(Received for publication, November 29, 1995; and in revised form, January 31, 1996)
From the
Caveolae are plasma membrane specializations that have been
implicated in signal transduction. Caveolin, a 21-24-kDa integral
membrane protein, is a principal structural component of caveolae
membranes in vivo. G protein subunits are concentrated
in purified preparations of caveolae membranes, and caveolin interacts
directly with multiple G protein
subunits, including
G
, G
, and G
. Mutational or
pharmacologic activation of G
subunits prevents the
interaction of caveolin with G proteins, indicating that inactive G
subunits preferentially interact with caveolin.
Here, we show that caveolin interacts with another well characterized
signal transducer, Ras. Using a detergent-free procedure for
purification of caveolin-rich membrane domains and a polyhistidine
tagged form of caveolin, we find that Ras and other classes of
lipid-modified signaling molecules co-fractionate and co-elute with
caveolin. The association of Ras with caveolin was further evaluated
using two distinct in vitro binding assays. Wild-type H-Ras
interacted with glutathione S-transferase (GST)-caveolin
fusion proteins but not with GST alone. Using a battery of GST fusion
proteins encoding distinct regions of caveolin, Ras binding activity
was localized to a 41amino acid membrane proximal region of the
cytosolic N-terminal domain of caveolin. In addition, reconstituted
caveolin-rich membranes (prepared with purified recombinant caveolin
and purified lipids) interacted with a soluble form of wild-type H-Ras
but failed to interact with mutationally activated soluble H-Ras
(G12V). Thus, a single amino acid change (G12V) that constitutively
activates Ras prevents or destabilizes this interaction. These results
clearly indicate that (i) caveolin is sufficient to recruit soluble Ras
onto lipid membranes and (ii) membrane-bound caveolin preferentially
interacts with inactive Ras proteins. In direct support of these in
vitro studies, we also show that recombinant overexpression of
caveolin in intact cells is sufficient to functionally recruit a
nonfarnesylated mutant of Ras (C186S) onto membranes, overcoming the
normal requirement for lipid modification of Ras. Taken together, these
observations suggest that caveolin may function as a scaffolding
protein to localize or sequester certain caveolin-interacting proteins,
such as wild-type Ras, within caveolin-rich microdomains of the plasma
membrane.
Caveolae are small bulb-shaped invaginations of the plasma membrane(1) . However, they may also assume different stages of invagination and may be present singly or in bunches like clusters of grapes(1, 2) . They represent a subcompartment or microdomain of the plasma membrane and are most abundant in certain cell types, including fibroblasts, adipocytes, endothelial cells, type I pneumocytes, epithelial cells, and smooth muscle cells (reviewed in (3) and (4) )). For example, in adipocytes caveolae may occupy up to 20% of the total plasma membrane surface area(5) .
Functionally, caveolae were first implicated in cellular transport processes (6, 7) and more recently in signal transduction related events(3, 8) . Membrane preparations enriched in caveolae contain (i) lipid-modified signaling molecules (such as heterotrimeric G proteins and Src-family tyrosine kinases)(8, 9, 10, 11, 12, 13) and (ii) caveolin, a transformation-dependent v-Src substrate(14, 15) . A ``caveolae signaling hypothesis'' states that caveolar localization of certain lipid modified signaling molecules could provide a compartmental basis for integrating certain transmembrane signaling events(3, 8) .
Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes and serves as a marker protein for the organelle(15) . At steady-state, greater than 90% of caveolin is both morphologically and biochemically localized within plasma membrane caveolae(15, 16, 17) . Caveolin is a 178-amino acid protein that contains three domains: a 101-amino acid N-terminal region; a 33-amino acid membrane-spanning segment; and a 44-amino acid C-terminal domain (18, 19, 20, 21) . Both the N- and C-terminal domains of caveolin face the cytosol, suggesting that its unusual membrane spanning segment may form a hairpin loop within the membrane(16, 22, 23) . In accordance with this cytoplasmic membrane topology, caveolin remains inaccessible to biotinylation probes that have been used to efficiently label proteins that face the extracellular environment(24) .
Caveolin
appears to be important for the formation of caveolae membranes.
Caveolin mRNA and protein expression levels are highest in cell types
that contain numerous caveolae, i.e. adipocytes, endothelial
cells, smooth muscle cells, and fibroblasts(9, 19) .
