endosomes, the Golgi apparatus, the endoplasmic
reticulum, and finally resorting to the
basolateral membrane (Lencer et al., 1999). However, this trafficking
scheme is not true for all caveolae-mediated endocytosis. It has
been shown that the budded caveolae containing SV40 eventually
merge with larger nonendosomal nonlysosomal caveolin-1 containing
organelles that target to the endoplasmic reticulum (Pelkmans et al.,
2001). These organelles were termed "caveosomes" and are the first
indication of distinct compartments formed from internalized caveolae
(Fig. 6). This is consistent with earlier data that under certain
conditions caveolins can either directly traffic from the plasma
membrane to ER/Golgi compartments or localize to nonendosomal vesicles (Conrad et al., 1996; Roy et al., 1999).
3. Mechanisms of Endocytosis/Transcytosis.
A variety of
proteins have been identified that comprise the vesicular
fission/fusion machinery required for endocytosis and transcytosis. In
a series of studies primarily by Schnitzer and colleagues, it was shown
that caveolae harbor the same components used for the budding and
docking of other vesicles (Schnitzer et al., 1995b). Early indications
for the presence of such classical transport machinery was based on the
observation that N-ethylmaleimide (NEM) can inhibit the
transcytosis/endocytosis of endothelial cell caveolae (Predescu et al.,
1994; Schnitzer et al., 1995a). Purification of these caveolar
microdomains showed that several components used by cells in general
vesicle formation, docking, and fusion (e.g., NSF, SNAP, VAMP, and
GTPases) are concentrated in caveolae and possibly associated with
caveolin-1 (Schnitzer et al., 1995b; Predescu et al., 2001) (see also
Table 2). Furthermore, cleavage of VAMP by specific neurotoxins
abrogates the endocytosis and fusion of caveolae with intracellular
compartments (McIntosh and Schnitzer, 1999). Therefore, it has become
clear that caveolae can co-opt the same mechanisms used in the
trafficking of other vesicles to transport cargo throughout the cell,
e.g., from the plasma membrane to internal compartments (Fig. 6).
). The GTP-dependent factor in the cytosol was shown to be
dynamin, previously implicated in the endocytosis of clathrin-coated pits (Henley et al., 1998; Oh et al., 1998). Microinjection of anti-dynamin antibodies into cells prevented the internalization of
clathrin-coated pits, as well as caveolae (Henley et al., 1998). In
vivo, it appears that dynamin localizes to the conspicuous annular
necks of membrane-bound caveolae seen by electron microscopy (Henley et
al., 1998; Oh et al., 1998). Given the role of dynamin in vesicular
budding, its position at the caveolar "necks" is not surprising
(Fig. 6).
Despite the involvement of the classical fission/fusion apparatus in
caveolar endocytosis, the steps leading to invagination and vesicle
formation remain fragmented. As mentioned before, caveolin-1 along with
cholesterol most likely provides the structural force for the
invagination of caveolae. However, the regulatory mechanisms mediating
the transition from membrane-bound caveolae to free intracellular
vesicles are at present unknown. How do caveolae sense the need to
undergo endocytosis or remain statically attached to the plasma
membrane, and what are the stimuli that trigger caveolar endocytosis?
Future work will be needed to provide the links between the caveolins,
dynamins, and such molecular sensors.
4. Potocytosis.
Caveolae have also been implicated in a
unique form of solute uptake, termed potocytosis (or "cellular
drinking"). First championed by Anderson and colleagues (1992),
potocytosis is the process by which cells can transport small molecules
(<1 kDa) without having to endocytose vesicles to internal
endosomal/lysosomal compartments. Using microscopy, they demonstrated
that the folate receptor, a GPI-linked protein, primarily localizes to
caveolae (Rothberg et al., 1990). Remarkably, caveolae then seem to
facilitate the uptake of folate by closing their necks, albeit
remaining associated with the plasma membrane (Anderson et al., 1992).
In this way, a highly concentrated pool of folate is created, which is
subsequently fluxed to the cytoplasm by a three-step process: 1)
lowering of caveolar pH by a caveolae-localized proton pump, 2) low pH
dissociation of folate from its receptor, and 3) flux into the
cytoplasm by a caveolae-localized anion carrier (Kamen et al., 1991)
(Fig. 6). At present, the mechanism of the dynamic closing and
opening of the caveolar "mouth" is not understood. Furthermore,
there is controversy as to whether cells even use potocytosis in the
uptake of small molecules or simply co-opt classical receptor-mediated
endocytic processes (Mayor et al., 1998). More detailed analysis of the
general applicability of caveolae to the uptake of other small
molecules needs to be conducted before potocytosis can be considered a
process distinct from caveolar endocytosis.
B. Cellular Cholesterol Homeostasis
As briefly described above, a unique relationship exists between
caveolins/caveolae and cholesterol. Ever since the discovery of
caveolin-1 as a marker of caveolae, it has been known that caveolae are
highly sensitive to cholesterol depletion; treatment of cells with
cholesterol binding agents flattens these structures and disaggregates
their caveolin-rich "striated coats" (Rothberg et al., 1992). The
first indication of a direct link between the caveolins and cholesterol
was demonstrated in vitro, where purified caveolin-1 was shown to
reconstitute only into lipid vesicles containing cholesterol (Murata et
al., 1995; Li et al., 1996c). Further analysis found that Cav-1 can
actually form a complex with cholesterol in these lipid mixtures, on
the order of ~1 to 2 cholesterol molecules per caveolin molecule
(Murata et al., 1995). Recently, a photo-activatable form of
cholesterol was shown to cross-link a ~21- to 24-kDa band in cells
(identified as Cav-1), thereby making caveolin-1 one of only a few
proteins with a demonstrated ability to bind cholesterol in vivo
(Thiele et al., 2000). Not surprisingly, the intricate relationship
with cholesterol renders Cav-1 highly sensitive to cholesterol
perturbations, both in terms of its subcellular localization and
transcriptional regulation.
1. The Effect of Cholesterol on Caveolin-1.
Treatment of
cells with cholesterol oxidase, which selectively converts caveolar
cholesterol to cholestenone, causes the movement of caveolin-1 to the
ER and subsequently to the Golgi compartment; removal of cholesterol
oxidase allows the return of Cav-1 and free cholesterol to caveolae
(Smart et al., 1994; Conrad et al., 1996). Although it is not known how
Cav-1 can retreat intracellularly, it is clear that its subcellular
transport is intricately linked to cholesterol transport.
). Rather than depleting cellular cholesterol,
Fielding and Fielding used cholesterol-loading to dissect this
regulation even further (Bist et al., 1997; Fielding et al., 1997,
1999). Incubation of fibroblasts with low density lipoproteins, a way
of loading cells with free cholesterol, led to an increase in Cav-1
mRNA levels (Fielding et al., 1997). Mutational/deletion analysis of
the 1-kb upstream caveolin promoter revealed that two putative sterol
regulatory elements (SREs) are necessary for robust transcriptional
activation by free cholesterol; electrophoretic mobility shift assays
further defined one particular SRE that is bound by the transcription
factor SRE-binding protein 1 (SREBP-1), a major mediator of
cholesterol-dependent transcriptional responses (Bist et al., 1997).
Taken together, the above results suggest a pivotal role for
caveolins/caveolae in the transport and regulation of cellular
cholesterol levels.
2. Intracellular Transport of de Novo Synthesized
cholesterol.
De novo synthesis of cholesterol occurs in the ER,
and upon membrane partitioning, it is rapidly transported to various
membrane compartments, of which the plasma membrane is a major site
(containing up to 90% of cellular cholesterol at steady state) (Simons
and Ikonen, 2000). However, much less is known about the molecular machinery and trafficking pathways mediating this cholesterol transport. Based on a variety of observations, it is believed that
cholesterol is predominantly transported to the cell surface by a
Golgi-independent route (DeGrella and Simoni, 1982; Kaplan and Simoni,
1985; Urbani and Simoni, 1990; Heino et al., 2000). Considering its
high binding capacity for cholesterol, caveolin-1 is a potential
mediator of this function.
were able to show
that newly synthesized cholesterol is distributed to the bulk plasma
membrane only after initial transport to the caveolae membrane system
(Fig. 7). Furthermore, this cholesterol transport was mediated by a nonvesicular protein complex, which includes caveolin-1 and several chaperone proteins of the cyclophilin and heat shock protein families (Uittenbogaard et al., 1998) (Fig. 7).
Interestingly, the three palmitoylation sites of Cav-1 were necessary
for the binding and delivery of cholesterol to caveolae (Uittenbogaard
and Smart, 2000). This is an important observation in light of the
previous finding that the palmitoylation of Cav-1 is not necessary for
its membrane binding or caveolar localization but rather for its
stability as an oligomer, a process intricately involved in its
cholesterol binding as well (Dietzen et al., 1995; Monier et al.,
1996). Based on these results, it is clear that cholesterol trafficking
to the cell surface utilizes the caveolin-1/caveolar system as a
"delivery device". It should be noted, however, that many cells
with an intact cholesterol transport apparatus (e.g., hepatocytes) have
extremely low levels of caveolin-1 (Calvo et al., 2001). Therefore,
under physiologic conditions, Cav-1 most likely only acts as a
facilitator/expeditor of cholesterol transport necessitating the
identification of other pathways in cholesterol trafficking. In line
with a facilitatory role, transfection of cells with the caveolin-1
cDNA leads to an ~4- to 5-fold enhancement of caveolar cholesterol
levels (Smart et al., 1996).
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3. Cholesterol Efflux from Cells.
Cellular cholesterol is
typically derived from two major sources: de novo synthesis (as
described above) or exogenous uptake primarily from serum low-density
lipoproteins (LDLs) (Fielding and Fielding, 1997). To maintain steady
levels intracellularly, peripheral cells can also efflux excess free
cholesterol from the exoplasmic leaflet of the plasma membrane to
high-density lipoproteins (HDLs) for delivery to the liver, a process
known as reverse cholesterol transport (Fielding and Fielding, 1997). In a series of studies by Fielding and Fielding, it appears that caveolae play an important role in this process as well. If the intracellular cholesterol pool is increased above baseline by incubating cells with cholesterol-donating LDL, excess cholesterol is
eventually observed in caveolae; caveolae then act as portals for the
efflux of this cholesterol upon incubation of cells with HDL (Fig. 7)
(Fielding and Fielding, 1995). Caveolin-1 regulates this process,
because antisense-mediated down-regulation of caveolin-1 decreases
cellular cholesterol efflux (Fielding et al., 1999; Arakawa et al.,
2000). In support of these data, in vivo expression of caveolin-1 in
the mouse liver via adenoviral-based strategies causes an increase in
plasma HDL cholesterol levels (Frank et al., 2001). Therefore, it
appears that Cav-1 enhances the availability and delivery of
cholesterol to plasma membrane caveolae for subsequent efflux. At
present however, the molecular mechanisms of this efflux process from
caveolae to HDL are not known, but most likely involve an association
between the HDL particle or its major apolipoprotein, ApoAI, and caveolae.
