From the Department of Molecular Pharmacology and the
Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New
York 10461, § Department of Pharmacology, School of
Medicine, University of Milan, 20129 Milan, Italy, ¶ Molecular
Pharmacology Group, Division of Biochemistry and Molecular Biology,
Institute of Biomedical and Life Sciences, University of Glasgow,
Glasgow G12 8QQ, Scotland, United Kingdom, and
Department of
Pathology, Washington University School of Medicine, St. Louis,
Missouri 63110
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ABSTRACT |
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Here we investigate the molecular
mechanisms that govern the targeting of G-protein The Because myristoylation is an irreversible modification, it has been
proposed that it may play only a passive role as a hydrophobic handle
or appendage for anchoring G Moreover, palmitoylation is required for a productive interaction
between G-protein-coupled receptors and G Recent evidence suggests that G-proteins are not homogeneously
distributed throughout the cell surface. Instead, they may be
concentrated within vesicle-like microdomains of the plasma membrane
termed caveolae that exist in most cell types. It has been proposed
that caveolae function as signaling depots where G-proteins and
G-protein-coupled receptors, as well as other signaling molecules, are
clustered (7-9).
Caveolae membranes fractionate as low buoyancy complexes on sucrose
density gradients and are resistant to solubilization by nonionic
detergents, such as Triton X-100, at low temperatures, due to their
specialized lipid composition that is rich in cholesterol and
glycosphingolipids (10, 11). The position of caveolae within these
gradients can be tracked by immunoblotting with specific antibodies
directed against the caveolin family of proteins, which are the
principal structural protein components of caveolae membranes. Caveolins interact with each other to form homo- and hetero-oligomeric complexes, bind cholesterol, and interact with a variety of signaling molecules. For example, caveolin binding to G Recent studies have indicated that point mutations that abolish
myristoylation and/or palmitoylation prevent the association of
Gi1 To evaluate the potential role of the NH2-terminal region
of G Materials--
The GFP cDNA was a gift of Dr. J. Pines
(Wellcome/CRC Institute, Cambridge, United Kingdom). It contains five
mutations (F64L, S65T, V163A, I167T, and S175G) resulting in improved
levels of fluorescence at 475 nm and is correctly folded at 37 °C.
The cDNA encoding nonpalmitoylated caveolin-1 was as described
previously (22). Antibodies and their sources were as follows: (i)
anti-caveolin-1 IgG (mAb 2297 and mAb 2234; gifts of Dr. J. R. Glenney, Transduction Laboratories); (ii) anti-GFP IgG (rabbit
polyclonal antibody; CLONTECH); and (iii) rabbit
anti-Gi1 Construction of Gi1 Cell Culture and Transient Transfection--
COS-7 and 293T
cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 2 mM
L-glutamine, 100 µg/ml streptomycin, and 100 units/ml
penicillin. Cells (30-50% confluent) were transfected using the
calcium phosphate precipitation method and routinely analyzed 48 h
after transfection.
Immunoblot Analysis--
Cellular proteins were resolved by
SDS-PAGE (12.5% acrylamide) and transferred to nitrocellulose
membranes. Blots were incubated for 2 h in TBST (10 mM
Tris-HCl, pH 8.0, 150 mM NaCl, and 0.2% Tween 20)
containing 2% powdered skim milk and 1% bovine serum albumin. After
three washes with TBST, membranes were incubated for 2 h with the
primary antibody (~1,000-fold dilution in TBST) and for 1 h with
horseradish peroxidase-conjugated goat anti-rabbit/mouse IgG
(~5,000-fold dilution). Proteins were detected using the ECL detection kit (Amersham). For cell fractionation, cells were harvested and resuspended in 0.5 ml of hypotonic buffer (5 mM
Tris-HCl, pH 7.5, 1 mM MgCl2, 1 mM
EGTA, and 0.1 mM EDTA with 0.067 TIU/ml aprotinin (Sigma)
and 0.2 mM phenylmethylsulfonyl fluoride (Sigma) as
protease inhibitors). Cell suspensions were incubated on ice for 30 min, freeze/thawed, and homogenized with a Teflon/glass homogenizer.
After a low speed centrifugation to remove unbroken cells and the
nuclear pellet, samples were centrifuged for 30 min at 200,000 × g at 4 °C in a Beckman TL100 centrifuge. The pellets
(particulate fraction) and the acetone-precipitated supernatants (soluble fraction) were separated by SDS-PAGE (12.5% acrylamide) and
analyzed by Western blotting.
