From the
As the first step in an investigation of roles played by fatty
acylation of G protein
Hetereotrimeric (
Myristoylation of
Excepting
Here we report
experiments designed to examine the effects of palmitoylation and
myristoylation on membrane attachment and signaling function of
Because the
molecular genetic approach to this problem requires easy detection and
specific quantitative immunoprecipitation of mutant
CHO K1 cells were propagated in MEM
S49 cyc
We introduced epitopes into
representatives (
Scanning
In contrast to epitopes at
site I, the EE epitope at site II consistently left signaling activity
intact. In response to receptor stimulation,
Mutant
The ability of
Extending an approach previously initiated with
The crystal structure of
The G2A
mutation could alter palmitoylation in several ways. First,
incorporation of radiolabel into
Second, the
G2A mutation could reduce incorporation of palmitate into
Lastly, the lower amount of radiolabel in
Blocking
myristoylation reduced the fraction of
The mechanism underlying this phenomenon is
obscure. The constitutive activity of
Alternatively, palmitoylation could decrease constitutive
activity by directing
Based on experiments
with
While it is reasonable
to postulate a palmitoylation cycle for
We thank Eva Schindler for dedicated technical
assistance and Susan Munemitsu, John Lyons, Bonnee Rubinfeld, and Phil
Wedegaertner for useful advice. We thank Pablo Garcia for the
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
chains in membrane targeting and signal
transmission, we inserted monoclonal antibody epitopes, hemagglutinin
(HA) or Glu-Glu (EE), at two internal sites in three
subunits. At
site I, only HA-tagged
and
functioned normally.
,
, and
subunits tagged at site II with the EE epitope showed
normal expression, membrane localization, and signaling activity. Using
epitope-tagged
, we investigated effects of mutations
in sites for fatty acylation. Mutational substitution of Ala for
Gly
(G2A) prevented incorporation of myristate and
decreased but did not abolish incorporation of palmitate. Substitution
of Ala for Cys
(C3A) prevented incorporation of palmitate
but had no effect on incorporation of myristate. Substitution of Ala
for both Gly
and Cys
(G2AC3A) prevented
incorporation of both myristate and palmitate. All three mutations
substantially disrupted association of
with the
particulate fraction. G
-mediated inhibition of adenylyl
cyclase, triggered by activation of the D2-dopamine receptor, was,
respectively, abolished (G2AC3A), impaired (G2A), and enhanced (C3A).
Constitutive inhibition of adenylyl cyclase by
was
unchanged (G2AC3A), strongly diminished (G2A), or strongly enhanced
(C3A). A nonacylated, mutationally activated
mutant
inhibited adenylyl cyclase, although less potently than normally
acylated, mutationally activated
. From these findings
we conclude: ( a) fatty acylations of
increase its association with membranes; ( b)
myristoylation is not required for palmitoylation of
or for its productive interactions with adenylyl cyclase;
( c) palmitoylation is not required for, but may instead
inhibit, signaling by
.
) G proteins transduce signals
from cell surface receptors to a variety of effectors, including
adenylyl cyclase, ion channels, and phospholipase C. Covalent
attachment of myristate and/or palmitate to G protein
subunits
greatly facilitates and is in some cases essential for their
association with the cytoplasmic surface of the plasma membrane and for
normal signaling function. These characteristics make G protein
subunits attractive models for investigating roles played by distinct
fatty acylations in membrane association and protein function.
subunits involves covalent addition of
myristate to a Gly
residue via an amide linkage, and takes
place during or immediately after translation. This modification is
usually irreversible, but not always
(1) and therefore an
unlikely target for regulation. Myristoylation is confined to members
of the
family, where it serves dual roles promoting
membrane attachment and increasing affinity of protein-protein
interactions. Mutations that abolish myristoylation substantially
impair attachment of
,
, and
to membranes
(2, 3, 4) .
Myristoylation promotes membrane attachment of
and
, at least in part, by increasing their affinity for
membrane bound
subunits
(5, 6) . Productive
interaction of
with adenylyl cyclase requires
myristoylation
(7) .
, all
subunits thus far examined are palmitoylated
(8, 9, 10) . In contrast to myristoylation,
palmitoylation is a dynamic, reversible modification involving
post-translational attachment of palmitate to a Cys residue by a
thioester linkage. For
and
, which
are modified solely by palmitate, this modification plays a critical
role: mutations that prevent palmitoylation markedly impair membrane
association
(8, 9, 11, 12) and
signaling functions
(12) of
and
. Moreover, palmitoylation is regulated
(13, 14, 15) . Following activation of
in intact cells, palmitate is removed, and
depalmitoylated
is found in the soluble compartment,
suggesting that activation-dependent depalmitoylation may serve to
decrease the amount of
available for subsequent
interactions with receptors and effectors
(15) . The function
and regulation of palmitate in
subunits that are modified by both
palmitate and myristate awaits elucidation.
. A member of the
family,
is expressed primarily in brain, adrenal gland, and
platelets
(16) . Like
,
can
mediate hormonal inhibition of adenylyl cyclase
(17) and is
both palmitoylated and myristoylated
(2, 4) . Unlike
,
is not uncoupled from receptor
activation by the action of pertussis toxin (PTX),
(
)
because it lacks the Cys residue modified by PTX.
Consequently, signals mediated by transfected
can be
assayed specifically and conveniently in cells treated with PTX. For
this reason, we chose
as an early target for a
molecular genetic investigation of the roles of attached palmitate and
myristate in modifying signaling by G proteins.
subunits, we
began by extending the epitope tagging strategy, first employed with
(18) . Here we report results of a search for
a common site in G protein
subunits suitable for epitope tagging.
Success of this search, with respect to
and
, as well as
, will facilitate
investigation of other G protein
subunits by strategies that
utilize mutation, immunoprecipitation, and immunofluorescence.