Caveolin expression levels directly correlate with the morphological
appearance of caveolae: (i) caveolin and caveolae are both induced
10-25-fold during the differentiation of 3T3-L1 fibroblasts
to the adipocyte form(25, 26) , and (ii) caveolin
levels are dramatically reduced and caveolae are morphologically absent
in NIH 3T3 cells transformed by various activated oncogenes
(v-abl, activated Ras and others)(27) . Furthermore,
recombinant expression of caveolin in caveolin-negative cell lines
results in the correct targeting of caveolin to caveolae-enriched
membrane fractions (23) and results in the de novo formation of caveolae(28) . These results indicate that
caveolin represents an important structural protein for directing the
formation of caveolae membranes.
Several structural properties of
caveolin are also consistent with a role for caveolin in orchestrating
the formation of caveolae microdomains. Caveolin exists as a high
molecular mass homo-oligomer of 350 kDa with
14-16
caveolin monomers per oligomer(24, 29) . These
caveolin homo-oligomers have the capacity to self-associate in
vitro into larger structures that resemble caveolae(24) .
In these structures, caveolin homo-oligomers exhibit side-by-side
packing, an indication of how caveolae membranes are
organized(24) . This would provide a ``caveolin platform
or scaffold'' for the recruitment of caveolin-interacting proteins
to caveolae microdomains of the plasma membrane(24) .
Recently, G protein subunits have been identified as
caveolin-interacting proteins(30) . G protein
subunits
are highly concentrated in preparations of caveolae membranes purified
from diverse
sources(8, 9, 10, 13, 30) .
Caveolin interacts directly with multiple G protein
subunits,
including G
, G
, and G
, and can
functionally suppress their basal activity by inhibiting GDP-GTP
exchange(30) . This interaction is abolished by mutational
activation of the G
subunit and does not require
co-expression of G
subunits(30) . Based on
the observation that caveolin is a cytoplasmically oriented integral
membrane protein and G
subunits cycle on and off the
membrane, it has been proposed that caveolin could function as a
scaffolding protein to recruit G protein
subunits to
caveolin-rich areas of the plasma membrane(24, 30) .
This hypothesis is further supported by the observation that
reconstituted caveolin-containing membranes (prepared with bacterially
expressed recombinant caveolin and purified lipids) are capable of
recruiting a soluble recombinant form of
G
(31) . We now present evidence that Ras,
another class of signal transducing GTPase, co-purifies with caveolin
and interacts directly with caveolin in a regulated fashion.
The corresponding constructs were transiently transfected into COS-7-cells grown in 100-mm dishes by the DEAE-dextran method(35) . 48 h post-transfection, cells were scraped into lysis buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100). Recombinant expression was analyzed by SDS-PAGE (15% acrylamide) followed by Western blotting with mAb 9E10. MDCK cells were stably transfected using a modification of the calcium-phosphate precipitation procedure(23, 34) . After selection in medium supplemented with 400 µg/ml hygromycin B, resistant colonies were picked by trypsinization using cloning rings. Individual clones were screened by immunofluorescence for recombinant expression of caveolin. Epitope-tagged forms of caveolin expressed in MDCK cells were detected using monoclonal antibody, 9E10, that recognizes the myc epitope (EQKLISEEDLN).
Figure 1:
Construction and
expression of a polyhistidine tagged form of caveolin. A,
schematic diagram summarizing the construction of recombinant caveolins
for expression in mammalian cells. A myc epitope tag was
placed at its C terminus (cav-myc) and a polyhistidine tag was
then placed following the myc tag
(cav-myc-H) for affinity purification by
Ni-NTA-agarose chromatography. B, expression of cav-myc and cav-myc-H
in COS-7 cells. Left,
COS-7 cells were transiently transfected with pCB7 vector alone and
pCB7 containing cav-myc or cav-myc-H
constructed as outlined in A. Note that
cav-myc-H
migrates at
29 kDa and is slightly
larger than cav-myc because it contains the polyhistidine tag. Right, COS-7 cell lysates containing equivalent amounts of
cav-myc or cav-myc-H
were incubated with
Ni-NTA-agarose, washed, and specifically eluted with imidazole. The
eluate was subjected to immunoblot analysis with mAb 9E10 that
recognizes the myc epitope (EQKLISEEDLN). Only
cav-myc-H
but not cav-myc was
specifically retained and eluted on
Ni-NTA-agarose.