Metabolic Labeling and Immunoprecipitation--
After
transfection, COS-7 cells were labeled for 4 h with
[9,10-3H]palmitic acid (150 µCi/ml) or
[9,10-3H]myristic acid (50 µCi/ml) in Dulbecco's
modified Eagle's medium supplemented with 5% dialyzed fetal bovine
serum, 5 mM sodium pyruvate, antibiotics, and
L-glutamine. After washing with PBS, cells were lysed in
0.2 ml of 1% (w/v) SDS. After breakage of DNA by repeated pipetting
and boiling for 4 min, 0.8 ml of the following mixture (Mix I) was
added to each sample: 1.25% (w/v) Triton X-100, 190 mM
NaCl, 6 mM EDTA, 50 mM Tris-HCl, pH 7.5, and
protease inhibitors as indicated above. After centrifugation at
13,000 × g for 10 min at 4 °C, 2 µl of GFP
antibody were added to each supernatant, and the samples were incubated
overnight at 4 °C. Twenty-five µl of a 1:1 suspension of protein
A-Sepharose CL-4B beads (Pharmacia Biotech Inc.) in Mix II (4 parts of
Mix I plus 1 part of 1% SDS) were added to each sample and incubated for ~5 h at 4 °C with continuous rotation. Immunoprecipitates were
washed three times for 15 min at 4 °C with Mix II (1 ml) and then
washed once with 50 mM Tris-HCl, pH 6.8, and heated in Laemmli sample buffer (23) containing 20 mM dithiothreitol. Samples were separated by SDS-PAGE, and gels were treated with 2,5-diphenyloxazole for fluorography (24). To maximize sensitivity, we
used a gel miniaturization procedure that involved soaking the
2,5-diphenyloxazole-impregnated gels after water washing in 50% (w/v)
polyethylene glycol 3000 (Sigma) at 70 °C for 15 min before drying
(25). Dried gels were exposed on Kodak X-Omat AR-5 films at
Fluorescence Microscopy--
Transfected 293T cells grown on
glass coverslips were washed three times with PBS and fixed for 30 min
at room temperature with 2% (w/v) paraformaldehyde in PBS. Fixed cells
were rinsed with PBS and treated with 25 mM
NH4Cl in PBS for 10 min at room temperature to quench free
aldehyde groups. Cells were washed three times with PBS, and slides
were mounted with Slow-Fade anti-fade reagent (Molecular Probes,
Eugene, OR) and observed under a Bio-Rad MR600 confocal microscope at
an excitation wavelength of 488 nm.
Preparation of Caveolin-enriched Membrane Fractions--
COS-7
cells were scraped into 2 ml of
2-(N-morpholino)ethanesulfonic acid-buffered saline (MBS; 25 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.5, and 0.15 M NaCl) containing 1% (v/v) Triton X-100. Homogenization was carried out with 10 strokes of a loose-fitting Dounce homogenizer. The homogenate was adjusted to 40% sucrose by the
addition of 2 ml of 80% sucrose prepared in MBS and placed at the
bottom of an ultracentrifuge tube. A 5-30% linear sucrose gradient
was formed above the homogenate and centrifuged at 39,000 rpm for
16-20 h in a SW41 rotor (Beckman Instruments). A light scattering band
confined to the 15-20% sucrose region was observed that contained
caveolin-1 but excluded most of the other cellular proteins. From the
top of each gradient, 1-ml gradient fractions were collected to yield a
total of 12 fractions. An equal volume of each gradient fraction was
separated by SDS-PAGE and subjected to immunoblot analysis.
Co-Immunoprecipitation--
Cells were washed twice with PBS and
lysed for 30 min at 4 °C in a buffer containing 10 mM
Tris, pH 8.0, 0.15 M NaCl, 5 mM EDTA, 1%
Triton X-100, and 60 mM octyl glucoside. Samples were pre-cleared for 1 h at 4 °C using protein A-Sepharose (20 µl; slurry, 1:1) and subjected to overnight immunoprecipitation at 4 °C
using anti-caveolin-1 antibody (10 µl; mAb 2234) and protein A-Sepharose (30 µl; slurry, 1:1). After three washes with the immunoprecipitation buffer, samples were separated by SDS-PAGE (12.5%
acrylamide) and transferred to nitrocellulose. Blots were then probed
with a polyclonal anti-GFP antibody.
The Dually Acylated NH2-terminal Region of
Gi1
Moreover, to define the role of fatty acylation in the specific
subcellular localization of the 32aa-GFP chimera, the known palmitoylation site was mutated by replacing cysteine 3 with serine (32aaC3S-GFP). Likewise, the known myristoylation site, glycine 2, was
mutated to alanine to generate a nonmyristoylated fusion protein
(32aaG2A-GFP). An additional fusion protein (CBD-GFP) was engineered by
attaching amino acids 186-200 of Gi1
The characteristics of all these engineered fusion proteins are
detailed in Fig. 1. The cDNAs
encoding wild type GFP and Gi1
Subsequently, fatty acylation patterns of the Gi1
The subcellular localization of GFP and each of the
Gi1
As expected, GFP was entirely soluble, whereas FL-GFP, 122aa-GFP, and
32aaWT-GFP were primarily concentrated in the particulate fraction
(80-90%; Fig. 4). In contrast, the amount of nonpalmitoylated 32aaC3S-GFP was greatly increased within the soluble fraction (>50%;
Fig. 4). An even greater shift was observed with 32aaG2A-GFP, which is
neither myristoylated nor palmitoylated, because ~90% of this
protein was detected in the soluble fraction, as was CBD-GFP (Fig. 4).
We conclude from these experiments that the NH2-terminal Gi1
To investigate the intracellular localization of the various
Gi1
Interestingly, FL-GFP, 122aa-GFP, and 32aaWT-GFP exhibited the same
pattern of cellular distribution as wild type Gi1 Lipid Modification of the NH2-terminal Region of
Gi1
Due to their high content of cholesterol and glycosphingolipids,
caveolar membranes are resistant to extraction at 4 °C by nonionic
detergents such as Triton X-100 and float on bottom-loaded sucrose
density gradients. Using a well-defined procedure that combines both of
these physicochemical properties to separate low density,
Triton-insoluble membranes containing caveolin-1 (7) from the bulk of
cellular membranes and cytosolic proteins, we analyzed by Western
blotting the content of GFP, Gi1
Caveolin-1, a structural protein of caveolar membranes, was used as a
marker protein for the distribution of these detergent-resistant membrane domains. As shown in Fig.
