From the Department of Pediatrics, United States
Department of Agriculture/Agricultural Research Service
Children's Nutrition Research Center, Baylor College of Medicine and
Texas Children's Hospital, Houston, Texas 77030 and the Departments of
¶ Ophthalmology and Visual Science and ** Pediatrics and the
Department of Pharmacology, Boyer Center for Molecular
Medicine, Yale University School of Medicine,
New Haven, Connecticut 06536
Received for publication, November 30, 2000, and in revised form, February 28, 2001
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ABSTRACT |
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The Hedgehog signaling pathway is involved in
early embryonic patterning as well as in cancer; however, little is
known about the subcellular localization of the Hedgehog receptor
complex of Patched and Smoothened. Since Hh has been found in lipid
rafts in Drosophila, we hypothesized that Patched and
Smoothened might also be found in these cholesterol-rich microdomains.
In this study, we demonstrate that both Smoothened and Patched are in caveolin-1-enriched/raft microdomains. Immunoprecipitation studies show that Patched specifically interacts with caveolin-1, whereas Smoothened does not. Fractionation studies show that Patched and caveolin-1 can be co-isolated from buoyant density fractions that represent caveolae/raft microdomains and that Patched and
caveolin-1 co-localize by confocal microscopy. Glutathione
S-transferase fusion protein experiments show that the
interaction between Patched and caveolin-1 involves the caveolin-1
scaffolding domain and a Patched consensus binding site.
Immunocytochemistry data and fractionation studies also show that
Patched seems to be required for transport of Smoothened to the
membrane. Depletion of plasmalemmal cholesterol influences the
distribution of the Hh receptor complex in the caveolin-enriched/raft
microdomains. These data suggest that caveolin-1 may be integral for
sequestering the Hh receptor complex in these caveolin-enriched
microdomains, which act as a scaffold for the interactions with the Hh protein.
The Hedgehog signaling pathway, first described in
Drosophila and conserved in vertebrates, is fundamental in
early embryonic patterning of many structures, including the neural
tube, axial skeleton, limbs, and lungs. Sonic Hedgehog (Shh),
the most studied of the three vertebrate homologs of
Drosophila Hedgehog, is a secreted protein that acts on
target cells to increase transcription of several genes, including
members of the Wnt and transforming growth factor- Little is known about the structure or function of the Hedgehog
receptor complex or the possible role of accessory proteins or lipids.
The Ptc protein is predicted to have at least 12 transmembrane domains,
and although it has little homology to other known receptors, it has
been shown to directly bind Shh. The transmembrane domains of Ptc have
a high degree of homology to the sterol-sensing domains of several
proteins involved in cholesterol processing and trafficking, including
NPC-1 (Niemann-Pick C
protein-1) (1, 2), 3-hydroxy-3-methylglutaryl-CoA reductase, and SCAP (sterol regulatory element-binding
protein cleavage-activating
protein). Smo encodes a serpentine, seven-transmembrane protein with characteristics of a G-protein-coupled receptor, including
a glycosylated extracellular N terminus. Smo, however, does not
directly bind Shh. The interaction with Ptc seems to be mediated
through the N-terminal domain and/or the first two transmembrane
domains of Smo,1 with most of
the signaling activity mediated through the third intracellular loop
and the seventh transmembrane domain (3). The exact mechanism through
which Smo transduces the Shh signal remains unclear, but it most
probably involves a conformational change in the receptor complex, as
the Ptc·Smo·Shh complex can be co-immunoprecipitated (4). The Shh
protein undergoes autoproteolytic cleavage with covalent attachment of
a cholesterol moiety to the N-terminal component of the protein. This
modified N-terminal product is responsible for most of the apparent
biological activity of the Shh protein. The cholesterol modification is
not absolutely required for binding to Ptc or for limited biological
activity, as several model systems have shown a response utilizing a
bacterially derived Shh-N fusion protein (5). It has been proposed,
however, that covalently linked cholesterol may modulate Shh activity, possibly through the sterol-sensing domains of Ptc, increasing the
efficiency of signal transduction (6).
Caveolae are non-clathrin-coated invaginations of the plasma membrane
that are important in endocytosis, cholesterol trafficking, and
sequestering various lipid-modified signaling molecules in discrete
microdomains. The caveolins, a family of three protein isoforms, are
the major coat proteins of caveolae. Caveolin-1 directly binds to and
transports cholesterol from the Golgi to the plasma membrane, and this
association is required for caveolar formation (7-11). Caveolae are
enriched in cholesterol and sphingolipids, are insoluble in nonionic
detergents such as Triton X-100, and can be isolated as low density
buoyant membranes in the absence of detergents. Associated with these
complexes are various lipid-modified signaling molecules, including
Ha-Ras, endothelial nitric-oxide synthase, serine/threonine
kinases, several G-protein Since very little is known about the cellular localization of members
of the Shh signaling pathway, we hypothesized that components of the
pathway were in caveolae or lipid raft domains of cells. Recent data
have shown that Hh is trafficked to lipid rafts in Drosophila, most probably through its association with
cholesterol (16). Given these recent data and our preliminary
observations of the Ptc trafficking pattern, we hypothesized that the
Shh receptor complex is also targeted to these cholesterol-rich
microdomains on the plasma membrane through an association between the
Shh receptor Ptc and perhaps caveolin-1. In this report, we show that Ptc and caveolin associate with each other in the caveolar/lipid raft
fraction of the plasma membrane, and we show data strongly implicating
cholesterol as a key player in the transport and, most likely, the
function of the Hedgehog receptor.
Constructs and Introduction of Mutations--
A full-length
human Ptc-GFP2
construct (cDNA gift from Rune Toftgard) was made by creating an
NheI/SalI cDNA fragment, ligating an 8-base
pair linker to create an EcoRI site at the 5'-end, and then
cloning this cDNA fragment into a mammalian GFP transfection vector
(GFP vector C2, CLONTECH). This placed the GFP
cDNA at the 5'-end of Ptc. The full-length human Smo-GFP construct
was made by creating a HindIII/KpnI cDNA
fragment (gift from Carol Wicking) and cloning it into a GFP
transfection vector (GFP vector N3, CLONTECH),
which placed the GFP cDNA at the 3'-end of Smo. The
NheI/SalI Ptc cDNA fragment was also cloned
into an expression vector (pCI-Neo mammalian expression vector,
Promega), and the HindIII/KpnI Smo cDNA
fragment was cloned into a FLAG mammalian transfection vector
(pFLAG-CMV5a, Sigma), which placed the FLAG tag at the 3'-end of the
Smoothened cDNA. All constructs were sequenced to confirm that the
GFP or FLAG tags were in frame. The Myc-tagged caveolin-1 construct was
a gift from Dr. Michael Lisanti (Albert Einstein College of Medicine).
