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Identification of Peptide and Protein Ligands for the
Caveolin-scaffolding Domain
IMPLICATIONS FOR THE INTERACTION OF CAVEOLIN WITH CAVEOLAE-ASSOCIATED PROTEINS*
(Received for publication, October 28, 1996)
Jacques Couet‡§, Shengwen Li‡
¶
, Takashi Okamoto
i
**, Tsuneya Ikezu
i
, and Michael P. Lisanti‡ ‡‡
From the ‡Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142-1479 and
i
Shriners Hospitals
for Crippled Children, Massachusetts General Hospital, Department of Anesthesia, Harvard Medical School,
Boston, Massachusetts 02114
Caveolin, a 21–24-kDa integral membrane protein, is a
principal component of caveolae membranes. We have
suggested that caveolin functions as a scaffolding pro-
tein to organize and concentrate certain caveolin-inter-
acting proteins within caveolae membranes. In this
regard, caveolin co-purifies with a variety of lipid-mod-
ified signaling molecules, including G-proteins, Src-like
kinases, Ha-Ras, and eNOS. Using several independent
approaches, it has been shown that a 20-amino acid
membrane proximal region of the cytosolic amino-ter-
minal domain of caveolin is sufficient to mediate these
interactions. For example, this domain interacts with
G-protein
a
subunits and Src-like kinases and can
functionally suppress their activity. This caveolin-
derived protein domain has been termed the caveolin-
scaffolding domain. However, it remains unknown
how the caveolin-scaffolding domain recognizes these
molecules.
Here, we have used the caveolin-scaffolding domain as
a receptor to select random peptide ligands from phage
display libraries. These caveolin-selected peptide li-
gands are rich in aromatic amino acids and have a char-
acteristic spacing in many cases. A known caveolin-in-
teracting protein, G
i2
a
, was used as a ligand to further
investigate the nature of this interaction. G
i2
a
and other
G-protein
a
subunits contain a single region that gener-
ally resembles the sequences derived from phage dis-
play. We show that this short peptide sequence derived
from G
i2
a
interacts directly with the caveolin-scaffold-
ing domain and competitively inhibits the interaction of
the caveolin-scaffolding domain with the appropriate
region of G
i2
a
. This interaction is strictly dependent on
the presence of aromatic residues within the peptide
ligand, as replacement of these residues with alanine or
glycine prevents their interaction with the caveolin-
scaffolding domain. In addition, we have used this inter-
action to define which residues within the caveolin-scaf-
folding domain are critical for recognizing these peptide
and protein ligands. Also, we find that the scaffolding
domains of caveolins 1 and 3 both recognize the same
peptide ligands, whereas the corresponding domain
within caveolin-2 fails to recognize these ligands under
the same conditions. These results serve to further dem-
onstrate the specificity of this interaction. The implica-
tions of our current findings are discussed regarding
other caveolin- and caveolae-associated proteins.
Caveolae are plasma membrane-attached vesicular or-
ganelles that have a characteristic diameter in the range of
50–100 nm (1, 2). Caveolae are present in most cell types but
are especially abundant in adipocytes, endothelial cells, fibro-
blasts, and smooth muscle cells (3). In adipocytes and smooth
muscle cells, they represent up to 20% of the total plasma
membrane surface area. Endothelial cells contain ;5,000–
10,000 caveolae/cell. Although they were originally implicated
in cellular transport processes (4), recent evidence suggests
that they may participate in signal transduction-related events
(5–11).
Caveolin, a 21–24-kDa protein, is a principal integral mem-
brane component of caveolae membranes in vivo (12, 13). Using
either Triton X-100-based methods or detergent-free methods,
caveolin co-purifies with certain lipid-modified signaling mole-
cules (such as G-proteins, Src family tyrosine kinases, and
Ha-Ras) (5, 6, 7, 9, 10, 14–16). In addition, caveolin was first
identified as a major v-Src substrate that undergoes tyrosine
phosphorylation in Rous sarcoma virus-transformed cells (17).
Based on these and other observations, we have proposed the
“caveolae signaling hypothesis,” which states that caveolar lo-
calization of certain signaling molecules could provide a com-
partmental basis for their actions and explain cross-talk be-
tween signaling pathways (18–20).
Several independent lines of evidence suggest that caveolin
may function as a scaffolding protein within caveolae mem-
branes: (i) both the amino- and carboxyl-terminal domains of
caveolin remain entirely cytoplasmic and are therefore acces-
sible for cytoplasmic protein-protein interactions (21); (ii)
caveolin forms high molecular mass homo-oligomers of ;350
kDa (22, 23) that have the capacity to interact with specific
lipids (cholesterol and glycosphingolipids; Refs. 24 and 25) and
lipid-modified signaling molecules (G-proteins, Src family ki-
nases, and Ha-Ras; Refs. 16 and 26–29); (iii) these caveolin
homo-oligomers can self-associate to form caveolae-like struc-
tures in vitro (22, 25); and (iv) recombinant overexpression of
caveolin in caveolin-negative cells (lymphocytes and Sf 21 in-
sect cells) is sufficient to drive the formation of caveolae-sized
* This work was supported in part by National Institutes of Health
FIRST Award GM-50443 (to M. P. L.), 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 be hereby marked “advertisement”inac-
cordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a postdoctoral fellowship from the Medical Research
Council of Canada.
