©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Co-purification and Direct Interaction of Ras with Caveolin, an Integral Membrane Protein of Caveolae Microdomains
DETERGENT-FREE PURIFICATION OF CAVEOLAE MEMBRANES (*)

(Received for publication, November 29, 1995; and in revised form, January 31, 1996)

Kenneth S. Song (1) Shengwen Li (1) Takashi Okamoto (2) Lawrence A. Quilliam (3) Massimo Sargiacomo (1)(§) Michael P. Lisanti (1)(¶)

From the  (1)Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142-1479, the (2)Shriners Hospitals for Crippled Children, Massachusetts General Hospital, Department of Anesthesia, Harvard Medical School, Boston, Massachusetts 02114, and the (3)Indiana University School of Medicine, Department of Biochemistry and Molecular Biology, Indianapolis, Indiana 46202-5122

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 alpha subunits are concentrated in purified preparations of caveolae membranes, and caveolin interacts directly with multiple G protein alpha subunits, including G(s), G(o), 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.


INTRODUCTION

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 alpha subunits have been identified as caveolin-interacting proteins(30) . G protein alpha 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 alpha subunits, including G(s), G(o), 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 alpha 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.


EXPERIMENTAL PROCEDURES

Materials

The cDNA for canine caveolin was obtained as we described previously(8) . Ni-NTA-agarose (^1)for purification of polyhistidine tagged proteins was from Qiagen. Antibodies and their sources were as follows: anti-caveolin IgG (mAb 2297; gift of Dr. John R. Glenney, Transduction Labs); anti-myc epitope IgG (mAb 9E10; Santa Cruz Biotech); anti-carbonic anhydrase IV (CAIV; gift of Dr. William S. Sly, St. Louis University); anti-G (Dupont NEN); anti-G (Transduction Labs); anti-Src (Oncogene Sciences); and anti-Ras (Transduction Labs). A recombinant baculovirus vector encoding H-Ras was as described(32) . Plasmids encoding GST-H-Ras and GST-H-Ras were a gift from Drs. M. White and M. Wigler (Cold Spring Harbor Laboratory). A variety of other reagents were purchased commercially: fetal bovine serum (JRH Biosciences); hygromycin B (Calbiochem); and pre-stained protein markers (Life Technologies, Inc.).

Cell Culture

MDCK cells were propagated as described(33) . The expression level of a given transfected antigen was increased by an overnight incubation with normal medium containing 10 mM sodium butyrate(34) .

Construction and Expression of Polyhistidine Tagged Caveolin

In order to purify caveolin after expression in mammalian cells, we incorporated the polyhistidine tag into the C terminus of the myc-tagged canine caveolin cDNA using PCR primers (caveolin-myc-H(7)). The C-terminally myc-tagged form of caveolin (caveolin-myc) used as the template was as we described previously(23) . The final construction was subcloned into the multiple cloning site (HindIII-BamHI) of the vector pCB7 (containing the hygro^R marker; gift of Dr. James E. Casanova, Massachusetts General Hospital) for transient expression in COS-7 cells or stable expression in MDCK cells.

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).

Detergent-free Purification of Caveolin-rich Membrane Fractions

MDCK cells grown to confluence in 150-mm dishes were used to prepare caveolin-enriched membrane fractions, essentially as we have described previously(8, 9, 23, 25, 30, 33) . However, two specific modifications were introduced to allow the purification of caveolin-rich domains without the use of detergent. Triton X-100 was replaced with sodium carbonate buffer, and a sonication step was introduced to more finely disrupt cellular membranes. After two washes with ice-cold phosphate-buffered saline, MDCK cells (two confluent 150-mm dishes) were scraped into 2 ml of 500 mM sodium carbonate, pH 11.0. Homogenization was carried out sequentially in the following order using a loose-fitting Dounce homogenizer (10 strokes), a Polytron tissue grinder (three 10-s bursts; Kinematica GmbH, Brinkmann Instruments, Westbury, NY), and a sonicator (three 20-s bursts; Branson Sonifier 250, Branson Ultrasonic Corp., Danbury, CT). The homogenate was then adjusted to 45% sucrose by the addition of 2 ml of 90% sucrose prepared in MBS (25 mM Mes, pH 6.5, 0.15 M NaCl) and placed at the bottom of an ultracentrifuge tube. A 5-35% discontinuous sucrose gradient was formed above (4 ml of 5% sucrose/4ml of 35% sucrose; both in MBS containing 250 mM sodium carbonate) and centrifuged at 39,000 rpms for 16-20 h in an SW41 rotor (Beckman Instruments, Palo Alto, CA). A light-scattering band confined to the 5-35% sucrose interface was observed that contained caveolin but excluded most other cellular proteins.