Materials
CHO K1 and HEK293 cells were obtained
from the American Type Culture Collection. cDNA constructs obtained as
described in Ref. 12. Receptor agonists were from Research Biochemicals
Inc. (quinpirole, UK-14304 as part of National Institute of Mental
Health Chemical Synthesis Program Contract 278-90-0007[BS]),
the National Pituitary Agency (human chorionic gonadotrophin), or Sigma
(isoproterenol). Antisera were the generous gifts of S. Mumby and A.
Gilman (G) and Paul Sternweiss (
).
[2-
H]Inositol and
[2-
H]adenine were obtained from Amersham Corp.
[9,10-
H]Palmitic acid and
[9,10-
H]myristic acid were from DuPont NEN.
Plasmid Construction
Single stranded DNA
corresponding to the antisense strand was prepared from pcDNA1
containing subunit constructs. Hemagglutinin (HA) or Glu-Glu (EE)
epitope sequences were introduced by the site-directed mutagenesis
protocol of Kunkel et al. (19) . Sense strand
oligonucleotides comprising the coding sequence for either epitope
flanked on both sides by 24 bases of coding sequence of the appropriate
subunit were used as primers. Coding sequences were
5`- gacgtccccgactacgcg for the 6-amino acid HA epitope DVPDYA
or 5`- tacgacgtccccgactacgcgtca for the 8-amino acid HA epitope
YDVPDYAS. Coding sequences for the EE epitope were
5`- gagtacatgccgatggag for the sequence EYMPME,
5`- gagtacatgccgatcgag for the sequence EYMPTE, or
5`- gcggagtacatgccgatctgag for the sequence AEYMPTE used in
EE-N. The G2A, C3A, and G2AC3A mutations were
constructed in a similar fashion using the antisense strand of pCDNA1
containing
EE-II. Mutagenesis was verified by DNA
sequencing. For transient expression in S49
cyc
cells,
constructs were subcloned into the expression vector pSR296
(20) by blunt-ended ligation at the unique PstI site.
Transient Transfection Protocols
HEK293 cells were
propagated in minimal essential medium (MEM) containing 10% fetal
bovine serum. Plasmids were transfected using Transfectam lipofectant
(Promega). A DNA-lipofectant mixture (ratio, 1 µg of DNA to 2
µl of Transfectam) was added to a 60-mm dish of subconfluent cells
bathed in 0.5 ml of serum-free MEM. After 2 h incubation, the medium
was replaced with 4 ml of MEM, 10% fetal bovine serum. For assaying
signaling activity of or
constructs, 1 µg of
R plasmid was
cotransfected with 3 µg of
or
plasmid.
containing
10% fetal bovine serum. Initially, CHO K1 cells were transfected
exactly as described for HEK293 cells except that MEM
was used and
the ratio of Transfectam to DNA was 4 µl/1 µg. In later
experiments plasmids were transfected using the adenovirus-mediated
transfection protocol
(21) . Plasmid DNAs were placed into 2.5
ml of bicarbonate-free, serum-free DME H16 medium supplemented with 20
m
M HEPES, nonessential amino acids, and 80 µg/ml DEAE. A
1:4 to 1:8 dilution of adenovirus stock was added to the tube and
mixed. The DNA-adenovirus solution was added to a 60-mm dish of
subconfluent cells. Following a 2-h incubation at 37 °C, the cells
were shocked for 2 min with 10% Me
SO in phosphate-buffered
saline and returned to bicarbonate-free DME H16 supplemented with 20
m
M HEPES, nonessential amino acids, and 10% fetal bovine
serum. For assaying signaling activity of
constructs,
1 µg of LHR plasmid and 2 µg of D
R plasmid were
cotransfected with 0-4 µg of
plasmid.
cells were propagated in
DME H21 medium supplemented with 10% heat-inactivated horse serum. For
transfection, cells in logarithmic growth were pelleted, washed once,
and resuspended in serum-free DME H21 at a concentration of 2
10
cells/ml. 225 µl of cells and 25 µl of 10
m
M Tris, 0.1 m
M EDTA, pH 8.0, containing 10 µg of
plasmid DNA were mixed in an electroporation chamber. Electroporation
was done in a Cell-Porator (BRL) at settings of 330 microfarads and 225
V. Cells were immediately transferred to 60-mm dishes containing 4 ml
of DME H21 with 10% horse serum.
cAMP Assay
HEK293 or CHO K1 cells, 24 h after
transfection, were reseeded into 6 wells of a 24-well plate and
[H]adenine (2 µCi/ml for HEK293, 6 µCi/ml
for CHO K1) was added. For assays involving
constructs, pertussis toxin (200 ng/ml) was also included. 24 h
later, wells were washed once with 0.5 ml of serum-free,
bicarbonate-free DME H16 medium containing 20 m
M HEPES (assay
medium), and then incubated with 0.5 ml of assay medium containing 1
m
M isobutylmethylxanthine with or without the appropriate
receptor agonists. After 30 min incubation at 37 °C, the cells were
lysed in 0.5 ml of 5% trichloroacetic acid containing 1 m
M
each cAMP and ATP. [
H]cAMP and
[
H]ATP were separated on Dowex and alumina
columns as described
(22) . S49
cyc
cells, 24 h after transfection,
were labeled with 6 µCi/ml [
H]adenine for 24
h. Cells were pelleted and resuspended in 2.8 ml of assay medium.
450-µl aliquots were added to 6 wells of a 24-well plate. 50 µl
of assay medium containing 10 m
M isobutylmethylxanthine with
or without 10 times concentration (10
M) of
isoproterenol was added. During the 30-min incubation at 37 °C, the
S49 cyc
cells adhered to the wells.
Reactions were terminated with 5% trichloroacetic acid as described
above. The remainder of the assay was identical to that described for
HEK293 cells.