6A, the bulk of cellular
proteins, including soluble polypeptides, were concentrated in the
bottom, high density fractions (40% sucrose layer of the gradient;
lanes 9-12), whereas caveolin-1 immunoreactivity was
exclusively detected in the caveolae fractions (~10-20% sucrose
layer of the gradient; lanes 4 and 5).
Fig. 6B shows that FL-GFP, 122aa-GFP, and 32aaWT-GFP fusions
exhibited a distribution pattern equivalent to that of wild type Gi1
Accordingly, fully soluble 32aaG2A-GFP and CBD-GFP fusions were
completely excluded from the caveolin-containing fractions and were
concentrated at the bottom of the gradient, behaving essentially as
soluble GFP alone. These observations suggest that amino acids 1-32 of
Gi1 Lipid Modifications Are Required for the Binding of
Gi1
In this study, we wanted to examine whether the NH2
terminus of Gi1
Fig. 7A shows that FL-GFP,
122aa-GFP, and 32aaWT-GFP clearly co-immunoprecipitated with
caveolin-1. In striking contrast, mutant fusions 32aaC3S-GFP,
32aaG2A-GFP, and GFP alone failed to co-immunoprecipitate with
caveolin-1. Therefore, even in the absence of the known
Gi1 Palmitoylation of Caveolin-1 Is Required for Its Recognition of the
Lipid-modified NH2-terminal Domain of
Gi1
To determine whether 32aaWT-GFP binds equally well to palmitoylated or
nonpalmitoylated forms of caveolin-1, we co-transfected COS-7 cells
with this GFP fusion together with either wild type caveolin-1 or a
mutant form of caveolin-1 that lacks all three palmitoylation sites
(C133S, C143S, and C156S). Extracts of the transfected cells were then
prepared and immunoprecipitated with a specific mAb directed against
the unique NH2 terminus of caveolin-1 (mAb 2234). These
caveolin-1 immunoprecipitates were separated by SDS-PAGE and subjected
to immunoblot analysis with a specific GFP antibody. Interestingly, we
observed that the 32aaWT-GFP fusion co-immunoprecipitated only from
cells expressing wild type caveolin-1 (Fig.
8A), suggesting that the
proper binding of Gi1 There is a general consensus that the dually acylated
NH2 terminus of
Gi/Go/Gz However, in the case of Lck and Fyn, the NH2-terminal
domain plays a more complex role than merely serving as a
membrane-anchoring device. Mutational analysis has demonstrated that
the association of Lck and Fyn with the
glycosylphosphatidylinositol-anchored membrane proteins mapped to the
first 10 amino acids of the NRTKs, and the addition of myristate and
palmitate is required for this reciprocal interaction (31). Subsequent
studies have highlighted the key role of palmitoylated cysteines in
targeting Lck and Fyn to Triton-insoluble, glycolipid-enriched,
membrane subdomains (DIGs; Refs. 32-35) and in sorting these
polypeptides to different subcellular membrane compartments (36). Thus,
the NH2-terminal domain of NRTKs accomplishes the dual
function of targeting these polypeptides to a final membrane
compartment and stabilizing their association with the cytoplasmic
leaflet of the lipid bilayer.
By analogy with NRTKs, a similar role for the dually acylated
NH2-terminal domain of
Gi/Go/Gz Here we consider the issue that the NH2 terminus of dually
acylated G The same distribution pattern was exhibited by 122aa-GFP and 32aaWT-GFP
fusions, indicating that the first 32 amino acids of Gi1 As with NRTKs, the cellular targeting of 32aaWT-GFP was strictly
dependent on fatty acylation. The 32aaG2A-GFP chimera, which lacked
both fatty acids, was diffusely localized within the cytoplasm and
behaved as wild type GFP. In contrast, the palmitoylation-defective 32aaC3S-GFP retained some membrane avidity due to the presence of
myristate and displayed a reduced association with caveolin-enriched sucrose density fractions.