¶
Recipient of National Institutes of Health Postdoctoral Fellowship
CA-71326 from the NCI.
** Recipient of fellowships from the Byotai-Taisha Foundation and
the Mochida Memorial Foundation.
‡‡ To whom correspondence should be addressed: Whitehead In-
stitute for Biomedical Research, Nine Cambridge Center, Cambridge,
MA 02142-1479. Tel.: 617-258-5225; Fax: 617-258-9872; E-mail:
lisanti@wi.mit.edu.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 10, Issue of March 7, pp. 6525–6533, 1997
© 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www-jbc.stanford.edu/jbc/ 6525
by guest, on February 14, 2013www.jbc.orgDownloaded from
vesicles (30, 31). Thus, it appears that the caveolin protein has
the capability of interacting with itself, specific lipids, and
other proteins and can serve to orchestrate caveolae formation.
Using a variety of domain-mapping approaches (deletion
mutagenesis, GST
1
fusion proteins, and synthetic peptides), a
region within caveolin has been defined that mediates the
interaction of caveolin with itself and other proteins (16, 26–
29). This cytoplasmic 41-amino acid membrane proximal region
of caveolin is sufficient to mediate the formation of caveolin
homo-oligomers (22), and the carboxyl-terminal half of this
region (20 amino acids, residues 82–101) mediates the interac-
tion of caveolin with G-protein
a
subunits and Src family tyro-
sine kinases (26–29). This interaction is sufficient to suppress
the GTPase activity of G-proteins and inhibits the autoactiva-
tion of Src family tyrosine kinases (29). As this caveolin domain
(residues 82–101) is critical for caveolin homo-oligomerization
and the interaction of caveolin with certain caveolin-associated
proteins (G-proteins, Ha-Ras, and Src family kinases), we have
previously termed this protein domain the caveolin-scaffolding
domain (29).
Here, we have used the caveolin-scaffolding domain and one
of its protein ligands (a G-protein
a
subunit, G
i2
a
) to explore the
possible protein sequence requirements that underlie this mo-
lecular recognition event. As a first step, a fusion protein car-
rying the caveolin-scaffolding domain was used to select ran-
dom peptide ligands from phage display libraries. Many of
these peptide ligands share two properties with a particular
region of the G-protein: (i) a preponderance of aromatic amino
acids in a short stretch; and (ii) a characteristic spacing be-
tween these aromatic residues. We show that this region of G
i2
a
interacts directly with the caveolin-scaffolding domain, and a
peptide encoding this G-protein domain competitively inhibits
the binding of the appropriate region of G
i2
a
to the caveolin-
scaffolding domain.
MATERIALS AND METHODS
Phage Display Library Selection—The 15-mer library and bacterial
strains were from Dr. George Smith (University of Missouri, Columbia,
MO), whereas the decapeptide (10-mer) library was constructed as
described elsewhere (32). The GST-caveolin-1-(61–101) fusion protein
was purified and immobilized on glutathione-agarose beads (Sigma) as
described previously (26). The incubation was performed using 6 3 10
10
transforming units of phage in presence of 50
m
l of beads in TNET
buffer (10 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.05% Tween 20)
at 4 °C under agitation for 16 h. Beads were then washed with 20 ml of
TNET before acidic elution with 100
m
lof50mMglycine, pH 2.2. The
eluate was neutralized with 0.1 volume of 1 M Tris, pH 10.0, and used
to infect Escherichia coli K91-kan cells for amplification. Purified phage
were prepared as described elsewhere (33). Four rounds of selection
were performed. Colonies from the last titering plate were picked to
inoculate 2 ml of Luria broth/tetracycline medium. The culture was
grown overnight, and phage DNA was prepared by polyethylene glycol
precipitation of the culture supernatant, phenol-chloroform extraction
and ethanol precipitation (33). The region corresponding to the selected
peptide was sequenced using a Sequenase II kit from U.S. Biochemical
Corp. An oligonucleotide specific for the fuse5 vector downstream of
the cloning site was used for sequencing (59-GCCTGTAGCATTCCA-
CAGACAA-39).
Phage Binding Assays—We used 96-well plates (Nunc Maxisorp) to
perform all ELISAs. Phage from selected clones were prepared as
described elsewhere (33). Purified phage in Tris-buffered saline (TBS)
were used for coating the plate, and the incubation proceeded at 4 °C
overnight. Plates were saturated for1hatroom temperature with
TBS/0.05% Tween 20 (TBST) containing 1% bovine serum albumin.