Inositol Phosphate Assay
HEK293 cells, 24 h after
transfection, were reseeded into 6 wells of a 24-well plate and
[H]inositol (2 µCi/ml) was added. 24 h later,
wells were washed once with 0.5 ml of assay medium and then incubated
with 0.5 ml of assay medium containing 10 m
M LiCl with or
without the receptor agonist, UK14304. After 30 min incubation at 37
°C, the cells were lysed in 0.75 ml of 20 m
M formic acid.
[
H]Inositol phosphate and
[
H]inositol were separated on Dowex columns as
described
(22) .
Metabolic Labeling
100-mm plates of CHO K1 cells,
24 h after transfection, were reseeded into two 60-mm plates. 24 h
later, the cells were washed twice with serum-free MEM and then
incubated for 2 h with 1 ml of serum-free MEM
containing 0.75 mCi
of either [
H]myristic acid or
[
H]palmitic acid. Cells were washed once with
ice-cold phosphate-buffered saline and lysed on ice for 60 min with 330
µl of 40 m
M Tris
HCl, pH 8.0, containing 1 m
M
EGTA, 2 m
M MgCl
, 1% sodium cholate, and 2
µg/ml aprotinin. Lysates were transferred to Eppendorf tubes on ice
and spun in a microcentrifuge at 4 °C at 3500 rpm for 3 min.
Soluble material was transferred to a new Eppendorf tube on ice. Triton
X-100 and SDS were added to final concentrations of 1 and 0.5%,
respectively, in a total volume of 400 µl. 30 µl of EE mAb
coupled to protein G-Sepharose beads (15 µl of packed beads) was
added to each tube and the tubes tumbled for 1 h at 4 °C. The beads
were pelleted by microcentrifugation and quickly washed with 500 µl
of ice-cold phosphate-buffered saline containing 1% sodium cholate, 1%
Triton X-100, and 0.5% SDS. Beads were resuspended in 100 µl of
SDS-PAGE sample buffer containing 10 m
M dithiothreitol and
heated to 65 °C for 3 min. 30-µl aliquots were resolved on two
separate 14% SDS-PA gels
(18) . Gels were fixed by three 15-min
washes in 50% MeOH, 10% acetic acid. After soaking for 15 min in 1
M Tris
HCl, pH 7.0, one gel was transferred to a fresh
solution of 1
M Tris
HCl, pH 7.0, and the other to a fresh
solution of 1
M hydroxylamine, pH 7.0, for 12 h. Gels were
then washed twice in 50% MeOH, 10% acetic acid and processed for
fluorography with Amplify (Amersham Corp.) according to the
manufacturer's instructions. An additional 30-µl aliquot was
resolved by SDS-PAGE and probed with biotinylated-EE mAb by Western
blotting.
Cell Fractionation
100-mm plates of CHO K1 cells,
48 h after transfection, were washed with ice-cold phosphate-buffered
saline. 300 µl of 40 m
M TrisHCl, pH 8.0, containing 1
m
M EGTA, 2 m
M MgCl
, and 2 µg/ml
aprotinin was added to each plate on ice. Cells were scraped off the
plates with a rubber policemen and transferred to Eppendorf tubes on
ice. Cells were disrupted by 10 passages through a 27-gauge needle.
Unlysed cells and nuclei were removed by microcentrifugation at 3,500
rpm for 5 min at 4 °C. Supernatants were separated into soluble and
particulate fractions by centrifugation for 30 min at 150,000
g. Particulate fractions were resuspended into equivalent
volumes of lysis buffer. Samples were analyzed by 14% SDS-PAGE followed
by Western blotting.
Miscellaneous Immunological Techniques
EE mAb was
obtained from Onyx Pharmaceuticals (Richmond, CA), 12CA5 mAb from
Berkeley Antibody Co. (Berkeley, CA). The septapeptide EEYMPME
corresponding to the sequence of the EE epitope was synthesized at the
Biomolecular Resource Facility at University of California at San
Francisco. EE mAb was coupled to protein G-Sepharose as described
(23) . EE mAb was biotinylated using the Fluoreporter Biotin-XX
labeling kit from Molecular Probes Inc. as described in the
manufacturer's instructions. For Western blotting the following
dilutions of primary antibodies were used: EE mAb, 3 µg/ml;
biotinylated-EE mAb,
1 µg/ml; 12CA5 mAb,
1 µg/ml;
anti-G
antiserum B600, 1/5,000 dilution; anti-
antiserum WO82, 1/250 dilution; anti-
antiserum
199.233, 1.2 µg/ml. Secondary antibodies with conjugated
horseradish peroxidase were used at a 1/10,000 dilution. Incubations
with primary antibodies were from 1 h to overnight, secondary
antibodies for 30 min. ECL (Amersham) was used to visualize
immunoreactive bands.
Rationale and Initial Studies
One of our major
goals was to identify a common site in G protein subunits for
introduction of well characterized monoclonal antibody epitopes, in
order to improve immunodetection and immunoprecipitation. We scanned
sequences of
subunits for regions of similar sequence, suitable
for introduction of epitopes and predictably accessible to antibodies;
we then introduced defined epitopes into these regions and assessed the
effects of epitope tags on
subunit function. We used the HA
(24) and EE
(25) epitopes (Fig. 1); these comprise small
contiguous stretches of amino acids, are well characterized and have
been used for epitope tagging of a variety of proteins
(26, 27) .
,
, and
) of three
subunit families. Signaling
activities of
subunit constructs were measured following
transient transfection in cell types with a null background, that is,
cells in which signaling activity of the corresponding endogenous
subunit could be blocked or was absent. For
constructs, S49 cyc
cells provide a
genetically null background and
signaling activity
can be assayed by measuring cAMP production following stimulation of
the endogenous
-adrenoreceptor (
-AR).
HEK293 cells provide null backgrounds for assaying coupling of
transiently expressed
or
to the
cotransfected
-adrenoreceptor (
-AR),
because the corresponding endogenous
subunits do not mediate
responses to the
-AR in these cells
(22, 28) .