The putative caveolin-binding motif found within the Patched protein
sequence (amino acids 788-798) was altered by polymerase chain
reaction-based site-directed mutagenesis (Stratagene) using conditions
recommended by the manufacturer. The peptide sequence YDFIAAQFKYF was altered
by replacing the underlined aromatic amino acids with alanine
(ADAIAAQAKYA).
Synthesis of an 82-base pair oligonucleotide (carried out at the Keck
Facility, Yale University/Howard Hughes Institute) that introduced
eight nucleotide changes into the wild-type sequence resulted in a
cDNA that would encode the altered protein. Sequencing of the
entire PtcBS-GFP construct (where PtcBS is the caveolin-binding site on
Ptc) confirmed the addition of the appropriate mutations. The altered
construct was cloned into GFP vector C2 as described above
(forward primer, 5'-GTACCTCGGGAAACCAGAGAA(T/G)(A/C)TGAC(T/G)(T/C)TATTGCTGCACAA(T/G)(T/C)CAAATAC(T/G)(T/C)TTCTTTCTACAACATGTATATAGTCACCC).
Cell Culture and Antibodies--
COS cells were maintained in
Dulbecco's modified Eagle's medium with 10% fetal calf serum and
antibiotics (penicillin and streptomycin). Chinese hamster ovary cells
were maintained in Ham's F-12 medium with 10% fetal calf serum and
antibiotics. An anti-Patched C terminus polyclonal antibody was a gift
from Dr. Allen Bale (Yale University, New Haven, CT). The monoclonal
antibodies were purchased as indicated: anti-Myc (Invitrogen),
anti-FLAG (Sigma), anti-caveolin-1 (Transduction Laboratories,
Lexington, KY), anti- Co-immunoprecipitation and Western Blotting--
COS cells were
grown to 60% confluence and then transiently transfected (Effectene,
QIAGEN Inc.) with the Patched-GFP and caveolin-Myc constructs under
conditions recommended by the manufacturer. Cells were allowed to
recover and express protein for 36 h and then lysed in lysis
buffer on ice (50 mM Tris (pH 8.0), 140 mM NaCl, 1% Triton X-100, 0.4% deoxycholate, and protease inhibitors (Complete mini-protease inhibitor mixture tablet, Roche Molecular Biochemicals)). The lysate was incubated on ice for 30 min and then
centrifuged at 12,000 × g for 2 min at 4 °C. The
anti-Patched polyclonal antibody (5 µl of serum) or the
anti-caveolin-1 monoclonal antibody (2.5 µg) was added to the lysate
and rotated for 1.5 h at 4 °C. Protein G-agarose (20 µl of a
50% suspension in PBS; Sigma) was added and rotated for 1 h at
4 °C. The beads were collected by centrifugation (brief pulse in a
microcentrifuge) and then washed three times with lysis buffer. The
beads were resuspended in 2× Laemmli loading buffer and boiled for 5 min, and the supernatant was loaded onto an SDS-polyacrylamide gel.
Samples were separated by SDS-polyacrylamide gel electrophoresis using
a 6% gel for the Patched and Smoothened proteins and a 15% gel for
detection of the caveolin protein. The samples were transferred to
nitrocellulose membrane (BiotraceTM, Gelman Instrument
Co.), incubated in blocking solution (PBS, 0.1% Tween, and 5% nonfat
dry milk) for 1 h at room temperature, and then washed twice with
PBS/Tween. The membrane was incubated with the primary antibody
(anti-caveolin-1 antibody or anti-Ptc polyclonal antibody) in blocking
solution, rotated for 1 h at room temperature, and then washed
three to four times with PBS/Tween. The membrane was then incubated
with the appropriate secondary antibody tagged with horseradish
peroxidase, and bands were detected by chemiluminescence using the ECL
detection system and reagents supplied by Amersham Pharmacia Biotech.
The membrane was exposed to film for up to 20 min and then developed in
an Eastman Kodak X-Omat M43A processor.
Confocal Microscopy--
COS cells were grown to 60%
confluence on 35-mm coverslip plates (Mattek Corp., Ashland, MA),
transiently transfected using Effectene transfection reagent, and
allowed to recover for 24-36 h. Cells were examined live under a
confocal microscope (Zeiss, 60× confocal objective; or Olympus, 60×
confocal objective) and assessed for localization and trafficking of
the Ptc protein. Multiple images were obtained from the same cell, and
a three-dimensional reconstruction of the cell was performed (NIH Image
software). Time series and photobleaching experiments were also performed.
Immunocytochemistry--
COS cells were grown to 60-80%
confluence in 6-well dishes containing untreated coverslips. They were
transiently transfected and allowed to recover for 36-48 h. The cells
were fixed and permeabilized with methanol at Subcellular Fractionation--
Fractionation experiments were
performed using a non-detergent method for isolation of
caveolin-1-enriched buoyant membranes (20) and modified as described
(39). 100-mm plates of COS cells were transiently transfected with
Ptc-GFP or PtcBSMut and allowed to recover and express protein for
48 h. To avoid interference from up-regulation of endogenous Ptc,
cells transfected with Smo-GFP alone were processed after 40 h.