Purified His-tagged Myc full-length caveolin-1 (1
m
g/well) (25) or GST-
caveolin-1-(61–101) fusion protein (1
m
g/well) (26) was then added in
TBST to the well for 2 h and washed several times with TBST. Mono-
clonal antibodies directed against the Myc epitope (Harvard Monoclonal
Antibody Facility, Cambridge, MA) (1:400) or GST (Santa Cruz Biotech-
nology) (1:1000) in TBST were then added for2hatroom temperature
followed, after washes, by horseradish peroxidase anti-mouse IgG (Am-
ersham Corp.) (1:2000). The reaction was revealed using 2,29-azino-di-
[3-ethylbenzthiazoline sulfonate (6)] (Boehringer Mannheim), and ab-
sorbance was measured on a microplate reader at 410 nm. The opposite
assay was also performed, in which purified full-length caveolin-1-
Myc-H
7
(1
m
g/well) or GST-caveolin-1-(61–101) fusion proteins (1
m
g/
well) were coated onto the wells in 100 mM sodium bicarbonate, pH 8.5,
and then incubated with phage. A biotinylated anti-fd polyclonal anti-
body (Sigma) (1:1000) was used in addition to horseradish peroxidase-
streptavidin (Zymed) (1:2000) and 2,29-azino-di-[3-ethylbenzthiazoline
sulfonate (6)]. For the ELISA using peptides, 500 pmol/well was used
for coating the wells in bicarbonate coating buffer. Peptide synthesis
was performed by the Biopolymers Facility at the Massachusetts
Institute of Technology (Cambridge, MA) and Research Genetics
(Huntsville, AL).
GST-G
i2
a
Fusion Proteins— Using the bovine G
i2
a
cDNA (provided
by Dr. Nukada) as a template, the polymerase chain reaction was
performed to construct GST-G
i2
a
-full length (1-355), GST-G
i2
a
-B (120-
238), and GST-G
i2
a
-C (239-355). The products were subcloned into the
EcoRI site of the pGEX-1
l
T vector; resulting constructions were sub-
jected to restriction analysis and sequencing via the Sanger method.
Purification of GST-G
i2
a
fusion proteins was essentially as described
1
The abbreviations used are: GST, glutathione S-transferase;
ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered sa-
line; TBST, TBS/Tween 20; FL, full-length.
FIG.1. Schematic representation of caveolin and the GST-
caveolin fusion protein used for selection of caveolin-binding
peptide ligands. A, diagram summarizing the cytoplasmic membrane
topology of caveolin. The known sites of palmitoylation within the
carboxyl-terminal domain of caveolin-1 are as indicated. G
a
subunits
are known to interact with a membrane-proximal region of caveolin
encoded by residues 82–101 of caveolin-1 (hatched boxes). This caveolin-
scaffolding domain also contains information that specifies the forma-
tion of high molecular mass homo-oligomers of caveolin-1 containing
;14–16 individual monomer units. For the purposes of illustration,
only a dimer of caveolin-1 is shown. B, schematic diagram summarizing
the overall domain structure of caveolin. The membrane-spanning do-
main is represented in black. The GST-caveolin fusion protein (hatched
box) used for the selection corresponds to a cytosolic membrane-proxi-
mal region of caveolin-1 (residues 61–101) and includes the caveolin-
scaffolding domain (residues 82–101) as depicted in C. C, alignment of
the caveolin-1-scaffolding domain with homologous regions from the
other members of the caveolin gene family, caveolins 2 and 3. Note that
in caveolin-1, this region is absolutely conserved from chicken to
human.
Caveolin, “Velcro” for Signal Transduction6526
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FIG.2.Relative abundance of the 20 different amino acids in 10- and 15-mer peptides displayed by their respective bacteriophage
libraries, before and after selection for caveolin binding. A and C, single-stranded DNA was purified from 25 (10-mer, 11 different clones)
and 55 (15-mer, 22 different clones) clones obtained after four rounds of selection. The sequences of the peptides displayed were deduced by DNA
sequencing. The percentage of occurrence for each amino acid calculated as the number observed divided by the total number of residues in the
peptides is shown. B and D, peptide sequence of 18 (10-mer) and 30 (15-mer) random bacteriophage clones from the unselected starting libraries,
respectively. Calculations were made as described for A and C. Amino acids are grouped according to the number of codons that specify them. The
frequency expected for each group is shown by the dotted line and assumes that all codons were used with equal efficiency.
T
ABLE I
Sequence, occurrence, and aromatic residue content of peptide ligands selected using a GST fusion protein containing the
caveolin-1-scaffolding domain
Single-stranded DNA was purified from phage clones after four rounds of selection for binding to the GST-caveolin-1-(61–101) fusion protein.
Peptide sequences were deduced from the DNA sequencing of the corresponding region of the bacteriophage genome.