, like
,
mediates receptor inhibition of adenylyl cyclase. Because
is resistant to PTX, treatment with PTX creates a null background
for
, by uncoupling endogenous G
from
activation by receptors
(17) . Activities of
mutants were therefore assayed by measuring
D
-dopamine receptor (D
R) mediated inhibition of
the cAMP accumulation generated by stimulation of the luteinizing
hormone receptor (LHR) in CHO K1 cells cotransfected with
, LHR, and D
R.
Epitope Tagging at Two Internal Sites
Previous
work from this laboratory showed that insertion of the HA epitope into
a site encoded by an alternative exon (exon 3) of the gene did not disrupt coupling to the
-AR or to
adenylyl cyclase
(18) . Epitope tags at the corresponding
positions of
and
, however, produced
non-functional proteins. In addition, appending epitopes at the
carboxyl termini of
and
impaired
function (data not shown).
subunit sequences revealed
two regions similar in amino acid composition to both epitopes. The
first (site I) is located approximately 125 residues from the amino
terminus. Site I sequences are well conserved among members of each
subunit family, although overall similarity among
subunits
at this site is not high (Fig. 1 A). This site appeared
suitable for both epitopes. The second region (site II) is located
seven residues upstream of the arginine residue in
that is ADP-ribosylated by cholera toxin
(Fig. 1 B). Site II sequences are highly conserved among
all
subunits and closely match the EE epitope sequence. In the
crystal structure of
(29) , site I and site
II are located in the
helical domain, in loops between helices B
and C and E and F, respectively.
Figure 1:
Sequences
of epitope-tagged subunits. The amino acid sequences of wild-type
and epitope-tagged
,
,
, and
at site I ( panel A)
and site II ( panel B) are shown. Numbers in
parentheses refer to the amino acid residues of the wild type
sequence that were mutated to create the epitope sequence. The
arrow in panel B points to the arginine residue in
that is covalently modified by cholera toxin.
Panel C lists the amino-terminal sequences of
and mutant
constructs. Epitope sequences are
underlined.
We introduced both epitopes into
site I in and measured the ability of epitope-tagged
to couple the
-AR to stimulation of
PI-PLC
in HEK293 cells. While the magnitude of stimulation of
PI-PLC
varied from transfection to transfection, both
HA-I and
EE-I stimulated PI-PLC
to the same extent as did untagged
(Fig.
2 A). Agonist dose-response curves for
HA-I
were similar to that for untagged
(Fig. 3 A).
Reproducibly, the dose-response curve for
EE-I was
shifted to the left (Fig. 3 B). Both epitope-tagged
proteins were detectable by Western blotting (data not shown).
Figure 3:
Agonist dose-response curves for
epitope-tagged subunits. Panels A-C, HEK 293 cells were
transfected with 1 µg of
-AR DNA and 3 µg of
pcDNA1 containing the indicated
constructs.
Production of inositol phosphates in response to varying concentrations
of the
R agonist, UK 14304, was measured as described
under ``Experimental Procedures.'' Panels D and
E, CHO K1 cells were transfected using lipofectant with 1
µg of LHR, 2 µg of D
R, and 1 µg of pcDNA1
containing the indicated
constructs. Production of
cAMP in response to 100 ng/ml human chorionic gonadotropin and various
concentrations of the D
R agonist, quinpirole, was measured
as described under ``Experimental Procedures.'' Quinpirole
inhibition of cAMP production for cells transfected with pcDNA1 vector
was less than 10% of the human chorionic gonadotropin-stimulated cAMP
signal (data not shown). Panel F, S49 cyc
cells were transfected with 10 µg of pSR296
containing
the indicated
constructs. Production of cAMP in
response to various concentrations of the
-AR agonist,
isoproterenol, was measured as described under ``Experimental
Procedures.'' For all panels, each point represents the mean
± S.D. of triplicate measurements. For each panel, at least two
additional experiments yielded similar
results.
HA-I mediated D
R inhibition of adenylyl
cyclase but was not detectable by Western blotting using the 12CA5
monoclonal antibody (data not shown).
HA-I probably
lacked sufficient determinants for recognition by the 12CA5 antibody,
because a second HA construct (denoted
HA
-I), comprising an additional two amino
acids of the original HA epitope, was recognized by the 12CA5 antibody
on Western blots (data not shown).
HA
-I
mediated D
R inhibition of adenylyl cyclase to the same
extent as did untagged
(Fig. 2 B) and
agonist dose-response curves were similar to those for untagged
(Fig. 3 D). In contrast to
HA
-I,
EE-I was expressed
but coupled weakly to the D
R (data not shown). At higher
levels of expression,
EE-I constitutively inhibited
adenylyl cyclase (as does untagged
), suggesting that
creation of the EE epitope at site I may perturb
activation, perhaps by impairing interactions with
and/or receptors.
Figure 2:
Signaling activity of subunits.
Untagged and epitope-tagged
subunits were transiently transfected
with the appropriate receptors as described under ``Experimental
Procedures'' and the legend of Fig. 3. Panel A,
; panel B,
; panel
C,
. Effector activities were measured in the
absence and presence of maximally stimulating concentrations of
receptor agonist and compared to controls transfected with pcDNAI
vector (see ``Experimental Procedures''). The extent of
effector activation for
and
constructs is normalized to effector activity in the absence of
receptor agonist. For
constructs, results are
expressed as percent inhibition of the cAMP signal produced by LH
receptor stimulation. Values represent the mean ± S.D. of three
separate experiments.
Introducing the HA epitope into site I of
completely disrupted its signaling activity in either
S49 cyc
cells or in HEK293 cells, even
though its expression in HEK293 cells was readily detected on Western
blots. Furthermore, cholera toxin failed to activate adenylyl cyclase
in S49 cyc
cells transfected with
HA-I (data not shown).