Interestingly, the binding of nonpalmitoylated 32aaC3S-GFP to
caveolin-1 was completely abrogated, suggesting that: (i)
co-fractionation with caveolin on sucrose gradients does not
necessarily imply a direct physical interaction between the two
polypeptides, and (ii) palmitoylation of cysteine 3 is an absolute
prerequisite for the binding of Gi1 Using the caveolin-1 scaffolding domain as a receptor to select
caveolin-binding peptide ligands from peptide sequences displayed at
the surface of the bacteriophage (phage display libraries), two related
caveolin-binding motifs have been identified
( subunits to the
plasma membrane. For this purpose, we used Gi1
as
a model dually acylated G-protein. We fused full-length
Gi1
or its extreme NH2-terminal domain (residues 1-32 or 1-122) to green fluorescent protein (GFP) and analyzed the subcellular localization of these fusion proteins. We show
that the first 32 amino acids of Gi1
are sufficient to
target GFP to caveolin-enriched domains of the plasma membrane in
vivo, as demonstrated by co-fractionation and
co-immunoprecipitation with caveolin-1. Interestingly, when dual
acylation of this 32-amino acid domain was blocked by specific point
mutations (G2A or C3S), the resulting GFP fusion proteins were
localized to the cytoplasm and excluded from caveolin-rich regions. The
myristoylated but nonpalmitoylated (C3S) chimera only partially
partitioned into caveolin-containing fractions. However, both
nonacylated GFP fusions (G2A and C3S) no longer co-immunoprecipitated
with caveolin-1. Taken together, these results indicate that lipid
modification of the NH2-terminal of Gi1
is
essential for targeting to its correct destination and interaction with
caveolin-1. Also, a caveolin-1 mutant lacking all three palmitoylation
sites (C133S, C143S, and C156S) was unable to co-immunoprecipitate
these dually acylated GFP-G-protein fusions. Thus, dual acylation of
the NH2-terminal domain of Gi1
and
palmitoylation of caveolin-1 are both required to stabilize and perhaps
regulate this reciprocal interaction at the plasma membrane in
vivo. Our results provide the first demonstration of a functional
role for caveolin-1 palmitoylation in its interaction with
signaling molecules.
INTRODUCTION
Top
Abstract
Introduction
References
subunits of heterotrimeric Gi, Go,
and Gz proteins contain a common
methionine-glycine-cysteine motif (MGC) at their extreme
NH2 terminus. This MGC motif is also shared by most members of the Src family of non-receptor tyrosine kinases
(NRTKs),1 with the exception
of Src itself and Blk (1, 2). This motif represents a consensus
sequence that specifies the covalent attachment of a 14-carbon lipid
(myristate) and a 16-carbon lipid (palmitate) to glycine 2 and cysteine
3, respectively. Moreover, some NRTKs may possess an additional
palmitoylated cysteine (e.g. cysteine 5 in Lck and cysteine
6 in Fyn). Point mutations that prevent myristoylation and/or
palmitoylation of G
subunits and NRTKs drastically reduce the
membrane avidity of these polypeptides, indicating that these lipid
modifications act cooperatively to anchor these proteins to the
cytoplasmic leaflet of the plasma membrane (3, 4).
subunits to membranes. In contrast,
palmitoylation is a reversible modification and is therefore potentially regulatable, suggesting that it may play a more complex role in this process. In support of this hypothesis, palmitoylated Gz
and Gi1
are totally refractory to the
GTPase-stimulating activities of the G
-interacting protein and the
regulator of G-protein signaling 4. Thus, palmitoylation may represent
a mechanism for prolonging or potentiating G-protein signaling (5).
subunits. The myristoylated but palmitoylation-negative C3S mutant of
Gi1
is unable to functionally couple with the
2A
adrenergic receptor co-expressed in transfected COS-7 cells, even when
the concentration of the mutant in the membrane is equivalent to that
of wild type Gi1
(6). One possible explanation for these
results is that the C3S mutant of Gi1
might be
segregated into a distinct plasma membrane compartment that is
separated from where the receptor is located.
subunits is sufficient to maintain them in the inactive GDP-liganded conformation (12-14). According to the "caveolae signaling hypothesis," G
subunits may
become activated when G-protein-coupled receptors are recruited to
caveolae upon agonist stimulation (15-17).
and other dually acylated proteins (such as NRTKs and endothelial nitric oxide synthase) with caveolin-enriched membrane
fractions (18-20). This raises the possibility that
NH2-terminal lipid modification can also specify membrane
compartmentalization of dually acylated molecules within caveolae by
acting as a targeting signal and/or through direct interactions with
the lipid or protein components of caveolae (e.g.
cholesterol, glycosphingolipids, or caveolins).
subunits and its lipid modification in targeting to the plasma membrane, we constructed a variety of lipid-modified and
non-lipid-modified fusion proteins using GFP. We examined their
subcellular distribution by immunofluorescence microscopy and their
ability to co-fractionate and co-immunoprecipitate with caveolin-1
using transient expression in COS-7 or 293T cells. In addition, we
studied the behavior of GFP fused to a short caveolin-binding domain
(21) recently identified within G
subunits (specifically, residues
186-200 in Gi1
).
EXPERIMENTAL PROCEDURES
polyclonal anti-serum (I1C; raised against a
synthetic peptide corresponding to amino acids 160-169 of the
Gi1
subunit). [9,10-3H]Palmitic acid
(40-60 Ci/mmol) and [9,10-3H]myristic acid (40-60
Ci/mmol) were from Amersham. All other biochemicals used were of the
highest purity available and were obtained from regular commercial sources.