Sequence
10-mer
Sequence
15-mer
Occurrence
W, F, or Y
content
Occurrence
W, F, or Y
content
MWHWEKRKWV 13 3 RNVPPIFNDVYWIAF 31 4
KWAWGLDRWV 23RHVAAAVFVGWAFSV 33
HWAWEVRMWR 23TEFLWGFRTVFHG 24
MWRWESCCWE 13TRWGESDSFRISPPG 12
MWVWEHNAWE 13PGAVRFTFGGSWHY 14
VWSWALRKWV 13GGWGQFRLFYGAPFD 15
VWHWAVSRFN 13CSSEYGVTYWVLCA 13
MWRWESSRWE 13IGRIVHHSLYSWPS 12
RWHWQSHMWL 13ECHFLFLLCRVWGR 13
KWLWGSSRWE 13WSVRYDYLVYPSLLP 14
RDWVGWVCL 12SSGFRDAFRGWDGSA 13
SDVHYIHAHWAVTSH 12
SAVSVLGYHSYFVFP 14
GSFIIFFLVLFMLV 14
PVRYGFSGPRLAILW 13
AARTLSFHPYGYPPY 14
GHGLYYWNFTYSSET 15
TEFLWFRTVLHG 13
LSGGFVWMGFRPSIG 13
RNQGGNWMRFMRCLL 12
VSWSFYRIFGHPGTD 14
LSWSIDYNRNTPSIG 12
Caveolin, “Velcro” for Signal Transduction 6527
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elsewhere (26, 34). To assess the quality of the purification, the fusion
proteins were analyzed by SDS-polyacrylamide gel electrophoresis (10%
acrylamide) and transferred to nitrocellulose. After transfer, nitrocel-
lulose sheets were stained with Ponceau S to visualize protein bands
and subjected to immunoblotting with anti-GST IgG (1:1000) to visual-
ize GST fusion proteins. For ELISAs, GST-G
i2
a
fusion proteins (;200
ng/well) were added to peptide-coated wells, and binding was revealed
using a monoclonal antibody directed against the GST moiety.
Peptide Competition of GST-G
i2
a
Fusion Protein Binding—To the
caveolin-1-(82–101) peptide-coated well, a mixture of ;120 ng of puri-
fied GST-G
i2
a
B fusion protein (;3 pmol) and increasing amounts of
competing peptide was added. The mixture was assembled just prior to
addition to the well. The procedure following was as described above.
We also used biotinylated peptides in several experiments, and binding
was revealed using horseradish peroxidase-streptavidin (1:2000).
RESULTS
Identification of Peptide Ligands for the Caveolin-scaffolding
Domain Using Phage Display—We used a GST-caveolin fusion
protein that contains the caveolin-scaffolding domain as a re-
ceptor to randomly select peptide ligands from two different
phage display libraries (Fig. 1). This GST-caveolin fusion pro-
tein contains caveolin residues 61–101 and has been previously
shown to be functionally sufficient to interact with G-protein
a
subunits (including G
i2
a
), c-Src, and Ha-Ras (26, 29).
After four rounds of selection, caveolin-binding phage clones
were subjected to DNA sequence analysis to reveal their pep-
tide sequences, as shown in Table I. Caveolin binding clones
from the 10-mer library were rich in tryptophan, and most
exhibited a characteristic spacing conforming to the sequence
WXWXXXXW. Random sequencing of the 10-mer library indi-
cated that tryptophan was enriched ;3-fold in caveolin-se-
lected clones (Fig. 2, A and B). In contrast, caveolin binding
clones from the 15-mer library did not exhibit any characteris-
tic spacing but were also enriched in aromatic amino acids.
More specifically, tryptophan, phenylalanine, and tyrosine
were enriched 3.8-, 1.5-, and 1.8-fold relative to the unselected
library population (Fig. 2, B and C). Also, a single 15-mer clone
(RNVPPIFNDVYWIAF) accounted for ;60% of all clones.
An ELISA was developed to evaluate the interaction of these
peptide ligands with various regions of caveolin. Nine caveolin-
derived peptides that correspond to regions of the cytoplasmic
amino-terminal domain of caveolin were used to as receptors to
capture these ligands (Fig. 3A). All phage clones tested only
interacted with the peptide that corresponds to the caveolin-
scaffolding domain (residues 82–101; Fig. 3A) and with recom-
binant full-length caveolin-1 purified from E. coli (not shown).
The clones tested include the three most abundant clones from
the 10- and 15-mer libraries (see Table I) and the clone con-
taining the sequence VWEWAVSRFN. Identical results were
obtained when a biotinylated peptide corresponding to the se-
quence of the most abundant 15-mer phage clone was tested.