EE-II,
EE-II, and
EE-II regulated activities
of their respective effectors to the same extent as did their untagged
counterparts (Fig. 2, A-C). Agonist dose-response curves for
EE-tagged constructs were also similar to those of their untagged
counterparts (Fig. 3, C, E, and F). All three
EE-tagged constructs were easily detected by Western blotting (Figs. 4
and 5). Site II appears to be a potential site for tagging all
subunits with the EE epitope, probably because highly conserved
sequences at this site closely resemble the sequence of the EE epitope.
Epitope Tags at Site II do Not Alter Membrane
Association
We fractionated cells transiently expressing
subunits into crude particulate and soluble fractions and measured
relative amounts of
subunit by immunoblotting. Transiently
transfected
EE-II,
EE-II, and
HA-I fractionate like their transiently expressed
untagged counterparts and express to the same degree ( Fig. 4and
data not shown). Transiently expressed
EE-II and
untagged
are found almost exclusively in the
particulate fraction, as is the endogenous
. A
substantial fraction of transiently expressed
EE-II
and untagged
is soluble, while endogenous
is primarily particulate. We suspect that overexpression per
se alters the distribution of
, because transient
transfection of larger amounts of
cDNA further
increases the fraction of soluble protein (data not shown). The
predominantly particulate localization of
EE-II
(Fig. 7) is similar to that reported by others for untagged
(4) . Thus these epitope tags do not appear to
alter expression or subcellular distribution of these three
subunits.
Figure 4:
Subcellular distribution of untagged and
epitope-tagged and
. CHO K1 cells (5
10
) were transfected with 10 µg of untagged
, untagged
,
EE-II,
EE-II, or pcDNA1 vector and fractionated into crude
particulate and soluble fractions 48 h later, as described under
``Experimental Procedures.'' Equivalent amounts of the
particulate ( P) and soluble ( S) fractions were
resolved by SDS-PAGE, transferred to nitrocellulose, and probed with an
antibody to a native
epitope (WO82) or
epitope (199.233). Immunoreactive bands detected in the
``vector'' lanes represent endogenous levels of
and
in CHO K1 cells.
Figure 7:
Subcellular distribution of
palmitoylation and myristoylation mutants. CHO K1
cells (5
10
) were transfected with 10 µg of
pcDNA1 containing the indicated
constructs. Crude
particulate ( P) and soluble ( S) fractions were
prepared following hypotonic lysis 48 h later as described under
``Experimental Procedures.'' Equal volumes of both fractions
were analyzed by SDS-PAGE and Western blotting with the EE
mAb.
Epitope-tagged
Previous work demonstrated that HA-tagged
Subunits Are Efficiently
Immunoprecipitated
and
were efficiently
immunoprecipitated by the HA monoclonal antibody
(12) . All
EE-II tagged
subunits immunoprecipitated in a nearly quantitative
fashion. A very small fraction of
EE-II remained in
the supernatant following immunoprecipitation (Fig. 5 A).
Efficient immunoprecipitation required the presence of sodium cholate
and SDS in addition to Triton X-100. These harsher detergents may be
required to effect complete denaturation of the EE epitope when it
occurs at sites other than the amino and carboxyl termini of the
protein. Immunoprecipitation was specific: no EE-tagged protein
immunoprecipitated when a nonspecific antibody was used (Fig.
5 B). Additionally, inclusion in the immunoprecipitation
solution of a 7-residue peptide comprising the EE epitope completely
blocked immunoprecipitation of
EE-II (Fig.
5 A).
subunits did not co-immunoprecipitate with
subunits tagged with either epitope (Fig. 5 C and data
not shown).
Figure 5:
Immunoprecipitation of epitope-tagged
. CHO K1 cells (
5
10
) were
transfected with 10 µg of
EE-II DNA. Crude
particulate fractions were prepared following hypotonic lysis 48 h
later, as described under ``Experimental Procedures.'' An
aliquot was solubilized in 1% cholate and following removal of
insoluble material by centrifugation was adjusted to 1% Triton X-100
and 0.5% SDS. In panel A, the soluble extract was divided into
3 equal aliquots. 2 aliquots were immunoprecipitated with EE mAb
without or with the presence of a septapeptide (50 µg/ml)
corresponding to the EE epitope sequence. Immunoprecipitated material
and the residual soluble extract were resolved by SDS-PAGE and probed
with biotinylated-EE mAb by Western blotting. Lane 1, starting
extract; lane 2, immunoprecipitate without septapeptide;
lane 3, residual extract without septapeptide; lane 4, immunoprecipitate with septapeptide; lane 5, residual
extract with septapeptide. Panels B and C, the
soluble extract was divided into 3 equal aliquots. 2 aliquots were
immunoprecipitated with EE mAb or 12CA5 mAb. Equivalent amounts of the
immunoprecipitates were resolved by SDS-PAGE and probed with
biotinylated EE mAb ( panel B) or G
antiserum ( panel
C) following Western blotting. Lane 1, starting extract;
lane 2, immunoprecipitate with EE mAb; lane 3, immunoprecipitate with 12CA5 mAb.
Myristoylation and Palmitoylation of Epitope-tagged
We constructed a series
of mutants of EE-II
EE-II (hereafter denoted
) in which we replaced with alanine residues the
Gly
and Cys
residues that are sites for
myristoylation and palmitoylation (Fig. 1 C). We examined
the effects of these mutations on the ability of
to
be myristoylated and palmitoylated, to attach to cell membranes, and to
transmit signals from receptors to effectors.
constructs, transiently expressed in CHO K1 cells, were
metabolically labeled with [
H]palmitate or
myristate. Incorporation of radiolabel into
subunits was detected
following immunoprecipitation, SDS-PAGE, and fluorography. Initial
experiments demonstrated that CHO K1 cells rather indiscriminately
incorporated radiolabel from both lipids into sites of myristoylation
and palmitoylation. The two sites can be distinguished by their
sensitivities to treatment with neutral hydroxylamine; the amide bond
linking myristate to glycine is resistant while the thioester bond
linking palmitate to cysteine is cleaved. All metabolic labeling
experiments, therefore, included treatments with hydroxylamine. As
controls for the specificity and completeness of the hydroxylamine
treatment, we included transfections with
EE-II, which
contains only a site for myristoylation, and
EE-II,
which contains only sites for palmitoylation. As anticipated,
radiolabel in
EE-II derived from either myristate or
palmitate was resistant to hydroxylamine, indicating that it was
incorporated at the site of myristoylation (Fig. 6, lane 1).