The plates were then washed three times with PBS on ice. Cells were
lysed in a solution containing 500 mM
Na2CO3 (pH 11.0) with protease inhibitors and
incubated on ice for 10 min. The cells were scraped from the plate and
homogenized in a Dounce homogenizer and then transferred to an
Eppendorf tube and centrifuged at 1000 × g for 10 min
at 4 °C. The supernatant was collected in a new Eppendorf tube and
sonicated on ice. The supernatant was mixed with an equal volume of an
85% (w/v) sucrose/MB solution (MB = 25 mM
A-morpholine-ethanesulfonic acid (pH 6.5) and 0.15 M NaCl),
allowed to equilibrate for 2 h at 4 °C, and then placed at the
bottom of a ultracentrifuge tube. An overlay of 6 ml of a 30% (w/v)
sucrose/MB solution and then 3.5 ml of a 5% (w/v) sucrose/MB solution
was added and centrifuged at 35,000 rpm for 18 h at 4 °C in a
Sorvall UltraPro-8 using a TH641 rotor. The gradient was fractionated
into 1-ml fractions taken from the top, mixed with an equal volume of
2× Laemmli loading buffer, and boiled for 5 min, and protein was
separated by SDS-polyacrylamide gel electrophoresis. The gels were
processed as described above, and the membranes were incubated with the
anti-Ptc C terminus polyclonal antibody to detect Patched, with
the anti-caveolin monoclonal antibody to detect caveolin-1, with the
anti-FLAG M2 monoclonal antibody to detect Smo, with the anti- GST Fusion Proteins--
GST fusion constructs of full-length
caveolin-1 and the caveolin-binding site (amino acids 81-101) were
made by creating two BamHI/EcoRI cDNA
fragments by polymerase chain reaction and cloning these fragments into
a GST vector (pGEX-2TK, Amersham Pharmacia Biotech). A GST fusion
protein of the putative PtcBS (amino acids 772-810) was made by
creating EcoRI/BamHI cDNA fragments by
polymerase chain reaction and cloning this fragment into the GST
vector. The constructs were sequenced to confirm that the GST cDNA
was in frame and then transformed into Escherichia coli
DH5
COS cells were transiently transfected with either the Patched or
caveolin-1 construct and lysed 36 h later as described above. The
GST beads (1-5 µg of protein) were incubated with cell lysate (300 µg of protein) for 2 h at 4 °C. The complexes were washed four times with wash buffer (50 mM Tris (pH 8.0), 400 mM NaCl, and 1 mM EDTA); the beads were
resuspended in 2× Laemmli loading buffer, boiled for 5 min, and
briefly centrifuged; and the supernatant was separated by
SDS-polyacrylamide gel electrophoresis. The protein was transferred to
a nitrocellulose membrane and processed as described above for
detection of the Patched or caveolin-1 proteins.
Patched Associates with Caveolin-1--
Ptc and Smo proteins are
difficult to study in untransfected mammalian cells due to their low
base-line levels of expression. Others who have studied endogenous Ptc
in Drosophila (17, 18) and epitope-tagged Ptc in mammalian
cells (19) have shown that the majority of Ptc is found in
intracellular vesicles, with a small proportion of the protein found at
the membrane. To assess base-line localization and trafficking patterns
for wild-type Ptc in our model system, we transiently transfected COS
cells with a Ptc-GFP construct and examined living cells by confocal microscopy. As shown in Fig.
1A, wild-type Ptc-GFP was
enriched in intracellular membranes, with the majority in the
perinuclear region, reminiscent of the ER/Golgi (see also Supplemental
Figs. 3 (lower panel) and 4 (upper
panel)). Using time-lapsed microscopy, these small packets
of protein trafficked from the Golgi to and along the cell membrane
(Ptc.mov). The vesicular pattern displayed by Ptc-GFP was
consistent across cell lines (Chinese hamster ovary, Madin-Darby canine
kidney, HepG2, and the medulloblastoma cell line Daoy) and was
consistent in cells transfected with untagged Ptc detected by
immunocytochemistry using the anti-Ptc C terminus antibody
(Supplemental Fig. 1A). A three-dimensional reconstruction of images obtained from a Z-series suggested that a portion
of the Ptc protein localized at or just under the plasma membrane. The
pattern was reminiscent of proteins associated with caveolae and
their major structural protein, caveolin-1.
Next, we performed studies to determine if Ptc and caveolin-1
co-localize. Immunofluorescent microscopy of cells transfected with the
Ptc-GFP cDNA (Fig. 1A, left panel) and
immunolabeled for endogenous caveolin-1 (middle panel)
demonstrated co-localization of the two proteins (right
panel). In addition, cotransfection of a Myc-tagged version of
caveolin-1 (caveolin-Myc) and Ptc-GFP constructs showed that the
proteins co-localized both within the cell and at the plasma membrane
(Fig. 1B). To examine if these proteins can interact
biochemically, we performed co-immunoprecipitation in COS cells
transiently transfected with Ptc-GFP and caveolin-Myc. As shown in Fig.
2 (A and B),
immunoprecipitation of caveolin-1 (left panel)
resulted in the co-association of Ptc, and immunoisolation of Ptc
(middle panel) resulted in the association of caveolin-1. To
investigate the potential interaction between Smo and caveolin-1, co-immunoprecipitation experiments were performed on lysates of COS-1
cells cotransfected with Smo-GFP and caveolin-Myc. Western blotting of
the immunoprecipitates (Fig. 2C) showed that there was no
association between these two proteins, and additional immunocytochemistry studies failed to show co-localization between the
two proteins (Supplemental Fig. 2).
Smoothened Interacts with Patched, but Does Not Interact with
Caveolin-1--
It is known that several G-protein-coupled receptors
are sequestered in a latent phase in caveolar or raft microdomains of the plasma membrane (9, 14, 15). Because Smo has significant homology
to G-protein-coupled receptors, we sought to determine if the presence
of Smo is necessary for the interaction between Ptc and caveolin-1.
Transfection of Smo-GFP into COS cells (Fig. 3A, right panel)
resulted in a more uniform intracellular distribution compared with Ptc
(left and middle panels). There was no detectable Smo protein at the membrane, suggesting that most of the expressed protein was in the ER and Golgi (Supplemental Figs. 3B and
4, upper panels). However, cotransfection of FLAG-tagged Smo
(Smo-FLAG) (Fig. 3C, second panel) with Ptc-GFP
(first panel) demonstrated that the Ptc and Smo proteins
co-localized within discrete vesicles within the cytosol and at the
membrane (third and fourth panels), suggesting
that Ptc is necessary for trafficking Smo to the membrane. Moreover,
Smo and Ptc were co-associated based on coprecipitation of the proteins
from transfected cells (Fig. 3B). Collectively, these data
suggest that the Ptc·Smo receptor complex forms prior to insertion in
the plasma membrane and traffics as a heteromeric complex after
synthesis in the ER.