To determine the feasibility of this
approach, both epitope-tagged forms of caveolin (cav-myc and
cav-myc-H) were transiently expressed in COS-7
cells and subjected to affinity purification on
Ni
-NTA resin. The imidazole eluate was then subjected
to immunoblot analysis with mAb 9E10 that recognizes the myc epitope. Only the cav-myc-H
bound and was
specifically eluted; no binding was observed with cav-myc lacking the polyhistidine tag (Fig. 1B). These
studies directly demonstrate the validity of this approach for
purification of caveolin and caveolin-associated proteins from
mammalian cells.
Figure 2:
A polyhistidine tagged form of caveolin
co-fractionates with endogenous caveolin using a Triton-based
fractionation scheme. MDCK cells stably expressing
cav-myc-H were derived and subjected to
subcellular fractionation after homogenization in buffer containing 1%
Triton X-100, as described
previously(8, 9, 23, 30, 33) .
The distribution of total cellular protein,
cav-myc-H
, and endogenous caveolin are shown.
Fractions were collected from the top, separated by SDS-PAGE (15%
acrylamide), and analyzed by Ponceau S staining/immunoblotting.
Immunoblot analysis was carried out with mAb 9E10 to selectively detect
cav-myc-H
and with anti-caveolin IgG (mAb 2297) to
detect both cav-myc-H
and endogenous caveolin.
Note that both
- and
-isoforms of endogenous caveolin are
detected, and they co-fractionate with cav-myc-H
.
The generation of
-caveolin results from an alternate translation
initiation site at amino acid 32 within the full-length caveolin
cDNA(23) . We and others have previously shown using this
fractionation scheme that caveolin (fractions 5-6) is purified
2000-fold relative to total MDCK cell
lysates(8, 23, 30, 33) . In
addition, caveolin is separated from greater than 99.95% of total MDCK
cellular proteins and compartment-specific markers for ER, Golgi,
lysosomes, mitochondria, and noncaveolar plasma membrane, which remain
in fractions 9-13 of these bottom-loaded sucrose density
gradients (8, 9, 12, 17, 23, 25, 30, 33).
Recently, it has been suggested that the inclusion of detergent in
the initial homogenization step results in the loss of resident
prenylated caveolin-associated proteins, such as G subunits (10) . To preserve these interactions, we
developed a detergent-free method for the purification of caveolin-rich
membrane domains. This slightly modified scheme replaces the detergent
Triton X-100 with sodium carbonate. Sodium carbonate extraction is
routinely used to determine if proteins are firmly attached to
membranes and caveolin is not solubilized by sodium
carbonate(8, 16, 39) . Fig. 3shows
that using this modified scheme endogenous caveolin and
cav-myc-H
were also recovered almost
quantitatively in fractions 5 and 6 while excluding most cellular
proteins. In addition, caveolin was separated from the GPI-linked
plasma membrane marker, carbonic anhydrase IV (Fig. 3). This is
consistent with recent observations that GPI-linked proteins are not
concentrated directly within caveolae but may reside in close proximity
to the ``neck regions'' of caveolae within intact
cells(40) .
Figure 3:
Distribution of total cellular proteins,
caveolin, and a GPI-linked protein using a carbonate-based
fractionation scheme. MDCK cells, stably expressing
cav-myc-H, were subjected to subcellular
fractionation after homogenization in a buffer containing sodium
carbonate (see under ``Experimental Procedures''). This
fractionation scheme is essentially the same as described in Fig. 2, except Triton X-100 is replaced by sodium carbonate.
Using this modified detergent-free scheme, (i) caveolin (fractions
5-6) is separated from most cellular proteins (fractions
8-12); (ii) endogenous caveolin and cav-myc-H
co-fractionate (fractions 5-6); and (iii) caveolin is
quantitatively separated from a GPI-linked plasma membrane marker (CA
IV, carbonic anhydrase IV; fractions 9-12). Endogenous caveolin
and cav-myc-H
were detected by immunoblot analysis
with anti-caveolin IgG (mAb 2297). They can be distinguished by
differences in their migration in SDS-PAGE gels (15% acrylamide);
cav-myc-H
migrates at
29 kDa, whereas
endogenous caveolin (
- and
-isoforms) migrates at 21 and 24
kDa.