Immunoblotting of Gradient Fractions

From the top of each gradient, 1-ml gradient fractions were collected to yield a total of 13 fractions. As shown previously, caveolin migrates mainly in fractions 5 and 6 of these sucrose density gradients(9, 23, 25, 30, 33) . Gradient fractions were separated by SDS-PAGE (15% acrylamide) and transferred to nitrocellulose. After transfer, nitrocellulose sheets were stained with Ponceau S to visualize protein bands and subjected to immunoblotting. For immunoblotting, incubation conditions were as described by the manufacturer (Promega, Amersham Corp.), except we supplemented our blocking solution with both 1% bovine serum albumin and 1% nonfat dry milk (Carnation).

Affinity purification of caveolin-myc-H(7) expressed in mammalian cells using Ni-NTA-agarose

The MDCK cell gradient fraction most enriched in caveolin (fraction 5) was collected and adjusted to pH 8.0 using a stock solution of concentrated Mes buffer. Ni-NTA-agarose (200 µl) was then pre-equilibrated with Tris-buffered saline (TBS, 10 mM Tris, pH 8.0, 0.15 M NaCl). One-half of fraction 5 was added to the resin and incubated rotating for 6 h at 4 °C. After binding, beads were allowed to gently settle by gravity (5 min on ice) and washed extensively (four times, 5 min each; twice with TBS and twice with TBS plus 30 mM imidazole). After washing, bound proteins were specifically eluted with TBS containing 200 mM imidazole, as per the manufacturer's instructions (Qiagen). Washes and eluates were then subjected to immunoblot analysis. COS-7 cell lysates were also processed in a similar manner, except COS-7 cells were directly lysed in TBS containing 1% Triton before binding and elution experiments. About 10% of a COS-7 cell lysate was used for binding and elution from Ni-NTA-agarose.

Interaction of Baculovirus-expressed H-Ras with GST-Caveolin Fusion Proteins

GST-caveolin fusion proteins were constructed and purified by affinity chromatography using glutathione-agarose, as described(30) . The interaction of GST-caveolin fusion proteins with baculovirus-expressed H-Ras was evaluated essentially as we described for the interaction of caveolin with baculovirus-expressed heterotrimeric G protein alpha subunits(30) . Briefly, GST or purified GST-caveolin fusion proteins bound to glutathione-agarose beads were extensively prewashed with phosphate-buffered saline (1times) and lysis buffer containing protease inhibitors (3times). These beads contained 100 pmol of a given fusion protein per 100 µl of packed volume. Approximately 100 µl of this material was incubated with 1 ml of precleared H-Ras lysates by rotating overnight at 4 °C. After binding, the beads were extensively washed (6-8times) with wash buffer containing 50 mM Hepes, pH 7.5, 120 mM NaCl, 1 mM EDTA, 0.5% CHAPS, and protease inhibitors. Finally, bound proteins were eluted with 100 µl of elution buffer containing 50 mM Tris, pH 8.0, 1 mM EDTA, 1% Triton X-100, 10 mM reduced glutathione, and protease inhibitors. The eluate was mixed 1:1 with 2times sample buffer and subjected to SDS-PAGE (10% acrylamide) and Western blot analysis with anti-Ras IgG (1:1000 dilution, Transduction Labs). Horseradish peroxidase-conjugated secondary antibodies (1:5000 dilution, Amersham Corp.) were used to visualize bound primary antibodies by ECL.

Incorporation of Purified Recombinant Caveolin into Lipid Membranes

Recombinant caveolin (cav-myc-H(7)) was expressed in Escherichia coli, purified by Ni-NTA-agarose chromatography, and reconstituted into liposomes as described elsewhere(31) . Briefly, recombinant caveolin was mixed with purified lipid components in 2 ml of MBS containing 60 mM octyl-glucoside and dialyzed overnight against MBS lacking detergent to allow the association of caveolin with lipids. More specifically, 85 µg of recombinant caveolin was added to 1.8 mg of a purified lipid extract (type VI; B1877; Sigma) dissolved in octyl-glucoside. After dialysis to remove octyl-glucoside, the dialysate was adjusted to 40% sucrose by the addition of 2 ml of 80% sucrose prepared in MBS and placed at the bottom of an ultracentrifuge tube. A 5-30% discontinuous sucrose gradient was formed above (4 ml of 5% sucrose/4 ml of 30% sucrose, both in MBS lacking detergent) and centrifuged at 39,000 rpms for 16-20 h in an SW41 rotor (Beckman Instruments, Palo Alto, CA). A light-scattering band at the 5-30% sucrose interface was collected by centrifugation and represents reconstituted caveolin-rich membranes (31) .