Radiolabel incorporated into
EE-II was, on the other
hand, completely sensitive to hydroxylamine, indicating its
incorporation via a thioester linkage (Fig. 6, lane 7).
Figure 6:
Palmitate and myristate labeling of mutant
constructs. CHO K1 cells (5
10
)
were transfected with 10 µg of the pcDNA1 constructs
EE-II ( lane 1),
EE-II
( lane 2),
EE-II G2A ( lane 3),
EE-II C3A ( lane 4),
EE-II
G2AC3A ( lane 5),
EE-N ( lane 6), or
EE-II ( lane 7). 48 h later cells were labeled
for 2 h with 0.75 mCi/ml [
H]palmitate or
[
H]myristate, as described under
``Experimental Procedures.'' Following detergent
solubilization, extracts were immunoprecipitated with EE antibody and
resuspended in SDS sample buffer. Proteins (one-third of each sample)
were resolved by SDS-PAGE. Following fixation, identical gels from each
labeling condition were soaked for 12 h in 1
M Tris
HCl,
pH 7.0 ( panels A and C), or 1
M
hydroxylamine, pH 7.0 ( panels B and D). Gels were
then processed for fluorography. The remaining one-third of each
immunoprecipitate was analyzed by SDS-PAGE and Western blotting with
biotinylated-EE mAb. A Western blot from the immunoprecipitate from
[
H]palmitate labeled cells is shown in panel
E. Western blots from myristate-labeled cells were similar (data
not shown). Autoradiograms exposed for 23
days.
incorporated radiolabel derived from both
myristate and palmitate (Fig. 6, lane 2). A sizeable
fraction of the radiolabel from palmitate resisted hydroxylamine
treatment, as did almost all of the radiolabel from myristate,
indicating that label was incorporated into both sites. Introducing the
G2A mutation resulted in incorporation of little or no radiolabel from
myristate (Fig. 6, lane 3). Reproducibily, however,
G2A still incorporated radiolabel from palmitate
(Fig. 6, lane 3) although the extent of labeling was
substantially less than that seen with
; all of the
incorporated radiolabel was completely sensitive to hydroxylamine,
indicating that it was attached through a thioester linkage.
C3A incorporated radiolabel from both myristate and
palmitate, at levels similar to
(Fig. 6,
lane 4). In contrast to
G2A, however, all
radiolabel remained attached to
C3A following
treatment with hydroxylamine, indicating that it was not attached by a
thioester linkage. Finally, as expected, mutations that simultaneously
abolished the sites for both myristoylation and palmitoylation
(
G2AC3A,
EE-N) completely abolished
incorporation of radiolabel from either lipid (Fig. 6, lanes
5 and 6). The simplest interpretation of the data is that
Gly
and Cys
are, respectively, the sites for
myristoylation and palmitoylation. Myristoylation does not require the
presence of the site of palmitoylation. Loss of the site of
myristoylation, however, reduces but does not prevent palmitoylation.
Removal of Sites for Palmitoylation and Myristoylation
Alters Association with Membranes
We compared the subcellular
localizations of epitope-tagged with those of the
G2A, C3A, and G2AC3A mutants. Crude 100,000
g particulate and cytosolic fractions were prepared from transiently
transfected CHO K1 cells and the relative distribution of
constructs assessed by Western blotting (Fig. 7). Wild type
is found almost exclusively in the particulate
fraction as is a mutationally activated
(
Q205L). In contrast, the G2A and G2AC3A mutants
are predominantly cytosolic, although a small amount remains in the
particulate fraction. The C3A mutant also is more soluble, although a
larger fraction remains in the particulate fraction than for the G2A
and G2AC3A mutants. Particulate fraction
and
mutants are almost completely solubilized by 1%
cholate (>90%, data not shown).
Removal of Sites for Palmitoylation and Myristoylation
Alters
We next examined
effects of these mutations on the ability of Signaling
to
mediate D
R inhibition of adenylyl cyclase, by comparing
responses in cells transfected with increasing amounts of DNA encoding
wild type
or the G2A, C3A, G2AC3A mutants (Fig. 8).
In the absence of D
R stimulation, increasing amounts of
constitutively inhibit cAMP production generated by
LHR activation. Stimulation of the D
R further inhibits cAMP
production; D
R-stimulated inhibition levels off at 2 µg
of
DNA (Fig. 8 A). Although all three
mutants mediate D
R inhibition of cAMP
production, their activities differ strikingly.
G2A,
which lacks the site of myristoylation, generates little constitutive
inhibition of cAMP production on its own, even though expression levels
are similar to
(expression data not shown); the
D
R inhibits cAMP production in the presence of
G2A, but the extent of inhibition is substantially
less than for wild type
(Fig. 8 B). In
contrast,
C3A, which lacks the site for
palmitoylation, constitutively inhibits cAMP production quite strongly,
and is consistently more effective than wild type
. At
low amounts of cDNA,
C3A when compared to wild type
produces a greater inhibition of adenylyl cyclase by
the D
R, although maximal inhibitions are similar (Fig.
8 C).
G2AC3A, lacking both sites of fatty
acylation, produces a modest constitutive inhibition of cAMP
production, which is barely augmented by stimulation of the
D
R receptor (Fig. 8 D).