; Graf et
al., 1999). SR-BI has been shown to act as an HDL receptor functioning
both in the transfer of free cholesterol from the plasma membrane to
HDL (Ji et al., 1997; de la Llera-Moya et al., 1999), while also acting
as a conduit for the selective uptake of cholesterol esters from HDL
into the cell (Acton et al., 1996). Therefore, the role of caveolae in
the efflux of free cholesterol could be largely due to the
compartmentalization of the SR-BI receptor.
Based on the dual functionality of SR-BI, caveolar localization would
also imply that caveolae are important sites of cholesterol ester
uptake. Indeed, it has been shown that SR-BI-mediated cholesterol ester
uptake predominantly occurs in caveolae (Graf et al., 1999) and that
caveolin-1 can regulate this process (Matveev et al., 1999, 2001; Frank
et al., 2001). However, many of these results need to be dissected
further to understand the physiological role of caveolins/caveolae in
tissues that utilize SR-BI for cholesterol influx/efflux processes.
C. Signal Transduction Mechanisms
1. Caveolae As Signalosomes: Compartmentalized
Signaling.
Caveolin-1 was not only the first protein to be
localized to caveolae but due to its apparent involvement in the
structural integrity of caveolae was also the first caveolar "marker
protein" (Rothberg et al., 1992). The issue that required
clarification, however, was whether caveolae could also serve as
platforms for the aggregation and/or concentration of other proteins.
Clearly, evidence for the presence of other caveolar resident proteins would be important in the understanding of caveolar function. In this
regard, Lisanti and coworkers were the first investigators to broadly
address this issue (Sargiacomo et al., 1993; Lisanti et al., 1994b).
Using the insolubility of caveolae in mild detergents and their
buoyancy in sucrose gradients, they were able to biochemically separate
caveolae membranes and, in turn, determine the identity of cosegregated
proteins. Of the numerous proteins identified in this manner, it was
surprising to find that a large majority were signal transduction
molecules, some at concentrations manyfold higher than the bulk plasma
membrane (Sargiacomo et al., 1993; Lisanti et al., 1994b). This
observation led Lisanti and colleagues to put forth the
"caveolae/raft signaling hypothesis": the compartmentalization of
such molecules has distinct advantages as it provides a mechanism for
the regulation of subsequent signaling events and explains cross-talk between different signaling pathways (Lisanti et
al., 1994a). subunits, and
endothelial nitric-oxide synthase (eNOS) all harbor one or several
myristoyl, palmitoyl, or prenyl groups (Li et al., 1995, 1995a; Feron
et al., 1996; Garcia-Cardena et al., 1996b; Song et al., 1996a, 1997a).
2. The Caveolins As Modulators of Signaling.
A topic
neglected by this signaling concept was the functional significance of
the caveolin proteins (were they simply structural bystanders or could
they actively contribute in the retention and modulation of signal
transduction proteins). After all, the concept of "scaffolding
proteins" is not a new one, and the literature is replete with
examples of molecules that act to restrict, organize, and/or regulate
the subcellular distribution of other signaling molecules (Pawson and
Scott, 1997). For example, a paradigm class of proteins with such
functions are the AKAPs (A-kinase anchor proteins), known to contain
the localization and activation of protein kinase A to different
subcellular compartments, thereby limiting the spurious activation of
this potent kinase (Colledge and Scott, 1999).
). Subsequent work has confirmed the
selectivity of this region for binding to and modulating the activity
of not only G-proteins but also a host of other signaling proteins (see
Table 2 for a complete list) including H-Ras, Src family tyrosine
kinases, PKC isoforms, EGF-R, Neu, and eNOS (Engelman et al., 1998c;
Okamoto et al., 1998; Smart et al., 1999). Because this region of the
caveolin molecule seems to be largely responsible for many of the
molecule's functional effects, it has been termed the caveolin
scaffolding domain (CSD) (see Fig. 8).
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). Interestingly, only a select group of peptides showed high-affinity binding to the CSD; compilation of these sequences
indicating that they almost invariably matched the following motifs:
XXXXX,
XXXXXX,
XXXXXXX, where is an
aromatic residue (Phe, Tyr, or Trp) and X is any amino acid
(Couet et al., 1997a). Furthermore, a search of known
caveolin-interacting molecules showed that at least one such motif
could be identified in their primary sequence, indicating that these
regions might be sites of direct interaction with the caveolin-1. Many
such motifs that are found in caveolin-interacting molecules are now
known as caveolin binding domains (CBDs) (see Fig. 8). However, the
presence of a CBD is not an absolute inclusive criteria for caveolin
binding, and in fact, many proteins with putative CBDs have not been
shown to interact with caveolin. Obviously, the solvent accessibility of such domains is a major factor in the ability of a protein to bind
caveolin. Thus far, support for the existence of such domains (namely
by demonstrating disrupted caveolin-1 binding upon targeted mutation of
a putative CBD) has been provided for only a few signaling molecules
(i.e., insulin receptor, eNOS, and GRKs) (Garcia-Cardena et al., 1997;
Carman et al., 1999; Nystrom et al., 1999).
Currently, the lack of structural information precludes an assessment
of the mechanism by which caveolin-1 and its CSD can recruit and bind
to CBD-containing molecules. However, the fact that both the CSD and
CBD peptide sequences are enriched in similarly spaced aromatic amino
acids (Couet et al., 1997a) suggests that such interactions could occur
by a linear juxtaposition of these hydrophobic-hydrophobic
interactions. In many respects, the CSD functions as a modular protein
domain (akin to SH2, SH3, and WW domains), recognizing motifs in
presumably solvent-accessible regions of signaling molecules.
With the exception of a few signaling molecules, interaction with the
CSD leads to inhibition of downstream signaling. In the list of
caveolin-interacting molecules shown in Table 2, most have been shown
to be inhibited by caveolin-1 by either biochemical or cell culture
experiments. In the case of tyrosine and serine/threonine kinases, such
functional interaction takes on practical significance. Nearly all
enzymes of this class harbor caveolin binding motifs located within the
active catalytic domain (namely kinase subdomain IX) (Couet et al.,
1997b), and a synthetic peptide corresponding to the CSD is sufficient
to inhibit their phosphotransferase activities in vitro. As many of
these protein kinases have thus far not been reported to have more
potent peptide inhibitors than the CSD, agents that mimic the binding
of kinases to caveolins are potentially useful as protein kinase
inhibitors and as lead compounds in drug development.
3. Caveolin-2 and -3 As Signaling Modulators.
Primarily due
to the overwhelming focus on the archetypal caveolin-1, the capacity of
the other members of the caveolin family to act as scaffolding proteins
remains unknown. Most of what is known about these proteins is by
analogy with Cav-1. A glance at the protein alignment in Fig. 3
illustrates that Cav-1 and Cav-3 have highly homologous CSDs
(especially in the spacing of the aromatic residues alluded to above),
whereas Cav-2 is largely divergent. Therefore, it can be surmised that
in muscle cells, where Cav-3 is selectively expressed, it can act as an
effective scaffolding protein in the absence of other caveolins.
Indeed, the few experiments that have been conducted with Cav-3 and its CSD seem to corroborate this idea (Li et al., 1995; Yamamoto et al.,
1998; Razani et al., 1999). At present however, the dearth of data on
the functions of caveolin-2 preclude its implication in any known
signaling pathway.
4. Signaling Spotlight: Modulation of Endothelial Nitric-Oxide
Synthase Function.
Probably the best studied of the signaling
molecules that are modulated by caveolin is eNOS. Acylation of eNOS
serves to target it to lipid-raft domains of the plasma membrane and
the Golgi apparatus (Garcia-Cardena et al., 1996b; Shaul et al., 1996;
Sowa et al., 2001; Fulton et al., 2002) (Fig.
9). eNOS localized to lipid
rafts/caveolae, but not interacting with Cav-1, has optimal enzymatic
function; however, interaction with Cav-1 inhibits eNOS function
(Garcia-Cardena et al., 1996a; Liu et al., 1996a; Michel et al., 1997a;
Sowa et al., 2001). The region of Cav-1 responsible for the inhibition
of eNOS has been mapped to the CSD (Garcia-Cardena et al., 1997; Ju et
al., 1997). Point mutations in the CBD of eNOS results in the loss of
Cav-1 binding without the loss of eNOS enzymatic activity
(Garcia-Cardena et al., 1997). Thus a model has been proposed in which
eNOS is targeted to caveolae/lipid rafts where the CSD/CBD interaction
serves to maintain eNOS inhibition until the CSD/CBD interaction is
disrupted due to an increase in cytosolic calcium followed by
calcium-calmodulin/eNOS interaction (Fig. 9) (Garcia-Cardena et al.,
1997; Michel et al., 1997a, b).
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).
Using ex vivo aortic rings, they were able to show that a
cell-permeable peptide containing the CSD potently inhibits acetylcholine (Ach)-induced NO production and vasodilation. The CSD was
also able to ameliorate the NO-mediated vascular compromise in mice
treated with pro-inflammatory agents (Bucci et al., 2000).
The emerging view is that caveolins may function as general negative
regulators to inhibit the basal activity of many signaling proteins.
Upon activation of a given signaling pathway, caveolin-mediated inhibition is released thus increasing the signal propagation (Okamoto
et al., 1998; Smart et al., 1999; Ostrom et al., 2000b). The logical
consequence of this view is the implication of Cav-1 as a tumor
suppressor based on the capacity of Cav-1 to inhibit the functional
activity of several proto-oncogenes (see below).
5. Signaling Spotlight: the Dynamic Relationship of
G-Protein-Coupled Receptors and Caveolae.
When G-protein coupled
receptors (GPCRs) and G-proteins were first isolated in low
buoyant-density sucrose gradients, it was postulated that caveolae may
serve as platforms for congregating these receptors with their
downstream effectors (Lisanti et al., 1994a, b). Since that time, a
more dynamic interaction between receptors and caveolae has emerged by
comparing the caveolar localization of proteins before and after
agonist stimulation.
adrenergic receptors can localize to
caveolae (Schwencke et al., 1999; Ostrom et al., 2000b; Rybin et al.,
2000). Further work has revealed that 2
adrenergic receptors are highly enriched in caveolae at rest but exit
caveolae upon adrenergic stimulation (Ostrom et al., 2001), where they
are thought to be targeted to clathrin-coated pits for internalization.
Importantly, the downstream effector, adenylyl cyclase type 6, also
localizes to caveolae where its proximity to activated
2 adrenergic receptors may facilitate
downstream signaling before receptor migration (Ostrom et al., 2000b,
2001). Adrenergic stimulation failed to cause the translocation of
1 adrenergic receptors out of caveolae, however (Ostrom et al., 2000b). The observed higher cAMP response to
1 adrenergic receptor-selective activation is
hypothesized to be due to the retention of the
1 adrenergic receptor in caveolae where it
remains in proximity to adenylyl cyclase type 6 (Ostrom et al., 2001).