Caveolin has been found associated with a number
of cytoplasmically oriented lipid-modified signaling molecules using
Triton-based methods (reviewed in Refs. 3, 4, and 33). These
caveolin-associated proteins include G protein subunits and
Src-family tyrosine
kinases(8, 9, 10, 11, 12, 30, 40) .
Using this detergent-free method, caveolin also co-fractionated with
these signaling molecules (G
, c-Src, Fig. 4A; Rap 1, Lyn, Fyn (not shown)). In addition,
prenylated proteins such as G
subunits and Ras
co-fractionated with caveolin, suggesting that prenylated proteins are
retained using this carbonate-based method (Fig. 4A).
This is the first demonstration that Ras is associated with caveolin,
because previous detergent-based methods showed that Ras did not
co-fractionate with caveolin(9) . As such, other prenylated
proteins may be associated with caveolin and caveolae in intact cells.
Figure 4:
Association of caveolin with Ras and other
lipid-modified signaling molecules. A, co-fractionation with
caveolin. Gradient fractions depicted in Fig. 3were subjected
to immunoblot analysis with specific antibodies directed against
signaling molecules that we and others have shown previously to
co-fractionate with caveolin using detergent-based
methods(8, 9, 10, 11, 12, 30, 40) .
In addition, we also examined the distribution of Ras. Our results
indicate that Ras, G, G
, and
c-Src all co-fractionate with caveolin when Triton X-100 is replaced
with sodium carbonate. Other G proteins (G
),
Src-family members (Lyn, Fyn), and Ras-related GTPases (Rap1) also
co-fractionated with caveolin in this detergent-free system (data not
shown). To detect G
subunits, an antibody directed
against G
was utilized as G
is always
tightly attached to G
, which is
lipid-modified(47) . B, co-elution with a
polyhistidine tagged form of caveolin. Fraction 5 of detergent-free
sucrose density gradients depicted in A was incubated with
Ni-NTA-agarose, washed (4
), and specifically eluted with
imidazole. Washes 3 and 4 and the eluate were then subjected to
immunoblot analysis. Note that Ras and other signaling molecules were
retained on Ni-NTA-agarose and co-elute with
cav-myc-H
. In addition to co-fractionation
depicted in A, co-elution provides a further indication that
caveolin and these other molecules are associated as a protein complex.
Cav-myc-H
was detected with mAb 9E10 that
recognizes the myc epitope
(EQKLISEEDLN).
In addition to co-fractionating with caveolin, Ras,
G, G
, and c-Src were all
retained on Ni
-NTA resin and were specifically
co-eluted with cav-myc-H
(Fig. 4B). This provides an indication that these
molecules are present within the same protein complex as caveolin and
could even interact directly with caveolin. In support of this notion,
caveolin interacts directly with G protein
subunits
(G
, G
, G
) as shown using a number
of different approaches, and this direct interaction is specifically
regulated by the activation state of the G
subunits(30) . Inactive GDP-bound G
subunits preferentially interact with caveolin(30) .
Figure 5: Interaction of Ras with GST-caveolin fusion proteins. A, schematic diagram summarizing each GST-caveolin fusion protein relative to a complete caveolin molecule. Numbers at end points reflect amino acid positions within caveolin. GST-caveolin fusion proteins were constructed and characterized, as we described previously(23, 24, 30) . B, upper panel, Ras interacts with full-length caveolin. GST alone or a GST-fusion protein containing the complete caveolin molecule (FL(1-178)) were incubated with detergent extracts of insect cells recombinantly expressing wild-type H-Ras. After extensive washing, GST fusion proteins were eluted from the beads with reduced glutathione and subjected to SDS-PAGE (15% acrylamide). Bound H-Ras was visualized by immunoblotting with a mAb directed against Ras. Wild-type H-Ras bound to full-length caveolin but not to GST alone. Equivalent amounts of GST and GST-FL-caveolin were used as the substrate for binding. Note that three forms of baculovirus-expresed H-Ras can be separated by 15% acrylamide gels. These correspond to different stages in the C-terminal farnesylation of Ras: u, unmodified; i, intermediate with cleaved CAAX sequence; m, mature farnesylated form(32) . As the unmodified form of H-Ras also bound to caveolin, this provides an indication that lipid modification is not absolutely required for this interaction. Lower panel, defining a region of caveolin that interacts with Ras. Using a number of GST-caveolin fusion proteins corresponding to the N-terminal (1-61, 1-81, and 61-101) or C-terminal(135-178) domains of caveolin, we systematically identified a 41-amino acid region of caveolin (residues 61-101) that is sufficient to interact with Ras. After SDS-PAGE (10% acrylamide) and transfer to nitrocellulose, bound H-Ras was visualized by immunoblotting with a mAb directed against Ras. Equivalent amounts of GST and GST-caveolin fusion proteins were utilized as the substrate for binding.