Patched Associates with Caveolin-1 in Cholesterol-rich Microdomains
of the Plasma Membrane--
Given the above data showing a biochemical
interaction between Ptc and caveolin-1 and co-localization of the
proteins and the recent experiments showing Hh in lipid rafts (16), it
seemed plausible that the Shh receptor complex might also be localized to these membrane microdomains. COS-1 cells were transiently
transfected with the Ptc-GFP cDNA, and caveolin-enriched
microdomains were isolated using a detergent-free purification method
(20, 21). Western blot analysis of sequential fractions from the
sucrose gradient showed the majority of endogenous caveolin-1 in
buoyant light membrane fractions 3 and 4 (Fig.
4A). Consistent with the confocal imaging studies, the majority of Ptc co-fractionated with
Golgi and ER markers (lanes 8-11); however, a significant amount of Ptc was also found in fractions 3 and 4, confirming that Ptc
localizes to the same microdomains as caveolin-1. Similar results were
obtained when these experiments were repeated on cells transfected with
both Ptc and caveolin-Myc constructs.
Because our confocal microscopy data suggested that Ptc and Smo
trafficked as a complex to the membrane, we hypothesized that Smo alone
would not be found in the caveolin-enriched buoyant fractions, whereas
Ptc and Smo would co-fractionate when both were transfected into cells.
COS-1 cells were transfected with either Smo-FLAG alone (40 h) or
Ptc-GFP and Smo-FLAG (48 h). The Smo-FLAG-alone cells were processed a
few hours earlier to avoid possible interference from up-regulated
endogenous Ptc, as Smo is a positive regulator of this pathway. Cells
were processed again by a non-detergent method and centrifuged through
a continuous sucrose gradient. Western blotting of sequential fractions
from cells transfected with Smo alone (Fig. 4B) showed that
the majority of the Smo protein was isolated in the heavier fractions
(fractions 9-11), whereas caveolin continued to be isolated in
the buoyant fractions (fraction 4). In cells that were
cotransfected, however, Smo co-fractionated with both Ptc and caveolin
in the buoyant fractions (Fig. 4C), with a significant
amount of protein still found in the ER/Golgi fractions as well. This
again supports the idea that Ptc and Smo traffic as a complex and can
be found together in the caveolin-enriched raft domains.
Interaction between Patched and Caveolin-1 Involves the Putative
Caveolin-1 Scaffolding Domain and a Consensus Binding Motif in
Patched--
Caveolin-1 interacts with various proteins through an
intracellular region termed the caveolin-1 scaffolding domain (amino acids 82-101) (reviewed in Ref. 12). To assess whether the interaction between Ptc and caveolin-1 is mediated through this scaffolding domain,
GST fusion proteins of the full-length caveolin-1 and the region of
caveolin encoding the scaffolding domain (amino acids 82-101) as well
as GST alone were incubated with lysates of COS-1 cells expressing the
Ptc-GFP construct. As shown in Fig. 5A, Ptc in cell lysates
interacted with full-length caveolin-1 and the caveolin-1 scaffolding
domain, but not with GST alone. Approximately 10-30% of input Ptc
interacted with the GST-caveolin fusion proteins.
Many proteins that can potentially bind to caveolin-1 contain a
specific caveolin-binding sequence motif that may facilitate interaction with the caveolin-1 scaffolding domain
(
To assess the function of this putative binding motif on Ptc, we
mutated the sequence by replacing the underlined aromatic amino acids
with the amino acid alanine. Previous studies have shown that these
substitutions inhibit the functional interaction between endothelial
nitric-oxide synthase and caveolin-1 (12). The altered Ptc construct,
PtcBSMut, was cloned into the GFP vector and transiently transfected
into COS cells. Cells were examined live under the confocal microscope
and compared with wild-type images. Unlike wild-type Ptc, which
accumulated in the perinuclear region and moved to and from the
membrane, the PtcBSMut protein accumulated in the same region, but
failed to traffic throughout the cell (Fig.
6A, left panel).
Immunocytochemistry studies of PtcBSMut and caveolin-Myc also failed to
show significant co-localization of the two proteins at the membrane,
but did show some overlap within the perinuclear region (Fig.
6A, right panel). Co-immunoprecipitation studies
on lysates derived from cells cotransfected with PtcBSMut and
caveolin-1 showed that, despite altering this putative binding site,
PtcBSMut and caveolin-1 continued to associate, but to a lesser degree
(Fig. 6B). Fractionation of these cells revealed some
PtcBSMut protein in the lipid raft component (Fig. 6C,
middle panel), but this seemed decreased in comparison with
wild-type Ptc-GFP (upper panel). This suggests that this
binding site is important for the association between Ptc and
caveolin-1, but that additional sites of interaction or accessory
proteins may be necessary for the interaction of the proteins in
vivo.
Role of Cholesterol in Trafficking of the Hedgehog Receptor to
Lipid Rafts on the Plasma Membrane--
Cholesterol is a key component
of lipid rafts in mammalian cells, and similar sterols seem to function
as a necessary component in Drosophila raft formation (16).
Prior studies in mammalian cells using methyl-
Detergent-free methods of caveolar isolation have shown that ~90% of
caveolin-1 in the cell is associated with lipid rafts (24, 25) and that
cholesterol is necessary for the insertion of caveolin-1 into these
rafts. We expected treatment with MBCD to cause a significant decrease
in the amount of Ptc and caveolin-1 recovered from the lipid raft
fraction of those cells. We performed detergent-free isolation and
sucrose gradient fractionation on lysates of Ptc-GFP-expressing COS-1
cells treated with MBCD or control medium containing serum. Fig.
8 shows that, in control cells, the
majority of caveolin-1 and a smaller proportion of Ptc-GFP were
recovered in the raft fractions. In cells treated with MBCD, both
caveolin-1 and Ptc were shifted from the raft fractions to the heavier
membrane fractions. This further suggests that cholesterol is important
for the correct trafficking of both Ptc and caveolin-1 to
caveolae/lipid rafts.
In this report, we provide evidence supporting the concept that
components of the Hh signaling pathway reside in caveolin-enriched microdomains. This assertion is supported by data demonstrating co-localization of Ptc with the caveolar coat protein caveolin-1, co-precipitation of Ptc (but not Smo) with caveolin-1, and
co-fractionation of Ptc and Smo with caveolin-1 in buoyant membrane
fractions containing caveolae/lipid raft microdomains. Furthermore,
depletion of plasma membrane cholesterol results in the decreased
amounts of both Ptc and caveolin-1 found in caveolae/lipid raft
microdomains, suggesting that the local concentration of cholesterol
influences the trafficking pattern of both proteins. Thus, our data, in
conjunction with recent results demonstrating enrichment of Shh in
lipid rafts, support the idea that Hh signaling resides in specialized,
cholesterol-enriched microdomains of the cell.