It is also thought that the localization of many endogenous
1 adrenergic receptors outside of caveolae
versus the complete enrichment of 2 adrenergic
receptors in caveolae may explain signaling differences as well (Ostrom
et al., 2000a, 2001; Steinberg 2001).
As opposed to trafficking out of caveolae, the
1 and 2 bradykinin
receptors both translocate into caveolae upon agonist stimulation
(Sabourin et al., 2002). Their ultimate cellular fate, however, is
different. The 2 bradykinin receptor moves
into caveolae and then is internalized, whereas the
1 bradykinin receptor is not internalized.
This difference in internalization may explain the previously observed
difference in the phosphorylation of the cytoplasmic tail of
2 bradykinin receptor versus the lack of phosphorylation of 1 bradykinin receptor
cytoplasmic tails. Further functional studies will be necessary to
determine how the localization of 1 and
2 bradykinin receptors effects their signaling
capability. Another receptor found to translocate into caveolae upon
agonist activation is the M2-muscarinic
acetylcholine receptor (Feron et al., 1997). It is thought that the
movement of the M2-muscarinic acetylcholine
receptor into caveolae may enhance its ability to activate eNOS, which
alters parasympathetic regulation.
The colocalization of GPCRs with their effectors in caveolae certainly
suggests a role for caveolae in signal transduction. The movement of
GPCRs into or out of caveolae upon agonist stimulation further
reinforces the view that caveolae are dynamic signaling domains.
D. Oncogenes and Tumorigenesis
1. Caveolae/Caveolins As Targets of Oncogenes.
The
association between caveolins and cancer dates back to the discovery of
caveolin-1 as a predominant phosphoprotein in v-src-transformed embryonic chicken fibroblasts (Glenney,
1989). However, follow-up work on the role of caveolae/caveolins in
tumorigenesis was not conducted until it was reported that the
oncogenic transformation of NIH 3T3 cells results in transcriptional
down-regulation of Cav-1 and ablation of morphologically identifiable
caveolae (Koleske et al., 1995). This observation along with several
subsequent studies in other tumor-derived cell lines and primary
carcinomas (Engelman et al., 1998b; Lee et al., 1998; Razani et al.,
2000a) emphasizes that regardless of the oncogenic process, caveolae and Cav-1 seem to be important targets in tumorigenesis.). The
fact that many tumor cells show down-regulation of caveolin-1 could
indicate that Cav-1 is a direct target of the activated oncogenes in
these cells. Indeed, this was first definitively demonstrated for the
activated form of H-Ras (G12V mutant): stable expression of H-Ras(G12V)
in NIH 3T3 cells leads to the down-regulation of Cav-1 (Koleske et al.,
1995), whereas treatment of these cells with an inhibitor of the
Ras-p42/44 MAP kinase cascade reverts Cav-1 expression to wild-type
levels (Engelman et al., 1997). Since these initial observations, a
growing list of other oncogenes have also been shown to down-regulate
Cav-1 protein expression at the transcriptional level (Table
3) (Koleske et al., 1995; Engelman et
al., 1997, 1998b; Razani et al., 2000a; Park et al., 2001). In all but
a few cases, the mechanism of this transcriptional suppression has not
been elucidated, a situation requiring detailed dissection of the
caveolin-1 promoter. Exceptions include the finding of functional
c-Myc-repressive and p53-responsive elements in the Cav-1 promoter,
thereby explaining the mechanism of Cav-1 down-regulation by
c-myc and the human papilloma virus oncogene E6, an oncogene
that targets p53 for degradation (Bist et al., 1997; Razani et al.,
2000a; Park et al., 2001).
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2. The Caveolins As Tumor Suppressors.
Because caveolin-1 has
been shown to act as a scaffolding protein with a capacity to inhibit
various signaling pathways, it can be surmised that the function of
certain proto-oncogenes and/or activated oncogenes might be similarly
regulated. This might provide a teleological reason as to why Cav-1 is
a common target among several activated oncogenes (see Table 3).
Overexpression experiments indicate that Cav-1 is indeed a potent
inhibitor of some of these pro-proliferative pathways. For example,
Cav-1 can interact with and suppress the function of the EGF receptor
and several members of the Ras-p42/44 MAP kinase cascade (Fig.
10) (Couet et al., 1997b; Engelman et
al., 1998a). Furthermore, antisense-mediated down-regulation of
caveolin-1 in NIH 3T3 is sufficient to hyperactivate the Ras-p42/44 MAP
kinase cascade and leads to cellular transformation (Galbiati et al.,
1998). Interestingly, ablation of caveolin-1 expression in
Caenorhabditis elegans via RNAi leads to hyperactivation of the meiotic cell cycle, a phenotype strikingly similar to uncontrolled Ras signaling (Scheel et al., 1999). Cav-1 has been found to have similar inhibitory effects on several other oncogenes, namely c-Neu and
c-Myc (Fig. 10) (Engelman et al., 1998b; Park et al., 2001).
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; Yamamoto et
al., 1999; Zundel et al., 2000). However, aside from this seemingly
pleiotropic suppression of plasma-membrane-initiated pro-survival
pathways, caveolae and the caveolins appear to have a highly
specialized role as well. Ceramide is an essential factor for the
commitment to apoptosis induced by several cellular stressors (Basu and
Kolesnick, 1998). Interestingly, sphingomyelin, the precursor to
ceramide generation, is one of the most abundant lipids in caveolae,
and sphingomyelinase, the ceramide-generating enzyme, has been
localized to caveolae microdomains (Liu and Anderson, 1995).
Furthermore, overexpression of Cav-1 sensitizes cells to
ceramide-induced cell death via a phosphatidylinositol
3-kinase-dependent mechanism (Zundel et al., 2000). Therefore, the
production of ceramide and its downstream actions seem to depend on
caveolar localization and caveolin-1 regulation. In support of these
results, overexpression of Cav-1 sensitizes cells toward apoptotic
stimuli, whereas antisense-mediated down-regulation of Cav-1 imparts
resistance to apoptosis (Liu et al., 2001).
The evidence in support of caveolin-1 as a tumor suppressor has for the
most part been elucidated in cell culture systems, with substantially
less research conducted in vivo. Intriguingly, it is known that the
caveolin-1 and -2 genetic loci are situated close to a region of
chromosome 7 (7q31.1) that is frequently deleted in numerous forms of
human cancer (Engelman et al., 1998d). Below, we will expand on this
observation and critically evaluate its relevance.
3. Relevance to Human Cancers.
LOH (loss of heterozygosity)
analysis has determined that the q31 region of human chromosome 7 is a
region of high deletion frequency in many types of human epithelial
tumors, including human primary breast (Zenklusen et al., 1994a),
prostate (Zenklusen et al., 1994b; Jenkins et al., 1998), ovarian (Kerr
et al., 1996), colon (Zenklusen et al., 1995a), and renal cell
carcinomas (Shridhar et al., 1997). More specifically, most of these
deletions have been found to be normally distributed around the D7S522
CA-repeat microsatellite, a marker that maps to the 7q31.1 region of
the human chromosome (Zenklusen et al., 1994b, 1995a,b; Lin et al., 1996). The frequent involvement of this region in different types of
cancers is highly suggestive for the presence of a previously uncharacterized tumor suppressor gene (Zenklusen et al., 1994b; Jenkins
et al., 1998); however, candidate genes mapping to this loci had never
been described.
). Further dissection of the loci revealed that D7S522 was
actually the closest microsatellite marker of any tested, located ~67
kb upstream of the CAV-2 gene, which in turn was followed ~19 kb
later by the CAV-1 gene (Fig. 11)
(Engelman et al., 1998d, 1999). At the time of these reports, the
caveolins were the first genes to be so closely localized to this
"tumor suppressor" locus; given its involvement in
proliferative/apoptotic pathways, Cav-1 was proposed to be the
candidate tumor suppressor hypothesized to be located in this region
(Engelman et al., 1998c, 1999).
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; Cui et al., 2001), indicating that
transcriptional silencing might be a mechanism of abrogating caveolin
function. In addition, a recent report found that the CAV-1 gene is
mutated in up to 16% of human breast cancer samples examined, with the majority of these being invasive carcinomas (Hayashi et al., 2001). Also, a Cav-1 cDNA harboring this point mutation (P132L) is sufficient to transform NIH 3T3 cells (Hayashi et al., 2001). Since an analogous mutation in the caveolin-3 gene (P104L) (see Fig. 3 for comparison) has
been implicated in several patients with an autosomal dominant limb-girdle muscular dystrophy (LGMD-1Cdescribed below) (Minetti et
al., 1998), it is likely that the P132L mutation behaves in a
dominant-negative fashion.
The role of Cav-1 as a tumor suppressor remains controversial. With the
availability of highly informative genomic sequences and previously
detailed physical mapping of the 7q31 region, Zenklusen and colleagues
(2001) recently attempted to definitively identify the culprit tumor
suppressor gene in this locus. They suggest instead that a gene called
ST7 is the long sought tumor suppressor gene, having identified ST7
mutations (premature stop codons) in primary tumors and cell lines. In
addition, they show that recombinant expression of ST7 in a human tumor
cell line can inhibit tumorigenicity in nude mice. Unfortunately, with
no analysis of the caveolin sequences, Zenklusen and colleagues dismiss
the idea that caveolin-1 is a functional tumor suppressor, stating
previous studies have found no mutations or promoter methylation
associated with these genes (CAV-1 and CAV-2). As described above, this
is clearly not true. It is very possible that the observed LOH in the
7q31 region could still be primarily due to Cav-1, with some contribution from the newly identified ST7. Undoubtedly, this issue
requires further analysis.
E. Specialized Functions: Caveolin-3 and Muscle Cells
Cav-3 is 65% identical and 85% similar to Cav-1 (see Fig. 3).
Given that Cav-3 is the only caveolin expressed in striated muscle
cells, it might be assumed that Cav-3 performs analogous functions to
those performed in other tissues by Cav-1. Akin to Cav-1-generated
caveolae, the Cav-3-generated caveolae of striated muscle have been
shown to be enriched in a variety of signaling molecules: eNOS; type B
atrial natriuretic factor receptor; m2 muscarinic acetylcholine
receptor; PKC, PKC
, PKC
; Erb 4; adenosine A(1) receptors;
Gs, Gi, Gq; 1,
1, 2 adrenergic
receptor; adenylyl cyclase, Src family kinases (Feron et al., 1996,
1997; Song et al., 1996b; Venema et al., 1997; Rybin et al., 1999,
2000; Lasley et al., 2000).