-GFP Fusion Proteins--
To
generate GFP fusion proteins, the cDNA encoding GFP was placed at
the 3' end of the full-length rat Gi1
sequence (amino acids 1-355; FL-GFP) and of the following sequences of wild type rat
Gi1
: (i) amino acids 1-32 (32aaWT-GFP), (ii) amino
acids 1-122 (122aa-GFP), and (iii) amino acids 186-200
(caveolin-binding domain; CBD-GFP). Two additional constructs were
generated by fusing GFP to the 1-32 NH2-terminal amino
acid sequence of Gi1
: (i) 32aaG2A-GFP (mutation at the
myristoylation site by replacing glycine 2 with alanine), and (ii)
32aaC3S-GFP (mutation at the palmitoylation site by substituting
cysteine 3 with serine). Finally, GFP fusions were constructed in which
the first 6 of 10 NH2-terminal amino acids of
Gi1
were fused to GFP. All Gi1
-GFP fusion proteins were generated by polymerase chain reaction amplifications using the appropriate primers and subsequently subcloned in pcDNA3 expression vector (Invitrogen). The correctness of intended base substitutions and the absence of unwanted mutations were verified by
DNA sequencing. All DNA manipulations, including ligations, bacterial
transformation, and plasmid purification, were carried out using
standard procedures.
70 °C.
RESULTS
Is Sufficient to Target Soluble GFP to the Plasma
Membrane in Transfected COS-7 and 293T Cells--
To date, all studies
examining the role of lipid modification in the subcellular
localization of Gi1
have relied on loss of function
mutations in the context of the full-length Gi1
protein.
To determine whether the acylated NH2 terminus of
Gi1
is sufficient to direct membrane localization, we
constructed fusion proteins consisting of full-length wild type
Gi1
or the first 6, 10, 32, or 122 amino acids of
Gi1
attached to the NH2 terminus of GFP, a
normally cytosolic protein.
to the
NH2 terminus of GFP. Amino acids 186-200 of
Gi1
contain the known caveolin-binding domain within
Gi1 (21).
-GFP fusions were
individually transiently transfected into COS-7 cells. Their expression
was analyzed by Western blotting of whole cell lysates using an
anti-GFP polyclonal antibody. Fig. 2
shows that 48 h after transfection, the cells produced comparable
levels of all of the fusion proteins. In striking contrast, fusion
proteins between GFP and the first 6 or 10 amino acids of
Gi1
were expressed at very low levels (data not shown)
and were not further characterized.
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Fig. 1.
Schematic illustration of
Gi1 -GFP fusion proteins used in this study. The
full-length sequence (FL) or the first 32 (32aaWT) or 122 (122aa) NH2-terminal
amino acids of Gi1
were fused to the NH2
terminus of GFP. In 32aaG2A-GFP and 32aaC3S-GFP, each of the 32 amino
acid sequences of Gi1
fused to GFP was mutagenized to
convert glycine 2 to alanine and cysteine 3 to serine, respectively
(substitutions are shown as bold letters). In the CBD-GFP
chimera, the caveolin-binding domain containing the phenylalanine
repeat (underlined; Ref. 21) was NH2 terminally
fused to GFP.
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Fig. 2.
Expression of GFP and Gi1 -GFP
fusion proteins in COS-7 cells. COS-7 were transiently transfected
with different constructs using the calcium phosphate precipitation
method. Forty-eight h after transfection, cells were harvested and
lysed in Laemmli sample buffer. Lysates were separated by 12.5%
SDS-PAGE and immunoblotted with a polyclonal anti-GFP antibody.
-GFP
fusions were determined by metabolic radiolabeling of transfected COS-7
cells with [3H]myristate or [3H]palmitate,
followed by immunoprecipitation of cell lysates with the GFP antibody.
As expected, GFP alone was neither myristoylated nor palmitoylated
(data not shown), whereas FL-GFP, 122aa-GFP, and 32aaWT-GFP
incorporated both fatty acids (Fig. 3).
The 32aaC3S-GFP fusion incorporated [3H]myristate but not
[3H]palmitate, whereas 32aaG2A-GFP lacked both fatty
acids, thus behaving as the corresponding full-length
Gi1
G2A mutant, in which myristoylated glycine was
required for efficient palmitoylation (26).
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Fig. 3.
Fatty acylation of the Gi1 -GFP
fusion proteins. Transfected COS-7 cells were metabolically
labeled for 4 h with either [3H]palmitic acid
(Pal) or [3H]myristic acid (Myr).
Cell lysates were immunoprecipitated with an anti-GFP antibody and
analyzed by SDS-PAGE and fluorography. To enhance signal detection,
fluorographed gels were miniaturized as described under "Experimental
Procedures."
-GFP fusion proteins was next examined by
fractionating postnuclear COS-7 cell homogenates into cytosolic (Fig.
4, S) and membrane (Fig. 4,
P) fractions by ultracentrifugation at 200,000 × g. Each fraction was then analyzed by immunoblotting with an
anti-GFP antibody.
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Fig. 4.
Subcellular distribution of GFP and
Gi1 -GFP fusion proteins in transfected COS-7 cells.
Postnuclear supernatants of transfected COS-7 cells were fractionated
by ultracentrifugation at 200,000 × g into soluble
(S) and particulate (P) fractions. Partitioning
of proteins between the fractions was determined by SDS-PAGE and
immunoblotting with an anti-GFP antibody.
sequence is sufficient to target a cytosolic protein to cellular membranes and that myristic and palmitic acids
cooperatively affect membrane binding. The results observed with
CBD-GFP fusion suggest that binding to caveolin-1 and anchorage to
cellular membranes are expressed by distinct domains of the
Gi1
protein. It is worth noting that the particulate
fraction in this assay contains total cell membranes, including plasma
membrane and intracellular membranes. Thus, changes in the distribution
among various cell membrane compartments will not be appreciated unless
a morphological analysis of transfected cells is performed.