Figure 8:
Signaling activity of
palmitoylation and myristoylation mutants. CHO K1 cells were
transfected with 1 µg of LHR DNA, 2 µg of D
R DNA,
and various amounts of pcDNA1 containing the indicated
EE-II constructs. 48 h later, cAMP production was
measured as described under ``Experimental Procedures'' on
cells treated with 20 ng/ml human chorionic gonadotropin alone
( filled circles) or with 20 ng/ml human chorionic gonadotropin
and 10 µ
M quinpirole ( open squares) ( panels
A-D). Each point represents the mean ± S.D. of triplicate
measurements. Axes in panels B-D are the same as in panel
A. Expression of
constructs was equivalent as
assessed by Western blotting (data not shown). Similar results were
obtained in three additional experiments.
We examined
whether the inhibition of adenylyl cyclase by might
be mediated by sequestration of otherwise free
complexes
that (in the absence of excess
) conditionally
activate adenylyl cyclase. We coexpressed LHR with
,
an
subunit that does not interact with adenylyl cyclase but can
sequester
complexes
(30) . Expression of
elevated slightly (up to 20%) cAMP levels following LHR
stimulation (data not shown). We conclude that free
complexes are not conditionally activating adenylyl cyclase following
LHR stimulation.
G2AC3A to inhibit
constitutively cAMP production suggested that neither the site of
myristoylation or palmitoylation was necessary for interaction with the
effector, adenylyl cyclase. To confirm this inference, both sites were
removed by introducing the EE epitope at the amino terminus of a
constitutively active
mutant,
Q205L,
and the ability of this mutant to inhibit cAMP production was compared
to that of
Q205L with the EE epitope at site II. Both
constructs inhibited production of cAMP to the same extent at the
highest amounts of DNA used for transfection. The mutant
lacking sites of lipid attachment produced less inhibition at
lower concentrations of transfected DNA (Fig. 9), indicating that while
fatty acylations enhance effector interactions of
,
they are not essential for inhibition of adenylyl cyclase.
, we have identified a site common to G protein
subunits that may be used for insertion of epitope tags. Using
epitope-tagged
mutants, our experiments establish the
critical importance of covalently attached fatty acids for
receptor-mediated signaling by
and extend and modify
previous observations defining the structural requirements for
palmitoylation. Finally, our results provide an experimental basis for
investigating the roles of palmitoylation and myristoylation in
regulating movement of
subunits among membrane compartments and
interactions with other signaling proteins.
Epitope Tagging
Availability of epitope-tagged
constructs will facilitate investigations of subunit structure
and function in intact cells. Our findings underline the importance of
assessing functional consequences of the introduction of heterologous
epitopes. Although individual
subunits could be tagged at site I
without disrupting function, only site II consistently tolerated
epitope tagging without altering expression, membrane localization, or
signaling activity. Although site II probably will prove to be a
generally useful site for tagging other
subunits with the EE
epitope, it will be important to assess function of each new
epitope-tagged
construct.
GTP assigns the locations of site I and site II
to loops between
helices in the
helical domain
(29) . While this domain contributes importantly to GTP binding
and GTP hydrolysis in
subunits
(31) , the altered
functions of
subunits tagged at site I hint at additional
functions for the helical domain. The shifted dose-response curve
produced by
EE-I for
-AR activation
of PLC-
and the substantial impairment of D
R mediated
inhibition of adenylyl cyclase by
EE-I suggest that
altering site I perturbs
interactions, GTP-induced
conformational change, or effector activation. Although the crystal
structures of
(29, 32) do not reveal
GTP-induced conformational changes at site I, a recent report of the
crystal structure of
notes in passing that this
region may undergo conformational change
(33) . The data
presented here argue strongly that the conformation of the region
encompassing site I in
,
, and
is functionally important.
Palmitoylation and Myristoylation of
Our results confirm previous observations that
is palmitoylated and myristoylated
(2, 4, 8) and agree with conclusions based on
experiments with other
subunits that myristoylation is not
affected by mutations that alter the site of palmitoylation
(14, 34) . In contrast to the results of others
(4, 14) , however, we find that palmitoylation can take
place in the absence of the site of myristoylation, although the G2A
mutation does reduce the amount of palmitate label detected.
G2A would be
decreased if Gly
or its attached myristate were an
important structural determinant for palmitoylation. This seems
unlikely, however, because both
and
are palmitoylated even though neither is myristoylated, and
lacks an amino-terminal Gly residue.
by impairing its localization to the compartment, presumably a
membrane compartment, that contains the relevant palmitoyl transferase.
In this view, myristoylation could play an important role in
palmitoylation by increasing associations of
with
cellular membranes directly or by increasing affinity of
for
complexes, which in turn present
to the membrane. The presence of myristate on
does increase its association with membranes (Fig. 7) and
myristate is reported to increase the affinities of both
(5) and
(6) for
.
G2A may
reflect more rapid depalmitoylation. Physiologically, Gly
and/or myristate may serve an important regulatory role in
depalmitoylation, although our data do not address this directly.
Gly
and/or myristate could also play an important but
nonphysiological role in stabilizing palmitate during the extraction
and immunoprecipitation procedure. Cell extracts contain palmitoyl
thioesterase activity; a substantial loss of palmitate from
can take place after breaking cells, during the process of
extraction and immunoprecipitation
(15) . If the G2A mutation
accelerates the rate of depalmitoylation, substantially less palmitate
will be detected by the end of the processing procedure, even though
the extent of palmitoylation in the intact cell may have been equal in
wild type and mutant
. This kind of nonphysiological
depalmitoylation could also explain the discrepancy between our results
and reports that G2A mutants are not palmitoylated. The conditions used
here represent a minimal time for extraction and complete
immunoprecipitation (2 h). Others, using transiently overexpressed
,
, or
in HEK293
and COS cells
(4, 14) , employed lengthier
immunoprecipitation protocols and did not detect palmitoylation in G2A
mutants. Our ability to detect palmitoylation of
G2A
in CHO K1 cells may reflect physiologically important cell-specific
differences, but may also indicate that extraction and
immunoprecipitation protocols are not always reliable for detecting the
presence of palmitate.