However, Cav-3 and the caveolae that they form appear to have a
specialized role, in addition to signaling compartmentalization, in
skeletal muscle tissue. Muscle caveolae contain a portion of the
cellular dystrophin-glycoprotein (DG) complexes. (Song et al., 1996b).
Although not an intrinsic member of the DG complex (Crosbie et al.,
1998), Cav-3 has been shown to coimmunoprecipitate with DG complex
proteins (i.e., -sarcoglyan, -dystroglycan, and dystrophin (Song
et al., 1996b; Sotgia et al., 2000), as well as to interact with other
proteins that associate with the DG complex (i.e., nNOS) (Brenman et
al., 1995). Cav-3 possesses a unique WW domain that binds to the PPXY
motif within -dystroglycan (Sotgia et al., 2000). Interestingly,
dystrophin also interacts with -dystroglycan via this motif, thus
suggesting a role for competitive binding between Cav-3 and dystrophin.
These molecular studies thus identify Cav-3 as a DG complex-interacting
protein (see Fig. 12 for an overview of
the DG complex).
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Initial EM analysis of caveolae in striated muscle led to the
hypothesis that the T-tubule system forms via the coalescence and
fusion of numerous caveolae (Ishikawa 1968; Parton et al., 1997). After
the identification of the Cav-3 protein and anti-Cav-3 antibody
generation (Song et al., 1996b; Tang et al., 1996), a multitude of
experimental evidence has emerged that supports the role of
Cav-3/caveolae in T-tubule development. Transcripts of Cav-3 mRNA are
first detectable in vivo at day 10 of gestation in developing somites
and heart (Biederer et al., 2000), well before T-tubule maturation.
Caveolae and Cav-3 were shown to be transiently associated with the
skeletal muscle T-tubule system during its developmental stages (Parton
et al., 1997) and are localized to sarcolemmal caveolae in fully
differentiated skeletal muscle (Song et al., 1996b). The T-tubule
system is an elaborate network of membrane invaginations that penetrate
the depth of muscle cells and allows for the rapid rise in
intracellular Ca2+ upon membrane depolarization
resulting in muscle contraction (Flucher et al., 1994). In vitro
experiments corroborate these studies, because antisense-mediated
down-regulation of Cav-3 in the skeletal myoblast cellline C2C12 is
sufficient to abrogate myoblast fusion and myotube formation (Galbiati
et al., 1999a).
The importance of Cav-3 for proper skeletal muscle function came to
light when Minetti, Lisanti, and colleagues identified two distinct
mutations in the Cav-3 gene that each result in an autosomal dominant
form of limb-girdle muscular dystrophy (LGMD-1C) (Minetti et al.,
1998). Patients with this disease present with calf hypertrophy, muscle
cramps, and mild-to-moderate proximal muscle weakness. Further analysis
shows elevated serum creatine kinase levels, typical of a pathological
muscle phenotype. Histological analysis reveals only moderate myopathic
changes but a near complete loss of Cav-3 protein expression. In vitro
studies in which the mutant Cav-3 proteins were overexpressed in a
heterologous cell system demonstrated that these LGMD-1C mutants
interact with normal Cav-3 proteins to form unstable aggregates, which
undergo ubiquitination and proteasomal degradation (Galbiati et al.,
1999b). The analysis of other patients with mutations in Cav-3 and
mouse models of caveolin-3-opathies has yielded an even greater
understanding of Cav-3 function in muscle (see relevant headings below).
F. Emerging Functions: Caveolins and Lipid Droplets
Lipid droplets, albeit traditionally associated with adipocytes,
are found to varying extents in almost all cells (Londos et al., 1999).
They are thought to consist of a core of neutral lipids (i.e.,
triglycerides and cholesterol esters) surrounded by a phospholipid
monolayer derived from the ER. Several proteins, including the highly
abundant perilipins (present in adipocytes and steroidogenic cells) and
adipocyte differentiation-related protein (ADRP; present in most other
lipid droplet-containing cells), coat this lipid core and maintain its
overall integrity (Londos et al., 1999).
Recently, several independent investigators have shown that the
caveolins can be redirected to lipid droplets under conditions where
they are localized to the ER (Ostermeyer et al., 2001; Pol et al.,
2001). Parton and colleagues showed that truncated versions of
caveolin-1, -2, and -3 mis-localize to intracellular cholesterol-rich vesicles, which they later identified as lipid droplets (Roy et al.,
1999; Pol et al., 2001). In a more direct experiment, Brown and
colleagues tagged the caveolin-1 protein with the ER-retrieval sequence, KKSL, and showed caveolin-1 accumulation in lipid droplets (Ostermeyer et al., 2001). The wild-type untagged Cav-1 protein can be
similarly redirected by treating cells with brefeldin A, which
collapses the Golgi/ER compartments or by incubation of cells with the
free fatty acid, oleate (Ostermeyer et al., 2001; Pol et al., 2001).
Interestingly, overexpression of the -isoform (not full-length
version) of caveolin-2 leads to its constitutive localization to lipid
droplets (Fujimoto et al., 2001). Taken together, these data suggest
that although the caveolins are not normally associated with internal
lipid compartments, they can certainly traffic to these locations under
certain conditions. As such, the caveolins are the first known integral
membrane protein components of lipid droplets. Although the
physiological relevance of this lipid droplet localization is not
known, these findings could be extremely valuable in the understanding
of caveolin/caveolae functions in adipocytes.
Based purely on ultrastructural comparisons and tissue expression
profiles, the adipocyte seems to have the highest concentrations of
caveolae and the highest levels of caveolin-1 and -2, i.e., more than
any other cell type (Scherer et al., 1994). Indeed, electron
micrographs of adipocytes dating back to 1963 show that caveolae
account for ~30% of the surface area of the adipocyte plasma
membrane (Napolitano 1963; Fan et al., 1983). Furthermore, in 3T3-L1
cells, a widely used model system for studying adipogenesis, the number
of caveolae increases ~9-fold, and caveolin-1 and -2 expression
increases ~20-fold during differentiation from the fibroblastic to
the adipocyte state (Fan et al., 1983; Scherer et al., 1994).
The uptake and storage of fatty acids as triglycerides is a major
function of adipocytes. As the flux of fatty acids into primary
adipocytes and 3T3-L1 cells follows saturable kinetics, facilitated
membrane transport has been proposed as the uptake mechanism (Abumrad
et al., 1984; Zhou et al., 1992). Interestingly, labeling of membrane
proteins with photoreactive long-chain fatty acids identifies
caveolin-1 as the major fatty acid binding protein in adipocytes
(Gerber et al., 1993; Trigatti et al., 1999). Together with the
association of the caveolins with lipid droplets, it is entirely
possible that caveolae and the caveolins act as portals for the uptake
and transport of fatty acids to lipid droplets. In addition to Cav-1,
several other proteins have been proposed to mediate this function
(Bernlohr et al., 1999). A true understanding of lipogenic processes
will require comparisons between this disparate group of fatty acid
binding proteins.
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VI. Animal Models in the Study of Caveolae and Caveolins |
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Although interest in caveolae and the caveolins has been steadily
increasing over the last 10 years, much of the research has been
limited to biochemical or cell culture analyses. Despite the successes
of this approach (namely the implication of caveolae/caveolins in
endocytosis, cholesterol trafficking, signal transduction, tumorigenesis, and other processes), it is not clear which of the
pleiotropic functions of caveolae are predominant in vivo and thus
physiologically relevant. In this regard, the recent series of reports
describing the various phenotypes of mice deficient in Cav-1, -2, or -3 are extremely enlightening (Drab et al., 2001; Galbiati et al., 2001a;
Razani et al., 2001a, 2002b).
Perhaps the most important initial observation made in these mice is that in the absence of Cav-1 or Cav-3, but not Cav-2, there is a concomitant loss of morphologically identifiable caveolae in tissues expressing those proteins. Therefore, the Cav-1 and -3 null mice are essentially tissue-specific caveolae-deficient mice, thus enabling the first controlled study of such vesicles in vivo. We will now discuss the previously proposed functions of caveolae in the context of these caveolin-deficient animal models and the relevance of these mouse models to the understanding of human disease.
A. Studies of Caveolin-1-Deficient Mice
The initial characterization of mice with a disruption of the
caveolin-1 (Cav-1) locus was reported simultaneously by two groups
(Drab et al., 2001; Razani et al., 2001a). At first glance, it is
surprising that mice lacking Cav-1 and caveolae would display no
"overt" phenotypic abnormalities. After all, it is hard to imagine
that certain tissues like the lung or adipose (where ~30-70% of the
membrane is composed of these organelles) can function in their
absence. However, more detailed histological and functional analysis
reveals a number of interesting abnormalities, which we will discuss
below (for a summary, see Table 4).
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1. Caveolin-1 and Caveolae Biogenesis.
As discussed above,
several overexpression studies point to Cav-1 as a mediator of caveolae
formation (Fra et al., 1995b; Engelman et al., 1997; Galbiati et al.,
1998; Li et al., 1998). In this regard, the generation of Cav-1 null
mice served as an important proof of principal. Indeed, there is a
complete ablation of morphologically identifiable caveolae in Cav-1
null tissues (endothelial and adipose) and primary cells derived from
Cav-1 null mice, firmly establishing that caveolin-1 is required for caveolar invagination (Fig. 13A) (Drab
et al., 2001; Razani et al., 2001a).
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). It
will be interesting to see if Cav-1 null cells still maintain the
normal composition of cholesterol/lipids in the plasma membrane and the
extent by which their lipid raft network is perturbed. Recent work by
Sotgia et al. (2002) demonstrates that Cav-1 null cells and tissues are
clearly not wild type in behavior, exhibiting defects in the sorting of
GPI-anchored and lipid-modified proteins. Further work in this area
will be rewarding not only in relation to caveolar biogenesis, but in
deciphering the role of caveolins in intracellular lipid/protein trafficking.
2. Interactions of Caveolin-1 with the Other Caveolins.
As
caveolin-1 is known to have very intricate interactions with its
closely related family member, Cav-2, Cav-1 null mice also provided the
opportunity to dissect this relationship in vivo. Intriguingly, in all
Cav-1 null tissues examined, the expression of Cav-2 is severely
reduced by ~90 to 95% (Drab et al., 2001; Razani et al., 2001a).
discovered that in the absence of Cav-1, Cav-2 is
destabilized and degraded by the proteasomal pathway. In Cav-1 null
cells, any residual Cav-2 protein is found in the Golgi apparatus;
however, reintroduction of Cav-1 in these cells rescues both the
expression and plasma membrane localization of Cav-2. The same effect
can be reproduced by the addition of proteasomal (but not lysosomal)
inhibitors, suggesting that Cav-2 likely misfolds at some point during
ER/Golgi transport and is targeted for degradation. The fact that at
steady state low levels of Cav-2 are localized to the Golgi most likely
reflects leakage from and/or inefficiencies in the ER quality control apparatus.