-GFP fusion proteins, we took advantage of the
spontaneous fluorescence of GFP. 293T cells expressing wild type GFP
and the Gi1
-GFP fusions were fixed and examined by laser
scanning confocal microscopy. The micrographs presented in Fig.
5 illustrate that although GFP was
cytosolic, it gained a plasma membrane localization after fusion with
full-length Gi1
or its NH2-terminal 122- and
32-amino acid sequences.
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Fig. 5.
Subcellular targeting of
Gi1 -GFP fusion proteins in transfected 293T cells.
293T cells expressing wild type GFP or Gi1
-GFP fusion
proteins were fixed with 2% (w/v) paraformaldehyde 48 h after
transfection. Cells expressing endogenous Gi1
were
stained with a rabbit antiserum (I1C) raised against a synthetic
peptide corresponding to amino acids 160-169 of the Gi1
subunit. GFP fluorescence and Gi1
indirect
immunofluorescence were visualized by confocal microscopy at an
excitation wavelength of 488 nm.
visualized by indirect immunofluorescence staining of 293T cells with a
Gi1
anti-serum. In contrast, nonacylated 32aaG2A-GFP showed a diffuse cytosolic staining (Fig. 5), whereas nonpalmitoylated 32aaC3S-GFP exhibited a hybrid distribution, as illustrated by the
staining of both cell edges and the cytoplasm. The fluorescence associated with the CBD-GFP fusion was diffuse, thus confirming its
localization primarily within the cytosol, in agreement with our
biochemical experiments (see Fig. 4). Mock-transfected 293T cells
showed no detectable fluorescence (data not shown). These results
clearly demonstrate that the first 32 amino acids of Gi1
contain information that is sufficient to specify a plasma membrane localization, provided that fatty acylation is successfully completed.
Is Required to Deliver a GFP Fusion Protein to
Caveolin-enriched Membrane Domains in COS-7 Cells--
Recently, it
has been shown that heterotrimeric G-protein subunits, as well as a
number of other signaling molecules, are concentrated within a distinct
subcompartment of the plasma membrane known as caveolae (7-9).
, and
Gi1
-GFP fusions in sucrose density gradient fractions obtained from transfected COS-7 cells.
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Fig. 6.
Subcellular fractionation of COS-7 cells
expressing Gi1 -GFP fusions. COS-7 cells transfected
with Gi1
, GFP, and each of the Gi1
-GFP
fusion proteins were extracted in 1% (v/v) Triton X-100 at 4 °C and
subjected to ultracentrifugation on sucrose density gradients. One-ml
fractions collected from the top of the gradients were separated by
SDS-PAGE and transferred to nitrocellulose. Fractions 1-4 (lanes
1-4) are the 5% sucrose layer, and fractions 5-8 (lanes
5-8) are the 30% sucrose layer. Fractions 9-12 (lanes
9-12), containing 40% sucrose, represent the "loading zone"
of these bottom-loaded flotation gradients and contain the bulk of the
cellular membrane and cytosolic proteins as described previously (14).
Upper panel, Ponceau S staining of the total cellular
proteins; lower panels, immunoblotting with anti-caveolin-1,
anti-Gi1
, or anti-GFP antibodies to visualize the
distribution of endogenously expressed caveolin-1 and transfected
Gi1
and Gi1
-GFP fusion proteins.
, distributing between both caveolin-containing
fractions as well as fractions 9-12 (lanes 9-12). The
palmitoylation-defective 32aaC3S-GFP fusion had a similar distribution;
however, compared with the wild type, significantly less of the fusion
protein was detected in the low density caveolae fractions and more was
present in the soluble, higher density fractions, as might be
anticipated by its increased solubility (see Fig. 4).
, which contain the myristoyl and palmitoyl
moieties, encode the complete address signal for subcellular targeting
of the Gi1
polypeptide.
to Caveolin--
Caveolin interacts directly with
G
subunits, preferentially recognizing their inactive conformation
(13). Thus, it has been suggested that caveolin functions as a
scaffolding protein to spatially organize certain caveolin-interacting
proteins within caveolae membranes. The scaffolding domain of caveolin
consists of a 20-amino acid stretch (residues 82-101) within the
NH2-terminal cytoplasmic domain of caveolin-1 (27). A
consensus sequence for caveolin binding has been deduced in many
caveolae-associated polypeptides, including G
subunits (21). Lipid
modification of G
subunits is not required for interaction with
caveolin-1 in vitro (13); however, it remains unknown
whether lipid modification of G
subunits is required for
interaction with caveolin-1 in vivo.
, although located a distance from the
caveolin-binding motif (residues 186-200 of Gi1
), might
influence interactions with caveolin-1 in vivo. For this
purpose, we immunoprecipitated lysates of COS-7 cells transfected with
various Gi1
-GFP fusions with a specific mAb probe that
recognizes the extreme NH2 terminus of caveolin-1 and
analyzed the composition of these precipitates by Western blotting with
a GFP-specific antibody.
caveolin-binding domain, as in 122aa-GFP and
32aaWT-GFP fusions, the NH2-terminal domain of
Gi1
played a key role in the association of
Gi1
with caveolin-1, provided that it is correctly
dually acylated.
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Fig. 7.