Purification of GST-H-Ras Proteins

An overnight culture of E. coli harboring plasmids encoding either GST-H-Ras or GST-H-Ras was diluted 1:100 into 500 ml of LB containing ampicillin. Expression of fusion proteins was induced with isopropyl-1-thio-beta-D-galactopyranoside (0.5 mM) when the A reached 0.5-0.6. After 2 h of shaking, bacteria were collected by centrifugation and resuspended in 5 ml of phosphate-buffered saline containing 20 mM EDTA, 2 mg/ml lysozyme, 100 µM GDP, and protease inhibitors. After incubation for 10 min at room temperature, bacteria were frozen and thawed three times in an ethanol-dry ice bath. The solution was then adjusted to 25 mM MgCl(2), 0.5 M NaCl, 1% Triton X-100, and 5 mM dithiothreitol. Cells were disrupted further by 10-15 passages through an 18 gauge needle. After centrifugation, the supernatant was collected, and 0.5 ml of a 50% slurry of glutathione-agarose (G4510; Sigma) was added. After 4 h rotating at 4 °C, beads were washed (4times) with 10 ml of Tris-HCl, pH 8.0, 50 mM NaCl, 5 mM MgCl(2), 20% glycerol, 1 mM dithiothreitol, 100 µM GDP. GST-H-Ras proteins were then eluted with 1 ml of the above buffer containing 10 mM reduced glutathione (G4251; Sigma). Eluates were dialyzed against 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 5 mM MgCl(2), 5% glycerol, and 1 mM dithiothreitol, adjusted to 50% glycerol and stored at -20 °C.

Recruitment of GST-H-Ras onto Reconstituted Caveolin-rich Membranes

Recombinant GST-H-Ras and GST-H-Ras were purified as described above. Equivalent amounts of purified soluble GST-H-Ras or GST-H-Ras (50-100 µg) were then incubated with one-tenth of a single preparation of reconstituted caveolin-rich membranes. After 2-4 h at 4 °C rotating end-over-end, soluble unbound GST-H-Ras was removed by reflotation in sucrose density gradients. Reconstituted caveolin-rich membranes used as the substrate for binding were prepared using a purified lipid extract and recombinant caveolin as detailed above. Bound GST-H-Ras was visualized by immunoblotting with anti-Ras antibodies.

Co-expression of H-Ras and Caveolin in 293T Cells

H-Ras (WT and C186S) and untagged caveolin were transiently co-expressed in 293T cells using the calcium-phosphate precipitation procedure(23, 34) . 48 h post-transfection, cells were fractionated as described above using a detergent-free method for purification of caveolae-enriched membrane fractions. The distributions of recombinantly expressed H-Ras (WT) and H-Ras (C186S) were visualized by immunoblotting with anti-Ras antibodies. pZIP-H-Ras (WT) and pZIP-H-Ras (C186S) were the generous gifts of Dr. Adrienne Cox (University of North Carolina, Chapel Hill) and were as described previously(36) . 293T cells were the generous gift of Drs. Anthony Koleske (MIT) and Kunxin Luo (Whitehead Institute).


RESULTS

Construction and Expression of His-tagged Caveolin in Mammalian Cells

A novel strategy was devised for the purification of caveolin and its associated proteins from mammalian cells. A polyhistidine tag was placed at the C terminus of an epitope-tagged form of caveolin to allow its purification by Ni-NTA affinity chromatography after expression in mammalian cells. The polyhistidine tag allows the affinity purification of the attached protein via Ni-NTA affinity chromatography(37) . Bound proteins are specifically eluted with imidazole that mimics the side chain of histidine(37) . As a control for nonspecific binding, an epitope tagged form of caveolin was also expressed that lacks the polyhistidine tag but contains the myc epitope. Both constructs are shown schematically in Fig. 1A.


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(7)) for affinity purification by Ni-NTA-agarose chromatography. B, expression of cav-myc and cav-myc-H(7) in COS-7 cells. Left, COS-7 cells were transiently transfected with pCB7 vector alone and pCB7 containing cav-myc or cav-myc-H(7) constructed as outlined in A. Note that cav-myc-H(7) 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(7) 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(7) 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(7)) 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(7) 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.

Detergent-free Purification of Caveolin-rich Domains: Association of Ras with Caveolin

Next, stable MDCK cell lines were derived expressing equivalent amounts of either cav-myc or cav-myc-H(7). These MDCK cell lines were then fractionated using an established protocol that separates caveolin from the bulk of cellular membranes and cytosolic proteins. This fractionation scheme is based on the specific buoyant density of caveolin-rich membrane domains and their resistance to solubilization by the noninonic detergent Triton X-100 at low temperatures (8-10, 12, 17, 23, 25, 30, 33, 38). Using this scheme, endogenous MDCK caveolin is purified 2,000-fold relative to total cell lysates; approximately 90-95% of caveolin is recovered in fractions 5 and 6 of these sucrose density gradients while excluding greater than 99.95% of total cellular proteins(8, 23, 30, 33) . Using this same fractionation scheme, cav-myc (not shown) and cav-myc-H(7) (Fig. 2) co-fractionated with endogenous caveolin, an indication that the myc and polyhistidine tags do not interfere with the correct targeting of caveolin. Similarly, we and others have previously shown that myc-epitope tagged caveolin behaves as endogenous caveolin and is correctly targeted(20, 22, 23) .