3. Caveolin-1 and Cellular Proliferation.
Based on several
lines of evidence discussed above (namely the inhibitory action of
Cav-1 on pro-proliferative/anti-apoptotic pathways), caveolin-1 has
been considered a candidate tumor suppressor. Thus, it would be
predicted that Cav-1-deficient cells would exhibit derangements in
proliferation and/or growth. Indeed, an analysis of the growth
properties of cultured primary MEFs reveals that Cav-1 null cells
display a more active cell cycle profile than their wild-type
counterparts (~30% increase in the S-phase) and attain nearly 3-fold
higher monolayer densities over a 10-day period (Razani et al., 2001a).
A number of prior studies support a role for Cav-1 in the suppression
of the Ras-MAP kinase pathway (Couet et al., 1997b; Engelman et al.,
1998a; Galbiati et al., 1998; Scheel et al., 1999). Therefore, of
the disinhibited signaling pathways that might be driving this
proliferation, the p42/44 MAP kinase cascade was thought to be the most
likely. Surprisingly, Cav-1 null MEFs show no hyperactivation of this
pathway (under both baseline and stimulated conditions) (Razani et al.,
2001a). Thus, to understand the basis for this hyperproliferation,
future studies will need to take into account a broader array of cell cycle regulators.
). However, it is possible that the loss of Cav-1
in conjunction with another tumor suppressor can act synergistically in
tumorigenesis. Such questions can be addressed by crossing Cav-1 null
mice with one or more of the many tumor-prone mice currently available.
4. Caveolin-1 and Endocytosis.
A large body of work suggests
that caveolae are the predominant route by which certain molecules
(e.g., cholera and tetanus toxins, albumin, GPI-anchored proteins),
viruses, and even bacteria are internalized by a cell (Montesano et
al., 1982; Anderson et al., 1992; Parton et al., 1994; Schnitzer et
al., 1994; Anderson, 1996; Shin et al., 2000; Stang et al., 1997). As
Cav-1-deficient mice also lack caveolae, especially in tissues with
central roles in endocytic processes (e.g., the endothelium), it is
possible to rigorously examine the actual contribution of caveolae in
this setting.
; Schubert et al.,
2001). Cav-1 null MEFs incubated with fluorophore-conjugated albumin
are entirely defective in membrane labeling and internalization, an
effect that can be reversed by transfection of the Cav-1 cDNA. In
contrast, the uptake of fluorophore-conjugated transferrin, a
clathrin-mediated event, remained unaffected in these cells (Razani et
al., 2001a). Infusion of gold-conjugated albumin into the pulmonary
vasculature of wild-type and Cav-1 null mice and subsequent electron
microscopy reveals that in the absence of caveolae, the albumin load
remains strictly luminal (Schubert et al., 2001). Similar results were
obtained in a more quantitative assay of endocytosis using
radioiodinated albumin (Schubert et al., 2001). Based on this evidence,
caveolae appear to be the predominant pathway by which a major serum
protein is endocytosed.
However, as albumin makes up ~60% of the serum protein mass and
serves as the major serum-transport protein for numerous endogenous molecules, it is difficult to reconcile the above data with the viability and overtly benign presentation of Cav-1-deficient mice. Obviously, other pathways must at least partially compensate for the
absence of caveolae in vivo. In fact, when Drab et al. (2001) measured
the albumin concentration in cerebrospinal fluid, a condition that has
been thought to depend on the caveolae-mediated transport of albumin
from the blood, they found it surprisingly to be normal. It is possible
that other undetermined defects in the endothelium of Cav-1-deficient
mice (e.g., more porous paracellular routes) allow for a small but
sufficient steady-state shunting of molecules.
5. Caveolin-1 in the Lung.
The architecture of the alveolar
septa (the primary site of gas exchange in lungs) is a thin cytoplasmic
extension of the type I pneumocyte, a negligible layer of basement
membrane/interstitial matrix, and the thin-walled endothelial cell.
Despite this dearth of tissue mass (~0.6-1 µm in thickness), up to
70% of the total plasma membrane in a typical septa is estimated to be
composed of caveolae (Gumbleton, 2001). This is mostly due to the fact that both endothelial cells and type I pneumocytes express Cav-1 and
Cav-2 at high levels; thus caveolae, in both plasmalemmal and fully
invaginated forms crowd the cytoplasmic extensions of both cell types
(Dormans, 1983). By sheer magnitude, these structures and their marker
proteins must serve an important role in lung physiology; yet, at the
present, researchers have very little insight into the possible
functions (proposed roles include involvement in gas exchange,
endocytosis, signal transduction, and/or responses to shear/mechanical
stress) (Lisanti et al., 1994b; Schnitzer et al., 1995c, 1996; Park et
al., 2000; Gumbleton 2001; Predescu et al., 2001). Unfortunately, none
of the studies have shown convincing lung-specific functions for
caveolae and the caveolins. In this respect, the finding of lung
defects in Cav-1-deficient mice was extremely enlightening.
; Razani et al.,
2001a). Immunohistochemical analysis using Ki67, a marker of cellular
proliferation, shows markedly increased cell proliferation in Cav-1
null lungs (Razani et al., 2001a). Similar analysis with an endothelial
marker, Flk-1 (vascular endothelial growth factor receptor), reveals an
increase in endothelial cell staining in Cav-1 null mouse lung as well.
Taken together, these data suggest an endothelial cell proliferation
phenotype in the Cav-1 KO lung. Cell cycle derangement might be
presumed to underlie this lung phenotype given the faster proliferation and increased S-phase of primary Cav-1 null MEFs. Why obvious hypercellularity is not observed in other Cav-1 null tissues remains to
be examined, although there are several possible explanations. For
example, caveolae may play a relatively specific role in lung tissue or
modifying factors may exist in other tissues that ameliorate the
effects due to Cav-1 loss. Alternatively, more indirect mechanisms may
be present, such as a compensatory increase in cell number due to the
ineffective function of the Cav-1 null lung parenchyma.
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noted increased trichrome-staining (consistent with
fibrosis/collagen deposition), whereas Razani et al. noted
increased reticulin staining (consistent with thickened basement
membranes) with negligible trichrome staining. This difference is
likely secondary to sample interpretation and will be resolved pending
other confirmatory histological stains.
Nevertheless, increases in cell number and extracellular matrix are
commonly seen in the restrictive lung diseases (a group of
etiologically diverse disorders where thickening of alveolar septa
results in reduced gas exchange and lung compliance, in turn leading to
ineffective pulmonary function and significant morbidity/mortality).
Examples of disorders with this manifestation include acute respiratory
distress syndrome, idiopathic pulmonary fibrosis, and pneumoconiosis.
Consistent with a restrictive picture, Cav-1-deficient mice are easily
fatigued and markedly exercise-intolerant in swimming assays of
exercise capacity (Drab et al., 2001; Razani et al., 2001a). Of course,
detailed insight into the phenotype of Cav-1 null lungs requires a more
rigorous assessment of lung physiology, including pulmonary function
testing, whole body plethysmography, measurements of arterial blood
gases, etc.
Given the extraordinary abundance of caveolae in the lungs and the
complete lack thereof in Cav-1 null mice, one can easily surmise that
such drastic pathology is directly due to a loss of morphologically
identifiable caveolae in the lungs. This attractive interpretation of
these phenotypes was complicated recently when we reported the
identical pathology in mice deficient in caveolin-2, the other caveolin
gene found highly expressed in all Cav-1-expressing tissues (Fig. 14)
(Razani et al., 2002b) (see Section VI.B.).
6. The Vascular Physiology of Caveolin-1-Deficient
Mice.
As mentioned above, the caveolin-1/eNOS signaling
connection has been well studied in the past few years with much
evidence supporting its physiological significance. Recent studies on
the vasoresponsiveness of isolated aortic rings from Cav-1-deficient mice have now bolstered this connection. Cav-1 null aortas have a
blunted response to phenylephrine-induced vasoconstriction and are
hyper-responsive to Ach-induced vasorelaxation (Fig. 13B) (Drab et al.,
2001; Razani et al., 2001a). Furthermore, the vasodilatory tendencies
of Cav-1 null aortas can be completely abrogated by the addition of
L-NAME, a potent eNOS inhibitor (Razani et al., 2001a).
Taken together, these data suggest that in the absence of caveolin-1,
eNOS not only retains a higher level of activity at baseline but lacks
the regulatory component by which it can be turned off after the
arrival of stimuli such as Ach (a mechanism akin to that proposed in
Fig. 9). In direct support of constitutive eNOS activation, Cav-1 null
vascular smooth muscle cells also have a severalfold increase in NO and
its downstream mediator, cGMP (Drab et al., 2001).
7. Caveolin-1 and Lipid Homeostasis.
Despite a strong
connection between caveolae, caveolin-1, and adipose function/lipid
storage, initial analysis of Cav-1 null mice showed no obvious
adipocyte pathology, with intraperitoneal fat pads remaining
histologically normal (Drab et al., 2001; Razani et al., 2001a).
However, if Cav-1 null mice are allowed to grow older, an interesting
observation is made: they have significantly smaller body sizes than
their wild-type counterparts. With some indication that this phenotype
might be related to alterations in adipose tissue, Razani et al.
(2002a) conducted a rigorous metabolic analysis of Cav-1-deficient mice.
assessed its in situ enzymatic activity in Cav-1 null
mice but found it to be normal. They also noted that irrespective of
the lipid perturbations, insulin and glucose levels remain normal in
these mice, suggesting that overt insulin resistance might not play a
role in the lipid abnormalities. However, they did not go beyond this
first-pass analysis, leaving several questions unanswered. Certainly,
in light of the previously discussed roles for Cav-1 in lipid
homeostasis (i.e., its ability to bind fatty acids, its targeting to
lipid droplets) or its role in signal transduction including the
insulin receptor signaling pathway (Yamamoto et al., 1998; Nystrom et
al., 1999), it is plausible that some or all of these pathways are
perturbed in caveolae-deficient adipocytes. Importantly, it should be
noted that Cav-2-deficient mice, which do not show any effects on
caveolae formation, have none of the adipocyte or lipid abnormalities
of the Cav-1 null mice (Razani et al., 2001a; unpublished
observations), indicating that Cav-1 and caveolae play a selective role
in the entire range of metabolic phenotypes observed here.
B. Studies of Caveolin-2-Deficient Mice
The near complete loss of Cav-2 protein levels in Cav-1 null
tissues leaves unresolved whether any of the phenotypes observed in the
Cav-1 null setting are related to the reduction of Cav-2. To approach
this question directly, we generated Cav-2-deficient mice (Razani et
al., 2002b). These mice are viable, fertile, and demonstrate no obvious
abnormalities upon general inspection. Interestingly, electron
microscopic analysis reveals that caveolae remain generally unaffected
by the loss of Cav-2, maintaining a morphology and cellular
distribution seen in wild-type animals. The generation of Cav-2 null
mice thus allows for the analysis of two issues: 1) identifying the
specific role of Cav-2; and 2) examining the functionality of caveolae
formed purely by Cav-1. As detailed below, the phenotypes observed in
Cav-2 null mice were anything but predictable (for a summary, see Table
4).