The Hh receptor complex is an interesting and unusual heterodimer in
that Ptc functions as the receptor domain for the Hh ligand, and Smo
functions as the signaling domain for the complex. Little is known
about the interactions between these two subunits, which compose the Hh
receptor complex. Analysis of tumors suggests that Ptc functions not
only at critical points in development, but also as a tumor suppressor
where loss of both copies is required for tumor formation (29-31).
Activating mutations in Smo have also been described that implicate it
as a potential oncogene (32, 33). Current knowledge has suggested that
Ptc and Smo exist as a complex on the membrane, where Ptc inhibits the
tonic activity of Smo in the absence of the Hh signal. Upon binding of
Hh to its receptor Ptc, this inhibition of Smo is released, through unknown mechanisms, and Smo is then free to transduce the Hh signal. It
is thought that, in the absence of Ptc, Smo alone is localized to the
plasma membrane, where its signal transduction is not under Hh control.
Our initial data from transfections of Ptc and Smo indicate that, in
the absence of Ptc, Smo does not traffic to the membrane at all, but
remains intracellular (Fig. 3). Confocal microscopy of living cells
transfected with Ptc alone shows that it is able to traffic effectively
to the membrane from the Golgi without Smo. The results of our
fractionation studies (Fig. 4) show that Ptc alone co-fractionates with
caveolin-1 in the lipid raft compartment, whereas Smo alone does not.
When Smo is cotransfected with Ptc, however, Smo is found to
co-fractionate with Ptc and caveolin in the buoyant membrane fractions.
Experiments with various Ptc mutants1 also show that
despite very abnormal trafficking patterns exhibited by some of the Ptc
mutants, Smo consistently co-localizes with Ptc. Collectively, these
data suggest that Ptc and Smo form a complex very early on, most
probably in the Golgi, and are trafficked intact to lipid-rich
microdomains on the plasma membrane (Fig. 9).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
families, and its receptor Patched. Patched (Ptc) is predicted to
encode a large transmembrane protein that acts as a negative regulator
of the pathway. It associates with a second transmembrane protein,
Smoothened (Smo), which is a positive regulator of the pathway. Prior
genetic and biochemical studies indicate that the two proteins form an
unusual complex at the membrane that is inactive in the absence of the
Shh ligand. Once the Shh protein binds to Ptc, this relieves the
inhibition of Smo (by unknown mechanisms) and allows transduction of
the Hedgehog signal.
-subunits, and Src tyrosine kinases
(reviewed in Ref. 12). It has been postulated that caveolae may be
signaling centers for multiple pathways and may regulate cross-talk
between different pathways. Caveolin, per se, may directly
influence signaling by serving as a molecular scaffold for signaling
complexes (13-15) or indirectly modulate signaling by influencing
cholesterol trafficking.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-COP (clone maD, Sigma), and anti-GRP78
(Transduction Laboratories).
20 °C for 2-4 min
and then washed five times with PBS. Slides were blocked with goat
serum; shaken for 1 h at room temperature; incubated with the
anti-caveolin monoclonal antibody (1:100) diluted in PBS, 2% bovine
serum albumin, and 10% goat serum; and shaken for 1 h at room
temperature. The slides were washed four to five times with PBS and
then incubated with Cy3-linked goat anti-mouse IgG secondary
antibody (1:2000; Amersham Pharmacia Biotech) for 30 min at room
temperature in the dark. Slides were washed five times with PBS and
once with distilled water and sealed with Crystal Mount. Slides
were then examined under the confocal microscope.
-COP
monoclonal antibody to detect the Golgi protein, and with the
anti-BIP/GRP78 antibody to detect the ER.
cells. A 1:100 dilution of an overnight culture of each
construct and the GST vector alone was grown in 100 ml of 2× yeast
tryptone medium for 3 h at 37 °C and then induced with
isopropyl-
-D-thiogalactopyranoside (0.5 mM)
for 4 h at 37 °C. The bacteria were pelleted at 7700 × g for 10 min and washed with 3 ml of STE buffer (150 mM NaCl, 7.5 mM Tris (pH 8.0), and 3 mM EDTA). The bacteria were resuspended in 3 ml of STE
buffer with lysozyme (100 µg/ml) and placed on ice for 15 min.
Dithiothreitol to a final concentration of 5 mM, phenylmethylsulfonyl fluoride to a final concentration of 100 µM, N-lauroylsarcosine to a final
concentration of 1.5%, and protease inhibitors were added, and the
mixture was incubated on ice for 30 min. The lysate was homogenized
with 15 strokes of a Dounce homogenizer and sonicated until clear. The
lysate was centrifuged at 15,000 rpm in a Sorvall SS34 rotor for 15 min
at 4 °C. The supernatant was removed, and Triton X-100 was added to
a final concentration of 2%. Glutathione beads (100 µl of a 50%
slurry in PBS) were added and incubated for 18 h at 4 °C. The
beads were collected by centrifugation at 500 × g for
5 min and washed five times with STE buffer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Co-localization of Ptc and caveolin-1 by
immunocytochemical staining. In A, COS-1 cells
were transfected with Ptc-GFP, fixed, and stained for native caveolin-1
(Cav) with the anti-caveolin-1 monoclonal antibody. Single
channel confocal imaging shows a vesicular pattern, which seems to be
typical of both Ptc and caveolin-1. Dual channel confocal imaging shows
co-localization of Ptc and caveolin-1 both within the cell and at the
plasma membrane (yellow). In B, COS cells
transfected with Ptc-GFP and a Myc-tagged form of caveolin-1
(CavMyc) again showed co-localization of the two proteins by
confocal microscopy.
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Fig. 2.
Caveolin-1 co-immunoprecipitates with Ptc,
but not with Smo. In A, lysates of COS cells
transiently transfected with Ptc-GFP and caveolin-Myc constructs were
immunoprecipitated (IP) with the anti-caveolin-1 monoclonal
antibody, and proteins were Western-blotted for Ptc (upper
panel) or caveolin-1 (Cav; lower panel).
Lane 1 shows co-immunoprecipitation of caveolin-1 and Ptc.