Interaction of Gi1 -GFP fusion
proteins with caveolin-1. A, detergent extracts of
transfected COS-7 cells were immunoprecipitated with anti-caveolin-1
mAb 2234 IgG and protein A-Sepharose beads. These immunoprecipitates
were subjected to SDS-PAGE and analyzed by Western blotting with
anti-GFP antibody. B, COS-7 cell extracts contained
equivalent amounts of each of the Gi1
-GFP fusion
proteins and of caveolin-1, as determined by Western blot analysis
before immunoprecipitation.
--
Caveolin-1 is palmitoylated on three
COOH-terminal cysteines, more specifically, residues 133, 143, and 156 (22). The loss of palmitoyl moieties does not affect the membrane
insertion of the caveolin-1 polypeptide, its targeting to caveolae
membranes, or its ability to form high molecular weight homo-oligomers
(22, 28). Palmitoylation of caveolin-1 protects caveolin-1
homo-oligomers from dissociation by SDS denaturation during boiling.
However, the physiological relevance of this finding remains unknown.
requires palmitoylation of
caveolin-1.
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Fig. 8.
Palmitoylation of caveolin-1 is required for
interaction with Gi1 . 32aaWT-GFP was co-transfected
in COS-7 cells with the wild type form of caveolin-1 (cav-1
WT) or with the palmitoylation-defective caveolin-1 mutant
(cav-1 Cys
). A, detergent extracts
of transfected cells were immunoprecipitated with anti-caveolin-1 mAb
2234 IgG and protein A-Sepharose beads. Immunoprecipitates were
subjected to SDS-PAGE and assayed by Western blotting with anti-GFP
antibody. B, Western blot analysis of cell extracts before
immunoprecipitation with anti-GFP and anti-caveolin-1 antibodies shows
that equivalent levels of 32aaWT-GFP and of wild type and
palmitoylation-defective caveolin-1 were expressed in COS-7 cells after
transfection.
DISCUSSION
subunits is required
for their attachment to the cytoplasmic leaflet of the plasma membrane.
This is based mainly on the observation that simultaneous mutations of
glycine 2 and cysteine 3, respectively, the acceptor sites for the
attachment of myristate and palmitate, considerably shift G
subunits
to the soluble fraction (26, 29, 30). Similarly, Src-like NRTKs (such
as Lck and Fyn) share the same NH2-terminal acylation motif with Gi/Go/Gz
subunits and an
extra palmitoylated cysteine at position 5 or 6, respectively, and
their membrane association is prevented by mutation of these key sites
of fatty acylation (3).
subunits in targeting
has been postulated. This view is supported by the finding that G
subunits, as well as Src-like NRTKs, can be found in flattened (termed
DIGs or rafts) or invaginated (termed caveolae) specializations of the
plasma membrane, which are rich in cholesterol and glycosphingolipids (7-9). In contrast, earlier reports have suggested that membrane anchoring and subcellular targeting of Gi
subunits are
contained within distinct protein domains. The COOH terminus plays a
role in the differential targeting of Gi2
and
Gi3
subunits to the plasma and Golgi membranes,
respectively, because intracellular localization of
Gi2
/Gi3
chimeras was dependent on which COOH-terminal half was used (37).
subunits also contributes to their subcellular targeting. We analyzed the ability of various NH2-terminal extensions
of Gi1
to act as a membrane-anchoring and targeting
signal for a normally soluble carrier protein, namely, GFP. The
subcellular targeting of each Gi1
-GFP fusion construct
was evaluated after transient expression in transfected cells using
confocal microscopy and biochemical techniques. The fluorescence
associated with the full-length Gi1
-GFP fusion protein
localized at the edges of transfected cells behaving as wild type
untagged Gi1
, suggesting that the GFP reporter did not
interfere with the proper localization of Gi1
. Moreover,
the full-length Gi1
-GFP fusion protein co-localized with
caveolin-1, a protein marker for caveolae membranes, in low buoyancy
fractions purified by sucrose gradient centrifugation and was
co-immunoprecipitated by caveolin-1 antibodies.
contain the signal to deliver polypeptides to low density,
caveolin-enriched, plasma membrane domains and to interact with
caveolin-1. We also attempted to define the targeting domain of
Gi1
in greater detail by fusing only the first 6 or 10 residues to GFP. Unfortunately, these GFP fusions were expressed at
much lower levels than 32aaWT-GFP in COS-7 cells (data not shown). This
may reflect the structural instability of these shorter Gi1
fusions because 6 or 10 amino acids would not be
long enough to allow the formation of the 30-amino acid residue
NH2-terminal
-helix present in wild type
Gi1
(38).
to caveolin in
vivo. This may explain the results of Huang et al. (9),
who failed to detect a substantial direct interaction between
recombinant myristoylated but palmitoylation-defective Go
and caveolin-1. However, it contrasts with the
observed in vitro interaction between caveolin-1 and
palmitoylated Go
or Gi2
expressed by
baculovirus-infected Sf9 cells (13).
X
XXXX
and
XXXX
XX
, with
representing aromatic
amino acids Trp, Phe, or Tyr) (21). These motifs exist within most
caveolae-associated proteins, including Gi1
(21).