1. Relationship with Caveolin-1.
In the absence of Cav-2,
Cav-1 protein expression and membrane localization is in large part
intact (Razani et al., 2002b). Caveolin-1 is able to homo-oligomerize,
traffic to the plasma membrane, and form caveolae. However, Cav-1
expression is decreased by as much as 2-fold in certain tissues (e.g.,
the lung), yet unaffected in others (e.g., adipose tissue). Although
this is not a dramatic alteration in expression, it likely signifies
slight instabilities in the caveolin oligomeric complex, albeit not
enough to disrupt caveolin homo-oligomerization or the structural
integrity of caveolae in vivo.
2. The Surprising Role of Caveolin-2 in the Lung.
Initial
characterization of Cav-2 null mice revealed that the absence of Cav-2
results in defects in only one major organ system, the lungs. Despite
an intact caveolae membrane system, the Cav-2 null lung parenchyma
shows hypercellularity with thickened alveolar septa and an increase in
the number of endothelial cells, a histological picture that is
virtually indistinguishable from that of the Cav-1 null mice (Fig. 14).
In addition, in the Cav-2-deficient setting, these mice are markedly
exercise-intolerant, to the same levels observed for the Cav-1 null
mice. As mentioned before, Cav-1 expression is diminished by ~2-fold
in these lungs; however, the observed pathology cannot be due to this
slight perturbation since Cav-1 heterozygous animals (who have a
similar decrease in Cav-1 expression) fail to exhibit any lung
pathology (Razani et al., 2002b). Thus, it appears that the morphologic
loss of caveolae is not required for proper lung function; rather, it is perturbations in the caveolar coat proteins that play a more significant role. This result is extremely important for two reasons: 1) it shows that the phenotypes present in the Cav-1 knock-out are not
all directly due to a loss of caveolae or the sole function of Cav-1;
and 2) it is the first time a specific role for caveolin-2, independent
of Cav-1, has ever been proposed.
). This suggests that Cav-1/Cav-2 hetero-oligomers play an important and distinct role from Cav-1 homo-oligomers in lung tissue.
C. Studies of Caveolin-3-Deficient Mice
To gain a better understanding of the role played by caveolin-3 in
a whole animal system, we and others have recently generated caveolin-3-deficient mice using standard homologous recombination techniques (Hagiwara et al., 2000; Galbiati et al., 2001a). These mice
are viable, fertile, and demonstrate no obvious abnormalities upon
general inspection. Electron microscopic analysis reveals a loss of
caveolae in all striated muscle cells, thus definitively demonstrating
the necessity of Cav-3 for caveolae formation in striated muscle (Fig.
15A) (Hagiwara et al., 2000; Galbiati
et al., 2001a; and unpublished data). Table 4 provides a summary of the
features of Cav-3 null mice that offers insights into the roles of
caveolae in these and other cell types.
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1. Caveolin-3 and Muscle Disease.
Mutations in Cav-3 that
cause muscle disease were first reported by Minetti et al. (1998) who
identified two families, each with different autosomal dominant
mutations, that result in LGMD-1C. Additional cases of LGMD-1C have
been reported since, making a total of five distinct Cav-3 mutations to
date that are causal for LGMD-1C (Herrmann et al., 2000; Matsuda et
al., 2001). Interestingly, mutations in Cav-3 have also been associated
with three other muscle diseases: rippling muscle disease, distal
myopathy, and idiopathic elevated creatine kinase levels (hyperCKemia)
(Carbone et al., 2000; Betz et al., 2001; Vorgerd et al., 2001;
Tateyama et al., 2002). It is thus clear that the genetic background in which mutant Cav-3 is expressed affects the ultimate phenotype as the
same mutation in Cav-3 can result in distinct muscle diseases. Common
to all these diseases, however, is a reduction or near elimination of
Cav-3 expression. Mice deficient for caveolin-3 thus represent an
animal model for diseases in which caveolin-3 is markedly reduced.
Histological analysis of skeletal muscle tissue from caveolin-3 null
mice reveals mild myopathic changes, with variability in the size of
muscle fibers, necrotic fibers, and a mononuclear cell infiltration,
consistent with the features of LGMD-1C pathology (Fig. 15B) (Hagiwara
et al., 2000; Galbiati et al., 2001a).
; Vaghy et al.,
1998). This indicates that proper control of Cav-3 expression may be
important for normal muscle homeostasis. In support of this conclusion,
transgenic mice overexpressing mutant or wild-type Cav-3 have severely
affected skeletal muscle both histologically and functionally (Sunada
et al., 2001).
2. Caveolin-3 and Transverse Tubules.
The coalescence of
caveolae to form the transverse tubule (T-tubule) system in muscle was
first indicated by EM studies that predated the identification of the
Cav-3 gene (Schiaffino et al., 1977). Later, Cav-3 was shown to be
transiently associated with the developing T-tubule system using
immunogold labeling with Cav-3 antibodies (Parton et al., 1997). The
generation of Cav-3-deficient mice provided the ideal system to
investigate the necessity of Cav-3 in T-tubule development/maintenance.
Immunostaining of mature Cav-3 null skeletal muscle with antibodies to
the dihydropyridine receptor-1 and ryanodine receptor, two well
characterized T-tubule markers, showed diffuse and disorganized
labeling, suggestive of structural abnormalities in the T-tubule system
(Galbiati et al., 2001a). EM analysis of ferrocyanate-stained mature
Cav-3 KO skeletal muscle showed dilated and disorganized/longitudinally oriented T-tubules (Fig. 15C). Longitudinally oriented T-tubules are
normally seen only in developing skeletal muscle. Interestingly, dilated and developmentally abnormal T-tubules have recently been observed in patients with LGMD-1C (Minetti et al., 2001). Thus, it
appears that the proper development and function of the skeletal T-tubule system requires caveolin-3. The detailed molecular
underpinnings of the T-tubule abnormality and its relevance to other
caveolin-3-deficient pathologies awaits further investigation.
3. Caveolin-3 and the Dystrophin-Glycoprotein
Complex.
Several components of the DG complex are concentrated in
caveolae (Doyle et al., 2000; Galbiati et al., 2001a). Although Cav-3 is not an integral member of the DG complex (Crosbie et al., 1998), Cav-3 can associate with the complex through a novel WW domain (Sotgia
et al., 2000). Examination of DG complex members in Cav-3-deficient animals reveals no alteration in their expression or membrane association; however, biochemical fractionation of the muscle cell
plasma membrane reveals dystrophin and several of its associated proteins to be excluded from lipid raft microdomains/caveolae (Galbiati
et al., 2001a). This indicates that caveolar localization of at least a
portion of the DG complex is critical for the maintenance of skeletal
muscle structural integrity. In future studies, it will be important to
further dissect the interactions between components of the dystrophin
complex in Cav-3 null mice.
D. Dominant-Negative Caveolin Mutations in Human Disease
There are 12 amino acid residues that are conserved within all
caveolins from worms to humans (Fig.
16). Each of the muscle diseases
associated with Cav-3 involves mutations in one of these residues. The
first to be discovered and best characterized are a Pro 
Leu
substitution at amino acid position 104 (P104L) and a deletion of 3 amino acids at positions 63-65 (
TFT) that result in LGMD-1C
(Minetti et al., 1998). These LGMD-1C patients demonstrated a ~95%
reduction in Cav-3 levels. The P104L and TFT mutant Cav-3 proteins
act in a dominant-negative fashion by hetero-oligomerizing with
wild-type Cav-3 and directing their proteasomal degradation (Galbiati
et al., 2000b). Two additional mutations in Cav-3 that result in
LGMD-1C have been identified: A45T and T64P (Herrmann et al., 2000).
Each of these mutations also results in a marked reduction in Cav-3
expression in skeletal muscle. Although a detailed molecular
characterization of these mutants has not been performed, given their
autosomal dominant inheritance, they too most likely act to sequester
wild-type Cav-3 in a dominant-negative fashion.
|
It is intriguing that some of the same mutations that result in LGMD-1C
have been shown to be associated with other muscle disorders. Multiple
families with autosomal dominant rippling muscle disease (RMD) harbor
Cav-3 mutations: R26Q, A45T, A45V, and P104L (Betz et al., 2001). A
sporadic case of RMD due to a R26Q Cav-3 mutation has also been
documented (Vorgerd et al., 2001). Recently, an R26Q Cav-3 mutation due
to a heterozygous nucleotide substitution was identified in a patient
with a distal myopathy (Tateyama et al., 2002). In both the RMD and
distal myopathy cases, a near elimination of Cav-3 expression is seen.
Finally, a sporadic R26Q mutation in Cav-3 was demonstrated in two
patients with elevated creatine kinase levels (hyperCKemia) (Carbone et al., 2000). Although a ~65% decrease in Cav-3 expression was shown in these patients, they did not manifest the paradigm symptoms of
LGMD-1C, RMD, or distal myopathy. Disease-causing mutations in Cav-3
are in residues that cause a dominant-negative phenotype, one in which
the Cav-3 mutant disrupts the normal functioning of the wild-type Cav-3
protein. How what appear to be a variety of dominant-negative mutations
in Cav-3 can manifest as distinctly different muscle dysfunctions
awaits further analysis.
Given the conservation across species of certain amino acid residues in
caveolins and the proven disease-causing mutations in these residues in
Cav-3, it might be predicted that mutations in the corresponding
residues of Cav-1 and -2 would be deleterious. In point of fact, it was
recently reported that the Cav-1 gene is mutated in up to 16% of human
breast cancer samples examined. The mutation results in a Pro Leu
amino acid substitution, Cav-1 (P132L). This mutation is analogous to
the Cav-3 (P104L) mutation (Fig. 16) (Hayashi et al., 2001).
Recombinant expression of the Cav-1 P132L mutant in NIH 3T3 cells was
sufficient to cause cellular transformation (Hayashi et al., 2001).
Anti-sense ablation of Cav-1 expression results in a similarly
transformed cellular phenotype (Galbiati et al., 1998). It is most
likely that Cav-1 P132L acts in a dominant-negative fashion. This first
description of a mutation in the Cav-1 gene is sure to be followed by
the identification of many similar dominant-negative caveolin mutations
in other human diseases.
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VII. Conclusions and Future Directions |
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A. Caveolae and Caveolins
Scientific research in a particular area is often marked by pivotal moments. The ultrastructural identification of caveolae in the 1950s and the cloning of each of the caveolins in the 1990s are two such moments in the caveolin field. The next era of caveolin research has now just begun with the generation and individual characterization of caveolin knock-out mice. These mice mark the first in vivo functional assessment of caveolins/caveolae. The phenotypes of the Cav-deficient mice have been everything from i) unpredicted, ii) to supportive of previous hypotheses, iii) to cause for reformulating prior notions.