Incubating lysates with a nonimmune mouse IgG (lane 2) did
not result in the precipitation of either caveolin-1 or Ptc. Lane
3 shows Ptc in total cell lysates. In B, lysates of COS
cells transiently transfected with Ptc-GFP and caveolin-Myc constructs
were immunoprecipitated with the anti-Ptc C terminus antibody
(Ptc CT Ab), and proteins were Western-blotted for
caveolin-1 (upper panel) or Ptc (lower panel).
Lane 1 again shows co-immunoprecipitation of Ptc and
caveolin-1, whereas there was no precipitation of either Ptc or
caveolin-1 using preimmune serum in lane 2. Lane
3 used the anti-caveolin-1 monoclonal antibody (MAb) to
detect caveolin-1 in these cell lysates. In C, lysates of
COS cells transiently transfected with the Smo-GFP and caveolin-Myc
constructs were immunoprecipitated with the anti-Smo C terminus
antibody, and proteins were Western-blotted for caveolin-1 (upper
panel) or Smo (lower panel). No co-immunoprecipitation
was detected between Smo and caveolin-1 in lane 1. The
negative control in lane 2 used preimmune serum to
precipitate nonspecific proteins. The positive control in lane
3 used the anti-caveolin-1 monoclonal antibody to detect
caveolin-1 in these cell lysates. These data are representative of at
least three experiments each.
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Fig. 3.
Ptc and Smo co-immunoprecipitate and form a
complex prior to transport to the plasma membrane. A,
confocal microscopy of living COS-1 cells transfected with Ptc-GFP or
Smo-GFP. Ptc-GFP alone (left panel) shows a distinctive
vesicular pattern, with the majority of protein localized to the
perinuclear region of the cell. A significant amount of Ptc protein can
also be seen within vesicles on or near the plasma membrane
(inset and middle panel). Smo-GFP alone localized
to the cytoplasm, with no detectable protein at the membrane
(right panel). B, immunoprecipitation of Smo-FLAG
results in co-immunoprecipitation of Ptc-GFP. Lane 1 shows
the Western blot for Ptc after precipitation with the anti-Smo
antibody. Lane 3 shows the Western blot for Smo, shown in
its two phosphorylation forms, after precipitation using the anti-Ptc C
terminus antibody (Ptc CT). Lanes 2 and
4 are negative controls using preimmune serum. In
C, COS-1 cells cotransfected with the Ptc-GFP (first
panel) and Smo-FLAG (second panel) constructs display a
vesicular pattern, with co-localization of Ptc and Smo seen both within
the cell and at the membrane (third and fourth
panels).
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Fig. 4.
Ptc co-distributes and recruits Smo into
caveolin-enriched buoyant membranes. A, shown are
the non-detergent isolation and sucrose gradient fractionation of
lysates from COS cells transiently transfected with Ptc-GFP alone for
48 h. Lanes 1-11 represent sequential (top to bottom)
fractions that were processed for Western blotting with the anti-Ptc C
terminus antibody (upper panel) and the anti-caveolin-1
monoclonal antibody (lower panel). Similar to prior studies,
>90% of native caveolin-1 (Cav) was isolated in the
buoyant density fractions 3 and 4. Ptc was also found to localize to
these buoyant density fractions, as well as layers that represent the
Golgi and ER fractions. In B, lysates from COS cells
transfected with Smo-GFP alone were processed by the above method after
40 h to avoid interference from up-regulated endogenous Ptc. The
fractions were Western-blotted for the presence of Smo (upper
panel) and caveolin-1 (lower panel). The majority of
the Smo protein was isolated in the heavy fractions (fractions 9-11),
whereas caveolin-1 was isolated in the buoyant fractions (fraction 4).
In C, fractions from COS cells expressing both Ptc-GFP and
Smo-FLAG show that Ptc (upper panel), Smo (middle
panel), and caveolin-1 (lower panel) can be found in
buoyant membrane fractions.
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Fig. 5.
Interaction of Ptc with the putative
caveolin-1 scaffolding domain in vitro. In
A, lysates from COS-1 cells expressing Ptc-GFP were
incubated with GST fusion proteins of full-length caveolin-1 (Cav
FL; amino acids 1-178) and the caveolin-1 scaffolding domain
(Cav SD; amino acids 81-101) or with GST alone bound to
glutathione beads. GST alone served as a control for nonspecific
binding. After extensive washing, GST fusion proteins were eluted and
subjected to SDS-polyacrylamide gel electrophoresis analysis. Western
blot analysis for the presence of Ptc was performed using the anti-Ptc
C terminus antibody (Ptc CT Ab). Ptc bound specifically to
both full-length caveolin-1 and the caveolin-1 scaffolding domain,
whereas there was no binding of Ptc to GST alone. Equivalent amounts of
cell lysates, GST fusion proteins, and GST were used in these
experiments. In B, lysates from COS-1 cells expressing
epitope-tagged full-length caveolin-1 were incubated with GST fusion
proteins of PtcBS (amino acids 788-798) or with GST alone. PtcBS was
sufficient to specifically bind full-length caveolin-1 in these cell
lysates, whereas there was no binding with GST alone. Endothelial cell
lysate was used as a positive (Pos) control for the presence
of caveolin-1.
X
XXXX
,
XXXX
XX
, or the composite
X
XXXX
XX
, where
is the
aromatic amino acid tryptophan, phenylalanine, or tyrosine, and
X represents any other amino acid) (23). We searched the Ptc
protein sequence for the presence of this motif and found a single
sequence that matched the consensus binding motif:
YDFIAAQFKYF (amino acid
788-798). This sequence is highly conserved in Ptc between species as
well as in the Ptc homolog Patched-2, but is not found in Smo. Thus, we
expressed the putative caveolin-binding site on Ptc (PtcBS) as a GST
fusion protein. As shown in Fig. 5B, PtcBS was able to isolate caveolin-1 from cell lysates expressing caveolin-1, whereas GST
did not.
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Fig. 6.