When the caveolin-scaffolding domain is divided into two
halves (residues 84–92 and 93–101), we have previously shown
that this inhibits its functional activity (26). Similarly, each of
FIG.3.Binding specificity of caveo-
lin-selected phage clones and pep-
tides. A, each of the caveolin-1 peptides
was coated in the bottom of wells of an
ELISA plate. The assay was performed as
described under “Materials and Meth-
ods.” A negative result was assumed for
an absorbance level (at 410 nm) less than
two times of that recorded for negative
controls (i.e. caveolin peptide incubated
without phage or peptide plus buffer
alone). In this case, a positive result (1)
indicates a level of absorbance more than
10 times of those with respective negative
controls. nd, not determined. Each deter-
mination was performed in duplicate. B,
left, relative binding of the most abundant
15-mer clone (phage or the corresponding
biotinylated peptide, biotin-RNVPPIF-
NDVYWIAF) to the scaffolding domain of
caveolin-1 (residues 82–101) and to the
same region divided into two peptides.
The assay was as described for A. Binding
to the caveolin-scaffolding domain was ar-
bitrarily set as 100%. Each determination
was performed in duplicate. Right, scaf-
folding domains for caveolin-1 (residues
82–101), caveolin-2 (Cav-2, residues 54–
73), and caveolin-3 (Cav-3, residues 55–
74) were compared for their relative bind-
ing to the most abundant 15-mer clone
(phage or the corresponding biotinylated
peptide). Binding to the scaffolding do-
main of caveolin-1 was arbitrarily set at
100%. All results represent the means 6
S.D. (bars) of triplicate determinations.
Caveolin, “Velcro” for Signal Transduction6528
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these halves was unable to interact with the caveolin-selected
phage clones and their corresponding biotinylated peptides
(Fig. 3B).
Recent studies have shown that caveolin is the first member
of a multigene family of related molecules (13, 27, 28, 35, 36);
caveolin has been retermed caveolin-1. Thus, we next tested
the interaction of these caveolin-selected phage clones with the
scaffolding domains of caveolins 1–3. Fig. 3B shows that these
peptide ligands only interact with the scaffolding domains of
caveolins 1 and 3 but fail to interact with the homologous
domain in caveolin-2. This suggests that the scaffolding do-
main of caveolin-2 has different protein sequence requirements
for its interaction with other molecules and further demon-
strates the selectivity of these interactions, as these peptide
ligands were selected using the scaffolding domain of
caveolin-1.
Interaction of G
i2
a
-derived Proteins and Peptide Domains
with the Caveolin-scaffolding Domain—By comparing the pep-
tide ligands obtained for the caveolin-scaffolding domain with
the protein sequences of a known class of caveolin-interacting
proteins (G-protein
a
subunits), we deduced two possible caveo-
lin-binding motifs: FXFXXXXF and FXXXXFXXF, where F is
an aromatic residue (Trp, Phe, or Tyr). These two motifs
correspond to the sequences of most 10-mer phage clones and
the most abundant 15-mer clone, respectively. Fig. 4A shows
that these two motifs are present in most G-protein
a
subunits
in a composite manner (FXFXXXXF1FXXXXFXXF 3
FXFXXXXFXXF).
To evaluate whether this region of G-protein
a
subunits can
serve as a ligand for the caveolin-scaffolding domain, we con-
structed: (i) a synthetic peptide containing this G-protein re-
gion (THFTFKLDLHFKMFDV), termed GP; and (ii) a variety
of GST-G
i2
a
fusion proteins, including full-length (FL) G
i2
a
and
two deletion mutants, termed B and C (Fig. 4B). Note that the
GP region is contained within two of these GST-G
i2
a
fusion
proteins (FL and B).
Fig. 5A shows that both the FL and B G
i2
a
fusion proteins
interacted preferentially with the caveolin-scaffolding domain,
but little or no interaction was observed with an adjacent
region of caveolin (residues 53–81). Also, the B region of G
i2
a
was only recognized by the scaffolding domains of caveolins 1
and 3 but not by the scaffolding domain of caveolin-2 (Fig. 5B).
This binding profile is exactly what we observed previously for
peptide ligands selected using the scaffolding domain of caveo-
lin-1 (See Fig. 3B), suggesting a similar mode of interaction.
Fig. 6A shows that the GP peptide interacts with the scaf-
folding domain of caveolins 1 and 3 and that this interaction is
strictly dependent on the presence of aromatic residues within
the GP peptide. When the phenylalanine residues of the GP
peptide (THFTFKLDLHFKMFDV) were changed to either al-
anine or glycine (THATAKLDLHAKMADV and THGTGKLD-
LHGKMGDV), binding of these peptides to the caveolin-scaf-
folding domain was almost completely abolished. This is
consistent with the idea that aromatic residues play a key role
in recognition by the caveolin-scaffolding domain. Also, it is
important to note that the GP peptide was only recognized by
the scaffolding domain of caveolins 1 and 3 but not by the
scaffolding domain of caveolin-2 (Fig. 6A).