To localize this Ras binding activity to a given region of the
caveolin molecule, GST-fusion proteins encoding portions of the N- or
C-terminal domains of caveolin were used as the substrate for Ras
binding. Only the fusion protein encoding caveolin residues
61-101 retained Ras binding activity (Fig. 5B, lower panel); this 41-amino acid stretch of caveolin residues
is located within a membrane proximal region of the cytosolic
N-terminal domain of caveolin. In addition, because a fusion encoding
caveolin residues 1-81 did not possess Ras binding activity, this
may suggest that caveolin residues 82-101 are especially critical
for interaction with Ras. Similarly, this region of caveolin has
recently been implicated in the binding and regulation of G protein
subunits(30) .
Reconstituted lipid membranes containing recombinant full-length
caveolin were briefly incubated with recombinant soluble GST-H-Ras
proteins. To stringently remove unbound GST-H-Ras proteins,
caveolin-containing lipid membranes were then reisolated by flotation
in sucrose density gradients. Thus, bound GST-H-Ras proteins must
attain the buoyancy of lipid membranes in this system. Fig. 6shows that a soluble form of wild-type Ras
(GST-H-Ras) preferentially bound to reconstituted
caveolin-rich membranes, but little or no binding was observed with a
mutationally activated form of Ras (GST-H-Ras
). These
results suggest that caveolin preferentially interacts with the
inactive form of Ras. Thus, a single amino acid change (G12V) that
constitutively activates Ras prevents this interaction. Similarly, a
single amino acid change (Q227L) that constitutively activates
G
also prevents its interaction with
caveolin(30) .
Figure 6:
Recruitment of GST-H-Ras onto
reconstituted caveolin-rich membranes is regulated by the activation
state of Ras. A, recombinant cav-myc-H was produced in E. coli and purified by Ni-NTA-agarose
chromatography. Recombinant cav-myc-H
was
biotinylated in vitro to assess its purity; it appeared as a
single band by streptavidin blotting, as described
previously(31) . In addition, the same band was immunoreactive
with anti-caveolin IgG (mAb 2234). B, interaction of GST-H-Ras
proteins with reconstituted caveolin-rich membranes. Purified
recombinant cav-myc-H
depicted in A was
incorporated into lipid membranes as described under
``Experimental Procedures.'' Reconstituted caveolin-rich
membranes were then incubated in solution with equivalent amounts of
purified recombinant GST-H-Ras
or GST-H-Ras
(see left panel). To stringently remove soluble unbound
GST-H-Ras proteins, membranes were then isolated by reflotation in
sucrose density gradients. After reflotation, fractions 5-6 of
each gradient (corresponding to the light-scattering band at the
5-30% sucrose interface) were collected, pooled, and subjected to
immunoblot analysis with anti-Ras IgG. Note that the binding of soluble
GST-H-Ras to reconstituted caveolin-rich membranes is dependent on the
activation state of Ras, because little or no binding was observed with
GST-H-Ras
(see right panel). GST-H-Ras proteins
migrate at
43 kDa as expected, because they should have the
cumulative molecular mass of GST (
25-26 kDa) plus H-Ras
(
20 kDa).
These results indicate that caveolin is
sufficient to recruit GST-H-Ras onto lipid membranes. Because H-Ras
normally undergoes farnesylation when expressed in mammalian
cells(41, 42) , our results indicate that these lipid
modifications, which do not occur in bacteria, are not absolutely
necessary for the caveolin-dependent recruitment of H-Ras onto
membranes. Similarly, lipid modification of G subunits
is not absolutely required for their interaction with caveolin in
vitro(31) . However, such lipid modifications may still
serve to critically modulate these interactions in vivo by
targeting G
subunits and Ras to the plasma membrane.