Interestingly, when residues 186-200 of Gi1
containing
the defined motif for interaction with caveolin-1 (21) were fused to
GFP, the resulting CBD-GFP chimera did not co-immunoprecipitate with
caveolin-1. This may suggest that the fatty acylation of
Gi1
, by promoting an interaction with lipid and/or
protein component(s) of the plasma membrane bilayer, must precede
caveolin-1 recognition by the caveolin-binding domain in
vivo. This implies a two-step mechanism for the caveolar targeting
of Gi1
. This proposed mechanism is summarized
schematically in Fig. 9. In the first
step, mutual interaction of palmitic acid residues in
Gi1
and caveolin-1 are responsible for the binding of
Gi1
to caveolin-1. In a second step, this interaction is
stabilized by the binding of the caveolin-binding domain of Gi1
to the scaffolding domain of caveolin-1. A similar
conclusion has been reached regarding the interaction of Ras with
caveolin-1 (39). Lipid modification of Ras by prenylation is required
for its correct targeting to caveolae in vivo but is not
required for its in vitro interaction with caveolin-1
(39).
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Fig. 9.
Schematic illustration of the caveolar
targeting of Gi1 . In Step 1, palmitic
acid of Gi1
is responsible for binding to the plasma
membrane bilayer and to caveolin-1 oligomers. Palmitic acid residues in
the COOH-terminal of caveolin-1 directly interact with palmitic acid
residues in Gi1
. In Step 2, the
caveolin-binding domain of Gi1
interacts with the
scaffolding domain of caveolin-1, stabilizing the mutual
interaction.
The unique role of palmitoylation in mediating the association of
Gi1 with caveolin-1 is important because, unlike
myristoylation, palmitoylation is a reversible modification. For
example, in the case of Gs
(40-42) and
Gq/11
(43), regulation of palmitoylation by direct or
receptor-mediated activation has been demonstrated experimentally. Our
results are also consistent with the previous observation that
mutationally activated Gs
showing an accelerated rate of
depalmitoylation (41) fails to interact with recombinant caveolin-1, as
compared with its wild type counterpart, nonmutated Gs
(13).
Caveolin-1 can associate with itself to form Triton-insoluble high
molecular mass homo-oligomers (300-400 kDa) in vivo as well
as in vitro (28, 44). It has been proposed that caveolin-1 oligomers complexed with cholesterol and glycosphingolipids can form a
matrix within membrane rafts, or caveolae, acting as a scaffold for
lipid-modified signaling proteins (17). Although the oligomerization
region of caveolin has been mapped to residues 61-101 of the
NH2-terminal cytoplasmic domain (44), recent observations (28) have shown that complete maturation of these high molecular mass
complexes requires the palmitoylation of three cysteine residues located at positions 133, 143, and 156 within the COOH-terminal cytoplasmic tail. This COOH-terminal region of caveolin-1 has also been
implicated in mediating interactions between individual caveolin-1
homo-oligomers, forming a caveolin-1 network or patch within the plasma
membrane (17). Here, we show that the 32aaWT-GFP chimera, when
co-expressed in COS-7 cells with a mutant form of caveolin-1 that lacks
all three palmitoylation sites, can no longer be co-immunoprecipitated
with caveolin-1 IgG, indicating a loss of interaction between the two
polypeptides. This finding suggests that palmitoylation of caveolin-1
oligomers is necessary for complex formation with inactive G, and
perhaps with other signaling molecules as well, within caveolae
membranes in vivo.
In conclusion, our results demonstrate that both membrane-anchoring and
targeting functions are encoded by the dually acylated NH2-terminal domain of Gi1 and that
palmitoylation of Gi1
cysteine 3 plays a key role by
facilitating the interaction with caveolin-1. A recent study (45) has
reached similar conclusions for Gz
, suggesting specific
roles for myristoylation and palmitoylation in membrane targeting. They
observed that the attachment of myristate cooperates with
dimer
binding to promote the stable association of Gz
with
membranes and that the addition of palmitate confers specific
localization at the plasma membrane. However, these authors did not
examine the interaction of Gz
with caveolae or
caveolins. Thus, regulated palmitoylation of G
, on one hand, and
caveolin-1, on the other, adds further potential handles to control
G-protein-mediated signal transduction.
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FOOTNOTES |
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* This work was supported by grants from the Consiglio Nazionale delle Ricerche (Target Project on Biotechnology) and Ministero dell'Università e Ricerca Scientifica e Tecnologica (to M. P.) and from the Wellcome trust (to G. M. and M. P.).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.
** Supported by National Institutes of Health Grant ROI-CA-80250 and by grants from the Charles E. Culpeper Foundation, the G. Harold and Leila Y. Mathers Charitable Foundation, and the Sidney Kimmel Foundation for Cancer Research.
To whom correspondence should be addressed: Dept. of
Pharmacology, School of Medicine, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy. Tel.: 39-2-70146282; Fax: 39-2-70146371; E-mail: marco.parenti{at}unimi.it.
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ABBREVIATIONS |
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The abbreviations used are:
NRTK, non-receptor
tyrosine kinase;
GFP, green fluorescent protein;
mAb, monoclonal
antibody;
PAGE, polyacrylamide gel electrophoresis;
TBST, Tris-buffered
saline/Tween 20;
G, G-protein
subunit;
PBS, phosphate-buffered
saline.
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REFERENCES |
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