Given that with the loss of Cav-1 or Cav-3, the cell loses not only a protein but an entire membrane domain in multiple organ systems, it might have been posited that Cav deficiency would result in the lethality of the mice. However, it must be considered that most disease-associated genes are not essential for viability. As mutations in Cav-1 and Cav-3 have been associated with human diseases, their proper expression is obviously not necessary for viability.
Despite the intricate role of Cav-1 in cholesterol trafficking and
homeostasis and its proposed roles in atherosclerosis, Cav-1 null mice
do not appear to have altered serum cholesterol levels (Drab et al.,
2001; Razani et al., 2002a). Obviously, a more rigorous analysis needs
to be conducted including lipid influx/efflux experiments, placement of
Cav-1 null cohorts on atherogenic diets, and double-crosses with
hypercholesterolemic mouse models.
Despite the down-regulation of Cav-1 in numerous tumors and its ability
to regulate several oncogenic pathways (both suggestive of a
tumor-suppressive role), Cav-1 null mice do not present with a higher
incidence of carcinomas (Razani et al., 2001a). However, they do
manifest hyperproliferative characteristics both in vitro and in vivo
(Drab et al., 2001; Razani et al., 2001a), indicating that full onset
tumorigenesis might require the synergistic targeted disruption of
Cav-1 and other classically known tumor suppressors. In this regard,
treatment of these mice with carcinogens and/or crosses with
tumor-prone mice will be an important next step.
Upon identifying multiple signaling molecules within the caveolar
fractions of cells, Lisanti and colleagues (1994a) proposed the
"caveolae signaling hypothesis". This hypothesis states that the
compartmentalization of signaling molecules allows for the regulation
of signal transduction by bringing interacting molecules into proximity
with one another and/or by sequestering molecules away from the rest of
the cellular pool. Through multiple in vitro experiments, eNOS has
perhaps been the best characterized of the caveolar localized
molecules. The exaggerated vasorelaxative response in the Cav-1 null
mice is a clear indication of the hyperactivation of eNOS due to loss
of Cav-1 inhibition (Drab et al., 2001; Razani et al., 2001a). The
analysis of other putative Cav-1 inhibited molecules awaits further
investigation (Smart et al., 1999).
Another fruitful area of future research is the characterization of mice with multiple caveolins knocked out. Cav-1 null mice still have caveolae in all their striated muscle cells and Cav-3 mice have caveolae remaining in their nonmuscle cells. Thus a truly "caveolae-less" mouse model has not been characterized. Whole organism ablation of caveolae will only come about by intercrossing Cav-1 KO and Cav-3 KO mice to yield a Cav-1/3 double knock-out mice. It will be especially interesting to examine tissues that abundantly express both Cav-1 and Cav-3, such as the heart, in Cav-1/3 double knock-out animals. The smooth muscle cell, which is the only known cell to express both Cav-1 and Cav-3 will also be of major interest. In addition, Cav-2/3 and Cav-1/2/3 knock-out crosses may illicit as yet unimagined phenotypes, which will further reveal the functional roles of the caveolins/caveolae.
This new era of caveolar research (brought to the forefront by the generation of caveolin-deficient mice) is sure to yield greater insight into the physiological and pathological roles of the different caveolin family members.
B. Modifiers of Raft Function
Several different classes of lipid rafts are now believed to coexist in a single mammalian cell. Besides classical lipid rafts, which lack structural protein components, liquid-ordered domains may be enriched in one particular structural protein component, which drastically changes the morphology and/or the function of the lipid raft. Here, we refer to this newly emerging class of structural proteins as MORFs (modifiers of raft function). The first MORF to be identified was the caveolin-1 protein. When caveolin-1 is integrated into the microenvironment of a lipid raft, these microdomains invaginate and form caveolae, i.e., 50- to 100-nm flask-shaped structures located at or near the plasma membrane.
Besides caveolins, several other protein families have been recently
reported to structurally and functionally modify lipid rafts. These
MORF proteins include the flotillins (FLO-1 and -2; a.k.a. reggies or
cavatellins), LAT/PAG, MAL/BENE, stomatins, and VIP36 (Galbiati et al.,
2001b). Each of these MORFs may be responsible for the formation of a
distinct class of lipid raft. Future studies will have to address the
detailed functions of this newly emerging class of raft
modifier integral membrane proteins.
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Acknowledgments |
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TopPreviousNextReferences
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This work was supported by grants from the National Institutes of Health, the Muscular Dystrophy Association, the American Heart Association (to M.P.L.). B.R. and S.E.W. were supported by a National Institutes of Health Medical Scientist Training Grant (T32-GM07288).
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Footnotes |
|---|
Address correspondence to: Dr. Michael P. Lisanti, Albert Einstein College of Medicine, Jack and Pearl Resnick Campus, 1300 Morris Park Ave., Bronx, NY 10461. E-mail: lisanti{at}aecom.yu.edu
| |
Abbreviations |
|---|
VIP-21, vesicular integral
protein of 21 kDa;
Cav, caveolin;
ER, endoplasmic reticulum;
N-MAD, N-terminal membrane attachment domain;
C-MAD, C-terminal membrane
attachment domain;
GPI, glycosylphosphatidylinositol;
SV40, simian
virus 40;
VAMP, vesicle-associated membrane protein;
kb, kilobase;
SRE, sterol regulatory element;
LDL, low-density lipoprotein;
HDL, high-density lipoprotein;
SR-BI, class B type I scavenger receptor;
eNOS, endothelial nitric-oxide synthase;
PKC, protein kinase C;
EGF-R, epidermal growth factor receptor;
CSD, caveolin scaffolding domain;
CBD, caveolin binding domain;
SH, Src homology;
NO, nitric oxide;
Ach, acetylcholine;
GPCR, G-protein-coupled receptor;
MAP, mitogen-activated
protein;
LOH, loss of heterozygosity;
LGMD-1C, limb-girdle muscular
dystrophy 1C;
DG, dystrophin-glycoprotein;
EM, electron microscopic;
MEF, mouse embryonic fibroblast;
KO, knock-out;
RMD, rippling muscle
disease;
L-NAME, N
-nitro-L-arginine methyl
ester;
MORF, modifier of raft function;
AKAP, A-kinase anchor protein;
SNAP, soluble NSF (N-ethylmaleimide-sensitive factor)
attachment protein;
SNARE, soluble NSF
(N-ethylmaleimide-sensitive factor) attachment protein
receptor.
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References |
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-isoform of caveolin is selectively phosphorylated by v-Src in vivo.
J Biol Chem
271:
3863-3868
0031-6997/02/5403-431-467$7.00
PHARMACOLOGICAL REVIEWS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
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T. Kamishima, T. Burdyga, J. A. Gallagher, and J. M. Quayle Caveolin-1 and caveolin-3 regulate Ca2+ homeostasis of single smooth muscle cells from rat cerebral resistance arteries Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H204 - H214. [Abstract] [Full Text] [PDF]
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 S. M. Storey, T. F. Gibbons, C. V. Williams, R. D. Parr, F. Schroeder, and J. M. Ball Full-Length, Glycosylated NSP4 Is Localized to Plasma Membrane Caveolae by a Novel Raft Isolation Technique J. Virol., June 1, 2007; 81(11): 5472 - 5483. [Abstract] [Full Text] [PDF]
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Z. Xiao, F. Schmitz, V. E. Pricolo, P. Biancani, and J. Behar Role of caveolae in the pathogenesis of cholesterol-induced gallbladder muscle hypomotility Am J Physiol Gastrointest Liver Physiol, June 1, 2007; 292(6): G1641 - G1649. [Abstract] [Full Text] [PDF]
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C. Nyalendo, M. Michaud, E. Beaulieu, C. Roghi, G. Murphy, D. Gingras, and R. Beliveau Src-dependent Phosphorylation of Membrane Type I Matrix Metalloproteinase on Cytoplasmic Tyrosine 573: ROLE IN ENDOTHELIAL AND TUMOR CELL MIGRATION J. Biol. Chem., May 25, 2007; 282(21): 15690 - 15699. [Abstract] [Full Text] [PDF]
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R. Gosens, G. Dueck, W. T. Gerthoffer, H. Unruh, J. Zaagsma, H. Meurs, and A. J. Halayko p42/p44 MAP kinase activation is localized to caveolae-free membrane domains in airway smooth muscle Am J Physiol Lung Cell Mol Physiol, May 1, 2007; 292(5): L1163 - L1172. [Abstract] [Full Text] [PDF]
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C. J. Clarke, V. Ohanian, and J. Ohanian Norepinephrine and endothelin activate diacylglycerol kinases in caveolae/rafts of rat mesenteric arteries: agonist-specific role of PI3-kinase Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2248 - H2256. [Abstract] [Full Text] [PDF]
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M. Xue, G. Hsieh, M. A. Raymond-Stintz, J. Pfeiffer, D. Roberts, S. L. Steinberg, J. M. Oliver, E. R. Prossnitz, D. S. Lidke, and B. S. Wilson Activated N-Formyl Peptide Receptor and High-Affinity IgE Receptor Occupy Common Domains for Signaling and Internalization Mol. Biol. Cell, April 1, 2007; 18(4): 1410 - 1420. [Abstract] [Full Text] [PDF]
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A. Adebiyi, G. Zhao, S. Y. Cheranov, A. Ahmed, and J. H. Jaggar Caveolin-1 abolishment attenuates the myogenic response in murine cerebral arteries Am J Physiol Heart Circ Physiol, March 1, 2007; 292(3): H1584 - H1592. [Abstract] [Full Text] [PDF]
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 W. Chen, D. B. Jump, W. J. Esselman, and J. V. Busik Inhibition of Cytokine Signaling in Human Retinal Endothelial Cells through Modification of Caveolae/Lipid Rafts by Docosahexaenoic Acid Invest. Ophthalmol. Vis. Sci., January 1, 2007; 48(1): 18 - 26. [Abstract] [Full Text] [PDF]