Mutation of PtcBS alters protein trafficking
and association with caveolin-1. A, shown is the
confocal imaging of fixed COS-1 cells expressing PtcBSMut-GFP
(left panel) and caveolin-Myc (CavMyc;
middle panel). PtcBSMut-GFP represents a Ptc-GFP fusion
construct in which the consensus binding site for caveolin-1 has been
altered. Single channel imaging shows that PtcBSMut was unable to
distribute from the perinuclear region to the plasma membrane, whereas
caveolin-1 retained its usual pattern of distribution. Dual channel
imaging shows some co-localization of PtcBSMut and caveolin-1 in this
perinuclear area (right panel), but no co-localization at
the plasma membrane. B, immunoprecipitation of caveolin-1 in
cells transfected with Ptc-GFP or PtcBSMut and caveolin-Myc was
performed using the anti-caveolin-1 monoclonal antibody. Lanes
1 and 3 show Western blot analysis for Ptc and
PtcBSMut, respectively, using the anti-Ptc C terminus antibody
(Ptc CT Ab; upper panel). The negative control in
lane 2 shows no binding using mouse IgG. Lanes 4 and 5 are Western blots of cell lysates probed with the
anti-Ptc C terminus antibody. Immunoprecipitation of the above cell
lysates was then performed using the anti-caveolin-1 monoclonal
antibody (MAb; lower panel). Lanes 1 and 3 show Western blot analysis for caveolin-1 using the
anti-caveolin-1 monoclonal antibody. The negative control in
lane 2 using preimmune serum shows no binding.
Lanes 4 and 5 represent Western blot analysis of
transfected cell lysates. Equal amounts of cell lysate were used in
each lane for both of these experiments. C, COS-1 cells
expressing either wild-type Ptc-GFP or PtcBSMut constructs were
processed by a non-detergent method for isolation of cellular membranes
and centrifuged through a continuous sucrose gradient. Lanes
1-11 represent sequential fractions (top to bottom) probed with
the anti-Ptc C terminus antibody on Western blots. The
middle panel shows decreased isolation of PtcBSMut in the
caveolin-1 (Cav-1)-enriched fractions compared with
wild-type Ptc in the upper panel, consistent with the
microscopy data in A.
-cyclodextrin (MBCD)
have shown that cholesterol depletion abrogates the formation of
caveolae, which is reversed upon cholesterol replacement (38). Due to
the similarities between Ptc and several other proteins involved in
cholesterol biosynthesis and transport (1, 2) and the role of
caveolin-1 in cholesterol trafficking (8-11), we hypothesized that
cholesterol might be involved in the transport of the
Ptc·Smo·caveolin-1 complex to caveolae and/or insertion into the
plasma membrane. We transfected COS cells with Ptc-GFP or PtcBSMut and
Smo-FLAG and then treated them with serum depletion and/or MBCD.
Confocal microscopy of cells transfected with wild-type Ptc-GFP and
Smo-FLAG showed the receptor complexes localized to discrete vesicles,
which remained intracellular and co-localized with Golgi markers (Fig.
7A, left and
middle panels). During live confocal imaging, there was
little movement of these vesicles to the plasma membrane in the
serum-depleted cell group and no movement of these vesicles in the
MBCD-treated group. This abnormal trafficking pattern was reversed
after serum repletion (Fig. 7A, right panel). In
contrast, cells transfected with PtcBSMut and Smo-FLAG showed the
formation of complexes in intracellular vesicles that were unable to
traffic to the membrane (Fig. 7B, left and
middle panels) even after cholesterol replacement (right panel). This indicates that cholesterol is most
likely involved in the transport of the Ptc·Smo complex to the
membrane. The failure of PtcBSMut to traffic correctly after
replacement of cholesterol may be due to the fact that the mutated
caveolin-1-binding site is also located in the sterol-sensing domain of
Ptc.
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Fig. 7.
Serum
depletion/methyl- -cyclodextrin treatment of
cell transfected with Ptc-GFP or PtcBSMut-GFP: role of cholesterol in
trafficking. In A, COS-1 cells expressing Ptc-GFP were
subjected to serum depletion (SD) or MBCD (CD)
treatment, followed by serum repletion. Cells were examined by confocal
microscopy under each of these conditions. After 24 h of serum
depletion, very little Ptc protein was seen trafficking to the plasma
membrane (left panel). After 4 h of MBCD treatment,
there was no evidence of Ptc protein trafficking from the Golgi to the
plasma membrane (middle panel). The effects on trafficking
seen with serum depletion or MBCD treatment were completely reversed
after serum repletion (right panel). In B, COS-1
cells expressing PtcBSMut were subjected to serum depletion or MBCD
treatment, followed by serum repletion. Similar to base-line studies
using PtcBSMut constructs, the protein was not seen to traffic out of
the Golgi to the plasma membrane (left and middle
panels). This pattern was not reversed by serum repletion
(right panel).
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Fig. 8.
MBCD treatment decreases the recovery of Ptc
and caveolin-1 in buoyant membrane fractions. In A,
COS-1 cells expressing Ptc-GFP were treated with normal growth medium
or MBCD and then processed by the non-detergent method for membrane
isolation and sucrose gradient centrifugation. Fractions were probed by
Western blotting for the presence of Ptc or caveolin-1
(Cav-1). The control panel (upper panel), treated
with growth medium, shows that ~90% of caveolin-1 was isolated in
fractions 3 and 4. Ptc was found in these buoyant fractions as well as
in fractions 9-11, which represent Golgi- and ER-enriched membranes,
as confirmed by -COP (
Cop) and
BIP/GRP78 markers (lower panel). MBCD treatment
(middle panel) resulted in a shift of the caveolin-1 and Ptc
proteins from the buoyant fractions into the non-caveolar
fractions.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Model for interactions of the Hh receptor
complex, caveolin-1, and cholesterol. Evidence to date suggests
that Ptc and Smo interact early on, most probably in the Golgi complex.
Our data also show a direct interaction between Ptc and caveolin-1
(Cav), which is probably integral for Hh receptor complex
targeting to caveolar membranes. Serum depletion and MBCD treatment
studies suggest that there is most likely a role for cholesterol
(CHOL) in the control of normal receptor trafficking and
possibly function.
The raft hypothesis proposed by Simons and Ikonen (26)
postulates that these lateral assemblies, composed of
glycosphingolipids and cholesterol, function to aggregate certain
proteins while excluding others. These rafts form a liquid order phase
whose formation is driven by the interactions of the specific lipids involved. Caveolae, on the other hand, are specialized raft structures that are flask-shaped invaginations of the plasma membrane and are
coated by caveolins. Indeed, caveolin-1 and -2 are necessary for the
assembly of caveolae (36). In this study, we have used a well described
detergent-free method for isolating caveolin-1-enriched membranes and
lipid rafts. The strength of this method is that it can discern between
buoyant membranes (caveolae/rafts) and heavy membranes (ER, Golgi, and
cytoskeleton); however, it cannot distinguish between proteins in
caveolae versus lipid rafts. Here we show that Ptc
co-segregates with caveolin-1 to these lipid-rich microdomains of the
plasma membrane, consistent with the trafficking of other important
signaling molecules such as Ha-Ras, endothelial nitric-oxide synthase,
Src tyrosine kinases, and -subunits of G-proteins (reviewed in Refs.