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(7) 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(7), 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(7) and with anti-caveolin IgG (mAb 2297) to detect both cav-myc-H(7) and endogenous caveolin. Note that both alpha- and beta-isoforms of endogenous caveolin are detected, and they co-fractionate with cav-myc-H(7). The generation of beta-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(7) 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(7), 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(7) 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(7) 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(7) migrates at 29 kDa, whereas endogenous caveolin (alpha- and beta-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 alpha 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 (4times), 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(7). 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(7) 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(7) (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 alpha subunits (G, G(s), G(o)) 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) .

Interaction of Ras with Caveolin

To evaluate whether Ras interacts directly with caveolin, we recombinantly expressed full-length caveolin and portions of caveolin as GST-fusion proteins in E. coli (Fig. 5A). These GST-caveolin fusion proteins were constructed and characterized previously(23, 24, 30) . After affinity purification using glutathione-agarose, these caveolin fusion proteins were then incubated with cellular extracts containing recombinant H-Ras. These extracts were prepared by infecting insect cells (Sf21) with a baculovirus-based expression vector encoding wild-type H-Ras. Using this in vitro binding assay, H-Ras bound specifically to full-length caveolin but not to GST alone (Fig. 5B, upper panel). In addition, lipid modification of H-Ras did not appear to be absolutely required for this in vitro interaction because an unmodified form of H-Ras also bound to full-length caveolin.


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 alpha subunits(30) .

Recruitment of Ras onto Reconstituted Caveolin-rich Membranes Is Regulated by the Activation State of Ras

Caveolin can be reconstituted into lipid membranes using purified lipids and a purified recombinant His-tagged form of caveolin expressed in E. coli(31) . These reconstituted caveolin-rich membranes can specifically interact directly with purified GST-G(31) but not GST alone. Because lipid modification of H-Ras was apparently not required for interaction with caveolin (see above; Fig. 5B), we tested whether GST-H-Ras or GST-H-Ras can be recruited onto reconstituted caveolin-rich membranes. Based on the observation that caveolin is a cytoplasmically oriented integral membrane, we have proposed the hypothesis that caveolin could function as a scaffolding protein or platform to recruit and sequester signaling molecules within caveolin-rich areas of the plasma membrane(24) .

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(7) was produced in E. coli and purified by Ni-NTA-agarose chromatography. Recombinant cav-myc-H(7) 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(7) 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.

Is Farnesylation of Ras Required for Its Caveolar Localization in Intact Cells?

Lipid modification of Ras by farnesylation of its C-terminal CAAX sequence is thought to play a critical role in targeting Ras to the plasma membrane(43, 44) . However, our in vitro studies indicate that lipid modification of Ras is not absolutely required for its interaction with caveolin. Thus, we next evaluated whether farnesylation of Ras is required for its caveolar targeting in intact cells.

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.


DISCUSSION

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. (^2)

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) .




FOOTNOTES

*
This work was supported in part by National Institutes of Health FIRST Award GM-50443 (to M. P. L.), National Institutes of Health FIRST Award CA-63139 (to L. A. Q.), a grant from the Elsa U. Pardee Foundation (to M. P. L.), and a grant from the W. M. Keck Foundation to the Whitehead Fellows program (to M. P. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Hematology and Oncology, Istituto Superiore di Sanita, Viale Regina Elena, 299, 00161 Rome, Italy.

To whom correspondence should be addressed: Whitehead Inst. for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142-1479. Tel.: 617-258-5225; Fax: 617-258-9872; lisanti{at}wi.mit.edu.

(^1)
The abbreviations used are: NTA, nitrilotriacetic acid; GST, glutathione S-transferase; MDCK, Madin-Darby canine kidney; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; Mes, 4-morpholineethanesulfonic acid; TBS, Tris-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

(^2)
T. Okamoto and M. P. Lisanti, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Harvey F. Lodish for enthusiasm and encouragement; Dr. Philipp Scherer for critical discussions; Dr. John R. Glenney for monoclonal antibodies (2297 and 2234) directed against caveolin; members of Dr. Robert A. Weinberg's laboratory, especially Drs. Barton Giddings and Sang Ho Park, for insightful discussions during the course of this study; Dr. Sang Seoh Koh for advice on the baculovirus expression system; Dr. Adrienne Cox for c-H-Ras mammalian expression constructs; Drs. Anthony Koleske and Kunxin Luo for 293T cells; and Marcia Glatt and other members of the Whitehead purchasing department for dedicated service.


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