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 F. Peng, D. Wu, A. J. Ingram, B. Zhang, B. Gao, and J. C. Krepinsky RhoA Activation in Mesangial Cells by Mechanical Strain Depends on Caveolae and Caveolin-1 Interaction J. Am. Soc. Nephrol., January 1, 2007; 18(1): 189 - 198. [Abstract] [Full Text] [PDF]
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 F. A. Medina, C. J. de Almeida, E. Dew, J. Li, G. Bonuccelli, T. M. Williams, A. W. Cohen, R. G. Pestell, P. G. Frank, H. B. Tanowitz, et al. Caveolin-1-Deficient Mice Show Defects in Innate Immunity and Inflammatory Immune Response during Salmonella enterica Serovar Typhimurium Infection Infect. Immun., December 1, 2006; 74(12): 6665 - 6674. [Abstract] [Full Text] [PDF]
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 D. Piwnica, I. Fernandez, N. Binart, P. Touraine, P. A. Kelly, and V. Goffin A New Mechanism for Prolactin Processing into 16K PRL by Secreted Cathepsin D Mol. Endocrinol., December 1, 2006; 20(12): 3263 - 3278. [Abstract] [Full Text] [PDF]
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 A. W. Norman Vitamin D Receptor: New Assignments for an Already Busy Receptor Endocrinology, December 1, 2006; 147(12): 5542 - 5548. [Abstract] [Full Text] [PDF]
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P.-K. Fang, K. R. Solomon, L. Zhuang, M. Qi, M. McKee, M. R. Freeman, and P. C. Yelick Caveolin-1{alpha} and -1{beta} Perform Nonredundant Roles in Early Vertebrate Development Am. J. Pathol., December 1, 2006; 169(6): 2209 - 2222. [Abstract] [Full Text] [PDF]
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 F. Sotgia, H. Rui, G. Bonuccelli, I. Mercier, R. G. Pestell, and M. P. Lisanti Caveolin-1, Mammary Stem Cells, and Estrogen-Dependent Breast Cancers. Cancer Res., November 15, 2006; 66(22): 10647 - 10651. [Abstract] [Full Text] [PDF]
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I. Hunter and G. F. Nixon Spatial Compartmentalization of Tumor Necrosis Factor (TNF) Receptor 1-dependent Signaling Pathways in Human Airway Smooth Muscle Cells: LIPID RAFTS ARE ESSENTIAL FOR TNF-{alpha}-MEDIATED ACTIVATION OF RhoA BUT DISPENSABLE FOR THE ACTIVATION OF THE NF-{kappa}B AND MAPK PATHWAYS J. Biol. Chem., November 10, 2006; 281(45): 34705 - 34715. [Abstract] [Full Text] [PDF]
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T. M. Williams, F. Sotgia, H. Lee, G. Hassan, D. Di Vizio, G. Bonuccelli, F. Capozza, I. Mercier, H. Rui, R. G. Pestell, et al. Stromal and Epithelial Caveolin-1 Both Confer a Protective Effect Against Mammary Hyperplasia and Tumorigenesis: Caveolin-1 Antagonizes Cyclin D1 Function in Mammary Epithelial Cells Am. J. Pathol., November 1, 2006; 169(5): 1784 - 1801. [Abstract] [Full Text] [PDF]
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L. Liu and A. Askari beta-Subunit of cardiac Na+-K+-ATPase dictates the concentration of the functional enzyme in caveolae Am J Physiol Cell Physiol, October 1, 2006; 291(4): C569 - C578. [Abstract] [Full Text] [PDF]
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R. Gosens, G. L. Stelmack, G. Dueck, K. D. McNeill, A. Yamasaki, W. T. Gerthoffer, H. Unruh, A. S. Gounni, J. Zaagsma, and A. J Halayko Role of caveolin-1 in p42/p44 MAP kinase activation and proliferation of human airway smooth muscle Am J Physiol Lung Cell Mol Physiol, September 1, 2006; 291(3): L523 - L534. [Abstract] [Full Text] [PDF]
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K. Gaus, S. Le Lay, N. Balasubramanian, and M. A. Schwartz Integrin-mediated adhesion regulates membrane order J. Cell Biol., August 28, 2006; 174(5): 725 - 734. [Abstract] [Full Text] [PDF]
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S. Batova, J. DeWever, T. Godfraind, J.-L. Balligand, C. Dessy, and O. Feron The calcium channel blocker amlodipine promotes the unclamping of eNOS from caveolin in endothelial cells Cardiovasc Res, August 1, 2006; 71(3): 478 - 485. [Abstract] [Full Text] [PDF]
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 C. C. Felder, A. K. Dickason-Chesterfield, and S. A. Moore Cannabinoids Biology: The Search for New Therapeutic Targets Mol. Interv., June 1, 2006; 6(3): 149 - 161. [Abstract] [Full Text] [PDF]
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 M. M. Vihanto, C. Vindis, V. Djonov, D. P. Cerretti, and U. Huynh-Do Caveolin-1 is required for signaling and membrane targeting of EphB1 receptor tyrosine kinase J. Cell Sci., June 1, 2006; 119(11): 2299 - 2309. [Abstract] [Full Text] [PDF]
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W. Xu, S.-I. Yoon, P. Huang, Y. Wang, C. Chen, P. L.-G. Chong, and L.-Y. Liu-Chen Localization of the {kappa} Opioid Receptor in Lipid Rafts J. Pharmacol. Exp. Ther., June 1, 2006; 317(3): 1295 - 1306. [Abstract] [Full Text] [PDF]
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 L. M. Smith, A. Nesterova, S. C. Alley, M. Y. Torgov, and P. J. Carter Potent cytotoxicity of an auristatin-containing antibody-drug conjugate targeting melanoma cells expressing melanotransferrin/p97. Mol. Cancer Ther., June 1, 2006; 5(6): 1474 - 1482. [Abstract] [Full Text] [PDF]
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G. S. Hassan, T. M. Williams, P. G. Frank, and M. P. Lisanti Caveolin-1-deficient aortic smooth muscle cells show cell autonomous abnormalities in proliferation, migration, and endothelin-based signal transduction Am J Physiol Heart Circ Physiol, June 1, 2006; 290(6): H2393 - H2401. [Abstract] [Full Text] [PDF]
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X. Cheng and J. H. Jaggar Genetic ablation of caveolin-1 modifies Ca2+ spark coupling in murine arterial smooth muscle cells Am J Physiol Heart Circ Physiol, June 1, 2006; 290(6): H2309 - H2319. [Abstract] [Full Text] [PDF]
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 E. Sbaa, J. DeWever, P. Martinive, C. Bouzin, F. Frerart, J.-L. Balligand, C. Dessy, and O. Feron Caveolin Plays a Central Role in Endothelial Progenitor Cell Mobilization and Homing in SDF-1-Driven Postischemic Vasculogenesis Circ. Res., May 12, 2006; 98(9): 1219 - 1227. [Abstract] [Full Text] [PDF]
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V. A. Torres, J. C. Tapia, D. A. Rodriguez, M. Parraga, P. Lisboa, M. Montoya, L. Leyton, and A. F. G. Quest Caveolin-1 controls cell proliferation and cell death by suppressing expression of the inhibitor of apoptosis protein survivin J. Cell Sci., May 1, 2006; 119(9): 1812 - 1823. [Abstract] [Full Text] [PDF]
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N. Ullrich, A. Caplanusi, B. Brone, D. Hermans, E. Lariviere, B. Nilius, W. Van Driessche, and J. Eggermont Stimulation by caveolin-1 of the hypotonicity-induced release of taurine and ATP at basolateral, but not apical, membrane of Caco-2 cells Am J Physiol Cell Physiol, May 1, 2006; 290(5): C1287 - C1296. [Abstract] [Full Text] [PDF]
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C. Schwencke, R. C. Braun-Dullaeus, C. Wunderlich, and R. H. Strasser Caveolae and caveolin in transmembrane signaling: Implications for human disease Cardiovasc Res, April 1, 2006; 70(1): 42 - 49. [Abstract] [Full Text] [PDF]
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R. C. Mora, V. L. Bonilha, B.-C. Shin, J. Hu, L. Cohen-Gould, D. Bok, and E. Rodriguez-Boulan Bipolar assembly of caveolae in retinal pigment epithelium Am J Physiol Cell Physiol, March 1, 2006; 290(3): C832 - C843. [Abstract] [Full Text] [PDF]
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S. Calaghan and E. White Caveolae modulate excitation-contraction coupling and {beta}2-adrenergic signalling in adult rat ventricular myocytes Cardiovasc Res, March 1, 2006; 69(4): 816 - 824. [Abstract] [Full Text] [PDF]
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A. F. El-Yazbi, W. J. Cho, G. Boddy, R. Schulz, and E. E. Daniel Impact of caveolin-1 knockout on NANC relaxation in circular muscles of the mouse small intestine compared with longitudinal muscles Am J Physiol Gastrointest Liver Physiol, February 1, 2006; 290(2): G394 - G403. [Abstract] [Full Text] [PDF]
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D. Mehta and A. B. Malik Signaling Mechanisms Regulating Endothelial Permeability Physiol Rev, January 1, 2006; 86(1): 279 - 367. [Abstract] [Full Text] [PDF]
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F. Sotgia, T. M. Williams, W. Schubert, F. Medina, C. Minetti, R. G. Pestell, and M. P. Lisanti Caveolin-1 Deficiency (-/-) Conveys Premalignant Alterations in Mammary Epithelia, with Abnormal Lumen Formation, Growth Factor Independence, and Cell Invasiveness Am. J. Pathol., January 1, 2006; 168(1): 292 - 309. [Abstract] [Full Text] [PDF]
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N. M. Urs, K. T. Jones, P. D. Salo, J. E. Severin, J. Trejo, and H. Radhakrishna A requirement for membrane cholesterol in the {beta}-arrestin- and clathrin-dependent endocytosis of LPA1 lysophosphatidic acid receptors J. Cell Sci., November 15, 2005; 118(22): 5291 - 5304. [Abstract] [Full Text] [PDF]
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C. Schwencke, A. Schmeisser, C. Walter, R. Wachter, S. Pannach, B. Weck, R. C. Braun-Dullaeus, M. Kasper, and R. H. Strasser Decreased caveolin-1 in atheroma: Loss of antiproliferative control of vascular smooth muscle cells in atherosclerosis Cardiovasc Res, October 1, 2005; 68(1): 128 - 135. [Abstract] [Full Text] [PDF]
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K. Ohnuma, T. Yamochi, M. Uchiyama, K. Nishibashi, S. Iwata, O. Hosono, H. Kawasaki, H. Tanaka, N. H. Dang, and C. Morimoto CD26 Mediates Dissociation of Tollip and IRAK-1 from Caveolin-1 and Induces Upregulation of CD86 on Antigen-Presenting Cells Mol. Cell. Biol., September 1, 2005; 25(17): 7743 - 7757. [Abstract] [Full Text] [PDF]
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) or up-regulation (+) of the indicated gene. DMD, Duchenne
muscular dystrophy; BMD, Becker muscular dystrophy; CMD, congenital
muscular dystrophy; LGMDs, multiple forms of limb-girdle muscular
dystrophy; LGMD-1C, limb-girdle muscular dystrophy, type 1C; nNOS,
neuronal form of nitric oxide synthase; CBD, caveolin binding motif.
Modified from Galbiati et al., 2001c
) and Cav-1 null (KO; 
) mice.
Points represent mean ± S.E.M of 5 (KO) or 6 (WT) rings from 3 mice each. 