For this purpose, we transiently co-expressed
wild-type Ras (WT) and the nonfarnesylated CAAX mutant (C186S)
in 293T cells and subjected these cells to fractionation. Wild-type Ras
(WT) and the nonfarnesylated CAAX mutant (C186S) are easily
distinguished by differences in their apparent migration in SDS-PAGE
gels. Conversion of Cys to Ser results in a well
characterized nonprocessed CAAX mutant that is not
farnesylated and therefore migrates with a slightly higher molecular
mass, as shown previously (Ref.. 36; See also Fig. 7A).
Figure 7: Caveolin-dependent recruitment of nonfarnesylated Ras onto membranes in an intact cell system. A, expression of c-H-Ras in 293T fibroblastic cells. Note that wild-type (WT) and the nonfarnesylated CAAX mutant (C186S) of c-H-Ras can be distinguished by their difference in apparent migration in SDS-PAGE gels. B, H-Ras (WT) and H-Ras (C186S) were co-expressed in 293T cells and subjected to detergent-free subcellular fractionation. Upper panel (minus sign), without co-expression of caveolin. Lower panel (plus sign), with co-expression of caveolin. Note that co-expression with caveolin results in the targeting of the nonfarnesylated CAAX mutant (C186S) to the caveolae-enriched membrane fraction (see arrow). Caveolin also migrated to the caveolae-enriched membrane fraction (not shown). Recombinant expression of Ras proteins was visualized by immunoblotting with anti-Ras antibodies.
Fig. 7B (upper panel) shows that although wild-type Ras (WT) was recovered within the caveolae-enriched membrane fraction, the nonfarnesylated CAAX mutant (C186S) was quantitatively excluded. Thus, it appears that farnesylation of Ras is normally required or greatly facilitates its caveolar localization. However, this requirement for lipid modification of Ras could be overcome by recombinant overexpression of caveolin (Fig. 7B, lower panel); both wild-type Ras (WT) and the nonfarnesylated CAAX mutant (C186S) were quantitatively recovered within the caveolae fraction when co-expressed with caveolin. Thus, it appears that caveolin can act as a scaffolding protein to functionally recruit nonfarnesylated Ras onto membranes both in vitro and in intact cells. Lipid modification may serve to bring Ras to the plasma membrane, facilitating its chance for interaction with caveolin.
Ras proteins are membrane-associated signal transducers that
bind and hydrolyze GTP(45, 46) . Mutated Ras proteins
have been detected in many human tumors. These mutations, such as G12V,
favor the generation of the active GTP-bound conformation of
Ras(45, 46) . In addition, lipid modification of Ras
by prenylation (in this case farnesylation) is required for its
attachment to membranes and its transforming
activity(43, 44) . G subunits
also undergo a form of prenylation(47) .
Given the central role of Ras proteins in transmembrane signaling and their strong association with cellular transformation, a major focus of research for over a decade has been the identification of factors that interact with Ras. This search has uncovered a number of cytosolic or soluble Ras-interacting proteins(48, 49) . These include nucleotide exchange factors such as SOS that stabilize Ras in the nucleotide-free state, thereby allowing Ras to bind GTP(50, 51, 52, 53) . Other additional factors, such as Grb-2, may act as a bridge between SOS and activated receptors of the tyrosine kinase family(54) . However, it is not yet known whether Ras interacts directly with any integral membrane proteins to stabilize its localization to the cytosolic face of the plasma membrane. The existence of such integral membrane co-factors or scaffolding proteins has been postulated because lipid modifications alone, such as acylation and prenylation, do not provide sufficient energy for stable membrane attachment(55) .
Our current studies indicate that Ras interacts directly with caveolin, an integral membrane component of caveolae membranes. Caveolae represent a subcompartment of the plasma membrane that has been previously implicated in signal transduction, including G protein coupled signaling events(8, 9, 10, 11, 12, 13, 56) . Here, we have shown that: (i) Ras and other classes of lipid-modified signaling molecules co-purify with caveolin in the absence of detergent; (ii) Ras interacts with a discrete region of the cytosolic N-terminal domain of caveolin; and (iii) caveolin can functionally recruit soluble Ras onto reconstituited lipid membranes in a regulated fashion. No binding of Ras to caveolin was observed when Ras was mutationally activated by a single amino acid change (G12V). Caveolin represents the first integral membrane protein identified thus far that directly interacts with Ras; previously, all other identified Ras-interacting proteins have been cytosolic molecules.