The GP peptide also competitively inhibits the binding of the
FIG.4.G
a
subunits and caveolin-selected peptide ligands. A,
alignment of the two caveolin-binding motifs with a conserved region of
various G
a
subunits. F represents amino acids Trp, Phe, and Tyr; X
denotes any amino acid. A peptide (designated GP) was designed for use
in binding studies, and its sequence corresponds to the one illustrated
here for the G
i2
a
subunit. B, schematic diagram summarizing the con-
struction of different GST-G
i2
a
fusion proteins used to examine inter-
actions with caveolin (FL, B, and C).
FIG.5.Interaction of GST-G
i2
a
fusion proteins with the caveo-
lin-scaffolding domain. A, Two peptides, constituting the entire re-
gion of caveolin-1 (residues 53–81 and 82–101) used for the selection of
caveolin-binding phage clones, were coated onto wells of an ELISA
plate. These peptides were allowed to interact with different GST-G
i2
a
fusion proteins (FL, B, and C; described in Fig. 4B), as well as with GST
alone as a control. Open bars, binding to caveolin-1 peptide 53–81; filled
bars, binding to caveolin-1 peptide 82–101 (the caveolin-scaffolding
domain). B, two GST-G
i2
a
fusion proteins representing the central third
(B) or the carboxyl-terminal third (C) of the G
i2
a
protein were examined
for their binding to the scaffolding domains of caveolins 1–3. Results are
expressed as the means 6 S.D. (bars) of triplicate determinations in
absorbance units at 410 nm.
Caveolin, “Velcro” for Signal Transduction 6529
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B region of G
i2
a
to the caveolin-scaffolding domain, and this
occurs in a dose-dependent manner (Fig. 6B). In this regard, it
is important to note that the B region contains the sequence
that corresponds to the GP peptide. Virtually identical results
were obtained using the peptide ligand selected from the 15-
mer phage display library (RNVPPIFNDVYWIAF), indicating
that this caveolin-selected peptide, the GP peptide, and G
i2
a
are all recognized by the caveolin-scaffolding domain in a sim-
ilar fashion. Importantly, mutated GP peptides lacking phenyl-
alanine failed to show any competition for binding.
Fig. 7 shows that the GP peptide also recognizes the full-
length intact caveolin-1 molecule (caveolin-1-Myc-H
7
). How-
ever, as predicted, the GP peptide was not recognized by
full-length caveolin-2 (caveolin-2-Myc-H
7
) under identical con-
ditions. These results provide an extraordinary demonstration
of the specificity of this interaction given the close protein
sequence homology between caveolins 1 and 2. Caveolin-2 is
58% similar and 38% identical to caveolin-1 (27).
As the protein sequence encoded by the GP peptide appears
to serve as a ligand for the caveolin-scaffolding domain, we
identified the location of this sequence within the known three-
dimensional structure of G-protein
a
subunits. The GP region
lies directly between switch I and switch II regions and pre-
cisely defines the space between where switch I ends and
switch II begins (Fig. 8). Interestingly, we have previously
shown that a Gln 3 Leu mutation (located 5 amino acid resi-
dues downstream from the end of the GP region) prevents the
interaction of G
s
a
with caveolin-1 (26). This mutation also locks
the G-protein
a
subunit in the GTP-liganded and -activated
conformation.
Mutational Analysis of the Caveolin-1-scaffolding Domain—
Two different approaches were used to define critical residues
within the caveolin-1-scaffolding domain that are required for
binding peptide ligands. First, deletion mutagenesis of the
82–101 region indicated that a minimal length of 16 amino
acids is required (residues 86–101) (Fig. 9A). Second, alanine-
scanning mutagenesis was then performed using this minimal
caveolin-1-scaffolding domain. Our results indicate that a cen-
tral core of four amino acids (
92
FTVT
95
) is strictly required for
interaction of the caveolin-1-scaffolding domain with both the
GP peptide and the corresponding region of G
i2
a
(Fig. 9B). This
region is FTVS in caveolin-3 and FEIS in caveolin-2. Thus, this
small difference may explain why these peptide ligands (library
peptides and GP peptide) and protein ligands (GST-G
i2
a
fusion
proteins) are recognized by the scaffolding domains of caveolins
FIG.6.The GP peptide interacts with the caveolin-scaffolding
domain and competitively inhibits the binding of a GST-G
i2
a
fusion protein. A, the biotinylated GP peptide was evaluated for its
ability to interact with the scaffolding domains of caveolins 1–3. The
binding of two mutated GP peptides was also evaluated in parallel. In
these mutant GP peptides, all four phenylalanine residues were
changed to glycine (Phe 3 Gly) or alanine (Phe 3 Ala). Binding to an
irrelevant region of caveolin-1 (residues 53–81) was included as a
negative control. Note that the wild-type GP peptide interacts with the
scaffolding domains of caveolins 1 and 3 but only weakly with the same
region of caveolin-2. In contrast, the mutant GP peptides showed little
or no interaction with any of the caveolin domains tested. B, affinity-
purified GST-G
i2
a
B fusion protein (120 ng) was allowed to interact with
the scaffolding domain of caveolin-1 in the absence or presence compet-
ing peptide. Two different peptides containing caveolin binding motifs
(the GP peptide and the 15-mer peptide (RNVPPIFNDVYWIAF)) com-
petitively inhibited the binding of the GST-G
i2
a
B fusion protein to
caveolin-1. In contrast, two mutant GP peptides were unable to inhibit
this interaction. All results are expressed as the means 6 S.D. (bars)of
triplicate determinations.