9 and 12).
The caveolin family consists of three distinct proteins that are
differentially expressed in certain cell types. Caveolin-1 is the major
structural protein in caveolae and is found in abundance in adipocytes,
endothelial cells, and fibroblasts (10). The caveolin gene family has
been conserved from Caenorhabditis elegans to humans,
underscoring its evolutionary importance. Caveolin proteins form homo-
and hetero-oligomers that directly bind and require cholesterol for
insertion into the membrane. Caveolin-1 forms a hairpin-like structure,
with the central portion of the protein inserted into the membrane and
the N and C termini located in the cytoplasm (10). Work on
heterotrimeric G-proteins as well as Ha-Ras, endothelial nitric-oxide
synthase, and others has shown that caveolin-1 interacts with these
proteins through an area termed the caveolin-1 scaffolding domain
(amino acids 82-101) (12-14). Our GST fusion protein studies have
shown similar interactions between Ptc and caveolin-1, in which the
full-length caveolin-1 protein and the caveolin-1 scaffolding domain
were able to bind Ptc from transfected cell lysates, whereas GST alone did not. Analysis of proteins that interact with caveolin through its
scaffolding domain have shown that these proteins contain a common
sequence motif (X
XXXX
,
XXXX
XX
, or
X
XXXX
XX
, where
is Trp,
Tyr, or Phe) (23). Sequence analysis of Ptc revealed that it contains
such a motif (
X
XXXX
) in the region of
its seventh transmembrane domain, within the region of its sterol-sensing domain. As shown in Fig. 6, mutation of this binding motif by replacement of the aromatic amino acids with alanine produced
a Ptc protein that was unable to traffic out of the Golgi complex to
the membrane. However, mutation of these residues did not completely
abrogate the association between Ptc and caveolin-1, indicating that
other domains in Ptc, other proteins, and/or lipids may be involved in
this interaction. It is known that caveolin-1 directly binds
cholesterol and requires cholesterol for insertion into the plasma
membrane and that its expression is regulated at the transcriptional
level by cholesterol (8, 11, 34, 35). Our initial studies using serum
depletion and MBCD treatment suggest that it is quite likely that
cholesterol is integral for the correct trafficking of the Hh receptor
complex to the plasma membrane as well. This hypothesis is also
supported by the similarities between Ptc and NPC-1 and the role of
NPC-1 in cholesterol trafficking (1, 2). The similarities between Ptc
and NPC-1 raise interesting ideas about how Ptc traffics to the
membrane and the role of its sterol-sensing domains in its interactions
with Hh. Others have postulated that the Ptc sterol-sensing domain is
probably important for binding of the Hh ligand, but it may also be
critical for trafficking and localization of the receptor complex to
caveolar domains of the plasma membrane.
Hh itself is an unusual morphogen in that it possesses an autocatalytic cleavage domain that cleaves the protein and covalently links cholesterol to the N terminus, forming the active portion. Recent data have shown that Hh is also modified by the addition of palmitate or other acyl residues to a Cys residue on the N terminus that seem to anchor Hh in the outer leaflet of the membrane bilayer, making it function like a glycosylphosphatidylinositol-anchored protein (16). This raises the conundrum of how a protein anchored in the lipid bilayer can function as a secreted morphogen. Sequestration of Ptc in these lipid-rich microdomains may be a method for the cell to separate this signaling pathway from others and to promote the interaction between Ptc and Shh.
Work on signaling molecules such as Ha-Ras, Src tyrosine kinases,
endothelial nitric-oxide synthase, and G-protein-coupled receptors has
shown that caveolin-1 plays an important role in holding these
signaling molecules in a latent phase (13-15). Other studies have
shown that this latent phase is also required for interaction of some
of these molecules with caveolin-1 and that this interaction can be
abolished by conversion of the signaling molecule to the activated form
(20, 37). Collectively, results from several labs suggest that Ptc
sequesters Smo in an inactive state in the absence of the Hh ligand. An
alternative interpretation, based on our studies, is that caveolin-1
may negatively regulate this heterodimeric receptor complex in a latent
phase and that binding of Hh to Ptc removes this association, allowing
Smo to transduce the signal. In addition, it is feasible that the Hh receptor complex is formed in the Golgi, associates with caveolin-1 and
cholesterol, and is trafficked through an exocytic pathway to the
plasma membrane. Additional studies examining the roles of caveolin-1
and cholesterol in signal transduction, stimulated by the Hh receptor
complex and the biogenesis of these components, are clearly warranted.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Michael Lisanti for the caveolin-1 cDNA, Dr. Allen Bale for the anti-Ptc C terminus antibody, Rune Toftgard for the full-length human Ptc cDNA, and Carol Wicking for the full-length Smo cDNA. We also thank Dr. Gzegorsz Sowa for initial help with fractionation studies.
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FOOTNOTES |
---|
* This work was supported by the National Institutes of Health, the Swebelius Foundation, and the American Cancer Society and in part by the United States Department of Agriculture/Agricultural Research Service under Cooperative Agreement 58-6250-6001. The confocal microscope at Yale University School of Medicine was supported by the Fanny Rippel Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Supplemental Figs. 1-4.
This paper is dedicated to the memory of Dr. Mae R. Gailani, who died on November 24, 1999, during work on this project. She will be remembered as a great clinician, a superb scientist, and a wonderful friend.
§ To whom correspondence should be addressed: Children's Nutrition Research Center, Baylor College of Medicine, 1100 Bates St., Suite 10070, Houston, TX 77030. Tel.: 713-798-7045; Fax: 713-798-7057; E-mail: hkarpen@bcm.tmc.edu.
Published, JBC Papers in Press, March 1, 2001, DOI 10.1074/jbc.M010832200
1 H. E. Karpen, J. T. Bukowski, and M. R. Gailani, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
GFP, green
fluorescent protein;
PBS, phosphate-buffered saline;
ER, endoplasmic
reticulum;
GST, glutathione S-transferase;
MBCD, methyl--cyclodextrin.
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