For these studies, we developed a novel detergent-free method for purifying caveolin-rich membrane domains from tissue cultured cells. Using this scheme, caveolin co-purifies with Ras and other lipid-modified signaling molecules, such as heterotrimeric G protein subunits and Src-like kinases. Ras and these other lipid-modified signaling molecules appear to be present within the same protein-lipid complexes as caveolin, as shown using a His-tagged form of caveolin recombinantly expressed in MDCK cells. Thus, caveolin and Ras must reside within close proximity in these specific microdomains of the plasma membranes of intact cells. However, other protein co-factors within caveolae or lipid modifications (farnesylation) may serve to hold or target Ras to caveolin-rich areas of the plasma membrane. This is the case for Src-like kinases, because myristoylation and palmitoylation both facilitate or are required for the correct localization of these tyrosine kinases within caveolae membranes (Refs. 11 and 12; reviewed in (3) and (4) ).
Recently, a report appeared
describing another method for detergent-free purification of caveolae
from tissue cultured cells (57) . These investigators showed
that caveolin co-purifies with multiple heterotrimeric G protein
subunits, including prenylated G subunits. They
confirm that prenylated proteins, such as G
subunits, may be removed from caveolae by initial homogenization
in Triton X-100. However, these authors did not examine whether Ras was
included in their preparations, nor did they express a His-tagged form
of caveolin to show that these proteins are actually present within the
same protein-lipid complexes. In addition, they only recover
1-5% of total caveolin in their preparations, whereas we
routinely recover
90-95% of total caveolin in fractions 5
and 6 of our sucrose gradients. Thus, the method described here has
several clear advantages.
What are the functional consequences of
the interaction of Ras with caveolin? One possibility is that caveolin
may serve as a plasma membrane platform or scaffolding protein to
sequester caveolin-interacting signaling molecules, such as Ras, within
caveolae membranes. First, lipid modification of Ras could serve to
increase its intrinsic affinity for the plasma membrane or other
cellular membranes. Second, the interaction of Ras with caveolin could
function to localize or sequester Ras within caveolin-rich areas of the
plasma membrane. This possibility is directly supported by: (i) in
vitro reconstitution experiments employing recombinant caveolin
inserted into lipid membranes and purified soluble Ras proteins (Fig. 6) and (ii) the observation that recombinant
overexpression of caveolin is sufficient to recruit a nonfarnesylated
form of Ras onto membranes in an intact cell system (Fig. 7).
Alternatively, caveolin may regulate the activation state of Ras,
either positively or negatively. However, our preliminary experiments
employing caveolin-derived polypeptides and recombinant purified
caveolin do not demonstrate an effect of caveolin on either GDP-GTP
exchange or GTPase activities of purified Ras proteins. ()
Perhaps surprisingly, the caveolin region we have
defined here that interacts with inactive Ras corresponds to the same
or a similar caveolin region we have previously identified that
interacts with inactive G subunits ((30) ; See Fig. 8). Thus, this caveolin region can interact specifically
with the inactive forms of two distinct classes of GTPases, both of
which are involved in signal transduction and both of which co-purify
with caveolin-rich membrane domains. As such, this caveolin domain
could provide a general mechanism for concentrating inactive
caveolin-interacting signaling molecules within caveolae microdomains
of the plasma membrane. In line with this proposal, inactive protein
kinase C
is also concentrated within purified caveolae
membranes, whereas activated protein kinase C
is
specifically excluded(58) .
Figure 8:
Caveolin: structure-function and domain
mapping. The overall domain organization of caveolin is shown at the top. Functional properties of caveolin are listed at left, and caveolin residues participating in these functions
are as indicated. These properties include the structure of caveolin
isoforms(23) , mAb epitopes(23) , G and
Ras binding activities ( (30) and this report), the caveolin
homo-oligomerization domain(24) , palmitoylation
sites(22) , and the Src tyrosine phosphorylation
site(59) . Note that differential serine and tyrosine
phosphorylation of caveolin isoforms is indicated at right(25, 59) .