FIG.7. The GP peptide interacts with full-length caveolin-1
but not with full-length caveolin-2. A, schematic representation of
full-length recombinant caveolins 1 and 2 (Cav-1-myc-His
7
and Cav-2-
myc-His
7
) used for this experiment. Both constructions contain: (i) a
Myc epitope tag for detection; and (ii) a polyhistidine tag for purification
from E. coli by Ni
21
-nitrilo-triacetic acid affinity chromatography, as we
have described previously (25). B, the two purified full-length caveolins
were coated onto wells of an ELISA plate (1
m
g/well). Only coating
buffer was used in control wells. The biotinylated GP peptide (1
m
g) was
allowed to interact with saturated control and caveolin-coated wells,
and then horseradish peroxidase-streptavidin was added as described
under “Materials and Methods.” Results are expressed as the means 6
S.D. (bars) of triplicate determinations in absorbance units at 410 nm.
Caveolin, “Velcro” for Signal Transduction6530
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1 and 3, but not by the scaffolding domain of caveolin-2 (as
shown in Figs. 3B,5B, and 6A).
DISCUSSION
Here, we have identified peptide and protein ligands for the
scaffolding domain of caveolin and characterized the sequence
requirements of this reciprocal interaction. Several independ-
ent lines of evidence indicate that this interaction is extremely
specific: (i) the identified peptide and protein ligands interacted
only with the caveolin-scaffolding domain, but not with other
regions of the caveolin protein; (ii) the interaction also occurred
with the purified full-length caveolin molecule expressed as a
polyhistidine-tagged protein; (iii) the interaction was sequence-
specific; mutation of critical phenylalanine residues within the
ligand prevented the binding of these ligands to the caveolin-
scaffolding domain; also, mutation of certain critical residues
within the caveolin-scaffolding domain abrogated the binding
of peptide and protein ligands; and (iv) these peptide and
protein ligands bound selectively to the scaffolding domains of
caveolins 1 and 3 but not to the scaffolding domain of caveo-
lin-2. This is despite the fact that these domains within caveo-
lins 1–3 are extremely homologous. Also, it is important to note
that these ligands were identified using the scaffolding domain
of caveolin-1, suggesting that other potential ligands may exist
that selectively recognize the scaffolding domain of caveolin-2.
These results are consistent with the previous observations
that the scaffolding domains of caveolins 1 and 3 can both act
as GDP dissociation inhibitors for heterotrimeric G-proteins,
and both inhibit the autoactivation of Src family kinases (16,
26–29). In contrast, the scaffolding domain of caveolin-2 exhib-
its GTPase-activating activity toward heterotrimeric G-pro-
teins and fails to affect the activity of Src family kinases
(27, 29).
What are the possible implications of these interactions?
Other modular protein domains (such as Src homology 2 and 3,
WW, and PID) have been previously defined and their corre-
sponding peptide and protein ligands identified using tech-
niques that are similar or identical to the ones used here for the
caveolin-scaffolding domain. Src homology 2 domains and PID
domains both recognize phosphotyrosine and 3 or 4 surround-
ing residues; Src homology 3 domains recognize proline-rich
sequences, especially with the consensus PXXP; and the WW
domain recognizes proline-rich sequences such as PPPY (see
Prosite data base for specific references). Protein kinases also
recognize short peptide sequences to direct phosphorylation of
specific peptide and protein substrates: protein kinase A (R-R-
X-[ST]); protein kinase C ([ST]-X-[RK]); casein kinase II ([ST]-
X-(2)-[DE]); and tyrosine kinases ([RK]-X-(2,3)-[DE]-X-(2,3)-Y).
This is also the case for other posttranslational modifications,
including: N-glycosylation (N-X-[ST]); N-myristoylation
(MG); dual acylation (MGC); and prenylation (C-aliphatic-ali-
phatic-X). Similarly, antibody binding depends on the recogni-
tion of specific epitopes that can be as small as 5–10 amino
acids in length. Thus, many diverse cellular processes rely
heavily on the recognition of short consensus peptide motifs.
However, not all of these motifs are recognized, as this depends
on whether a given motif is cytoplasmic or extracellular or is
exposed on the surface of the protein; exposure on the surface
of the protein may even be conformation-specific. Also, recog-
nition may depend on the subcellular localization of the protein
and the potential interacting partner, e.g. nuclear, cytoplasmic,
plasma membrane, or Golgi-associated. These added criteria
for interaction thus strictly modulate whether the interaction
may or may not take place and greatly increase the specificity
of these interactions. Of course, these same constraints would
apply to the recognition of peptide and protein ligands by the
caveolin-scaffolding domain.
FIG.8. Three-dimensional repre-
sentation of a G
a
subunit in its GDP-
liganded conformation. A, the GP pep-
tide (caveolin binding domain) is
highlighted in yellow; switch and linker
regions are indicated in red and green,
respectively. The GDP molecule is at the
center of the image. Modified from
SwissProt (accession number P10824;
G
i1
a
). B, the caveolin binding domain de-
fined within G
i2
a
is shown relative to the
position of switch I and switch II regions.
Note that a point mutation (Gln 3 Leu)
within the switch II region, which is 5
residues downstream from the caveolin
binding domain, has been shown: (i) to
constitutively activate G
a
subunits (47,
48); and (ii) to abolish G
a
binding to
caveolin (26). G
a
residues have been color-
coded for the purpose of illustration: blue,
switch regions I and II; red, caveolin bind-
ing domain; white, Gln 3 Leu mutation.
Caveolin, “Velcro” for Signal Transduction 6531
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An increasing number of reports suggest that many different
classes of molecules are concentrated within caveolin-rich
membrane domains. Thus, we searched the protein sequences
of known caveolin- or caveolae-associated proteins for aromat-
ic-rich sequences that contain a specific spacing (FXFXXXXF,
FXXXXFXXF,orFXFXXXXFXXF, where F5Trp, Phe, or
Tyr), as defined here using the caveolin-scaffolding domain.
Most molecules reported to be associated with caveolin or
caveolae contain cytoplasmically accessible sequences that re-
semble those that we have defined here as peptide and protein
ligands for the caveolin-scaffolding domain (summarized in
Table II). Although it is not yet known whether all these
molecules interact directly with caveolin, our studies provide a
rational and systematic basis for investigating whether these
protein sequences are indeed recognized as cytoplasmic ligands
by the caveolin-scaffolding domain. This type of interaction
could provide a simple mechanism for sequestration of a di-
verse group of molecules within caveolin-rich regions of the
plasma membrane.
The caveolin-binding motif is reminiscent of another motif
identified using the chaperone BiP for selection: Hy(W/X)HyX-
HyXHyXHy, where Hy is a large hydrophobic amino acid (most
frequently Trp, Leu, or Phe) and X is any amino acid (37). In
fact, the best peptides tested for binding to BiP contain one Phe
and three Trp, which suggests that they could also bind to
caveolin. However, for BiP, no single peptide sequence was
enriched over others. This is in contrast to the present situa-
tion, in which strong enrichments were observed for particular
caveolin-selected peptide sequences. The apparent similarity
between BiP and caveolin in this matter may suggest a poten-
tial role for caveolin as a chaperone. Although to our knowledge
no chaperone described thus far is an integral membrane pro-
tein, caveolin could fulfill the role of a “membrane-bound chap-
erone” by interacting with signaling molecules and maintain-
ing them in an inactive conformation.
Acknowledgments—We thank Drs. Gerald R. Fink, Peter S. Kim, and
Harvey F. Lodish for their enthusiasm and encouragement; Dr. Ton
Schumacher (Kim Laboratory) for help with the phage display experi-
ments and for critical discussions; Dr. Philipp Scherer for critical dis-
cussions; Dr. John R. Glenney for monoclonal antibodies (2297 and
2234) directed against caveolin; and members of the Kim and the
Lisanti laboratories for insightful discussions during the course of this
study.
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A selection of known caveolin and/or caveolae-associated proteins
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Protein
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1st residue
b
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FXFXXXXFXXF
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Actin-cytoskeleton related
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827 WPWMKLYF (6)
Dystrophin
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2108 FHYDIKIFNQW (42)
G-protein-coupled receptors
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-AR
c
359 FVFFNWLGY (21)
Endothelin R (ET
A
) 146 WPFDHNDFGVF (8)
mAcR
c
422 WTIGYWLCY (18)
Growth-factor receptors
EGF-R 898
WSYGVTVW (15)
Insulin-R 1220
WSFGVVFW (43)
PDGF-R
c
887 WSFGILLWEIF (44)
Channels
Aquaporin-1
c
210 WIFWVGPF (45)
IP
3
-sensitive Ca
21
channel
2452
YLFSIVGYLFF (46)
Others
Caveolin
c
92 FTVTKYWFY (12, 13)
NSF 138
FSFNEKLF (38)
Cholera toxin A 138
YGWYRVHF (18)
subunit
a
PKC, protein kinase C; HC, heavy chain; AR, adrenergic receptor;
ET
A
, endothelin A; EGF-R, epidermal growth factor receptor; insulin-R,
insulin receptor; PDGF-R, platelet-derived growth factor receptor; IP
3
,
inositol triphosphate.
b
First residue of the motif in the protein sequence.
c
The caveolin binding motif is present in other members of the
protein family.
d
More than one possible caveolin binding motif in the protein.
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