Membrane Attachment and Signaling Activity
The
simultaneous abolition of myristoylation and palmitoylation in
produces effects similar to abolition of
palmitoylation in
(12) . The subcellular
localization of both
subunit constructs shifts to the soluble
fraction; only a small amount remains in the particulate fraction.
Receptor mediated interactions with adenylyl cyclase are severely
impaired or abolished. Constitutively activating forms of both
subunits, however, still productively interact with adenylyl cyclase,
albeit less efficiently. The more pronounced functional impairment seen
with receptor activation may reflect the critical importance of fatty
acid acylation for interactions with
complexes which in turn
are required for receptor-catalyzed GDP/GTP exchange.
associated
with the particulate fraction and impaired but did not prevent signal
transmission, which requires productive interactions of
with
, receptor, and effector. These results were
surprising because myristoylation of some
family
members greatly increases their affinity for
(5, 6) and is required for productive interaction of
with adenylyl cyclase
(7) ; this latter finding is in
agreement with results of our own experiments with a constitutively
active form of
in intact cells.
(
)
Activation of non-myristoylated
, whether
by receptor (
G2A) or by a constitutively activating
mutation (
EE-N Q205L), still resulted in inhibition of
adenylyl cyclase. Larger amounts of the nonacylated, mutationally
activated
were required for maximal inhibition of
adenylyl cyclase (Fig. 9). Presumably, myristate increases either
the affinity of
for adenylyl cyclase or the amount of
at the plasma membrane, thereby increasing its
effective concentration.
Figure 9:
Signaling activity of
Q205L palmitoylation and myristoylation mutant. CHO K1
cells were transfected with 1 µg of LHR DNA and various amounts of
pcDNA1 containing
EE-II ( open squares),
EE-II Q205L ( filled squares), or
EE-N Q205L ( filled circles) constructs. 48 h
later, cAMP production was measured as described under
``Experimental Procedures'' on cells treated with 20 ng/ml
human chorionic gonadotropin. Each point represents the mean ±
S.D. of triplicate measurements. Expression of
constructs was equivalent as assessed by Western blotting (data
not shown). An additional experiment yielded similar
results.
Mutation that prevents palmitoylation
decreases substantially the fraction of associated
with the particulate fraction but surprisingly this has no discernible
effect on D
R-mediated inhibition of adenylyl cyclase
(compare D
R inhibition of cAMP production for
versus
C3A, Fig. 8, A versus C). Even more surprisingly, preventing
palmitoylation of
enhances its constitutive
inhibition of adenylyl cyclase. This effect is seen most clearly in
comparing myristoylated forms of
(compare LHR
stimulation of cAMP production in the presence of
versus
C3A, Fig. 8, A versus C), but also occurs in the absence of
myristoylation (compare LHR stimulation of cAMP production in the
presence of
G2A versus
G2AC3A, Fig. 8, B versus D).
could reflect
the presence of
GTP that arises from non-receptor
catalyzed exchange of GDP for GTP. In this context, palmitoylation
could decrease the rate of GDP/GTP exchange directly by generating a
more stable conformation of
GDP or indirectly by
increasing the affinity of
for
. Although
myristoylation can indirectly slow exchange by increasing the affinity
of
for
, no evidence yet implicates palmitoylation or
any other fatty acylation of
subunits in modulating GDP/GTP
exchange.
to a membrane domain that
regulates interactions with effector. Increasing numbers of signaling
molecules, including G protein
subunits, are being found to
concentrate in caveolae
(35, 36) , plasmalemmal domains
that play roles in intra- and intercellular communication
(37) .
Palmitoylation is a signal that directs otherwise cytosolic proteins to
caveolae, as shown most clearly for kinases of the Src family
(38) . It is attractive to hypothesize that palmitoylation of
localizes it to caveolae and that this localization
prevents spurious interactions with effector.
(12, 15, 18) ,
Wedegaertner and Bourne
(15) proposed that the dynamic
palmitoylation cycle regulates signaling by G
and,
possibly, by other G proteins. Depalmitoylation of
follows its activation by receptor and correlates with its
movement from the membrane into the soluble compartment. After
hydrolysis of GTP,
GDP is repalmitoylated, binds
, and returns to the membrane for another cycle of receptor
activation (although the temporal sequence of events that follows GTP
hydrolysis is unknown). Depalmitoylated
is
functionally uncoupled from receptor activation, so that
depalmitoylation may be viewed as a potential mechanism for decreasing
the signaling activity of
.
,
depalmitoylation does not seem likely to decrease
-dependent signaling, at least in relation to adenylyl
cyclase. First, the subcellular distribution of mutationally activated
is virtually identical to that of non-activated
; in contrast, constitutive activation shifts a large
fraction of
into the soluble compartment
(12) . Second, receptor mediated signaling by
is enhanced by mutation that prevents palmitoylation, again in
contrast to the effect this mutation has on
signaling. Third, mutational removal of the palmitoylation site
in
produces a protein whose ability to constitutively
inhibit adenylyl cyclase is actually increased. Dynamic palmitoylation
of
may serve to shuttle it between membrane
compartments, rather than to decrease its activity.
Summary
Our results sketch a low resolution
picture of the roles that palmitate and myristate play in the membrane
association and signaling properties of . The lack of
resolution reflects the fact that our assays are all-inclusive
measurements of signaling function, and do not distinguish individual
steps during receptor-mediated signal transduction, such as
binding to
and
binding to receptor.
Nevertheless our results clarify the distinct and different effects of
palmitoylation and myristoylation on different G protein
subunits, and raise new questions which can be tackled with
epitope-tagged constructs.
-AR,
-adrenergic receptor;
LHR, luteinizing hormone receptor; D
R,
D
-dopamine receptor;
-AR,
-adrenergic receptor; PI-PLC,
phosphoinositide-specific phospholipase C; MEM, minimal essential
medium; mAb, monoclonal antibody; PAGE, polyacrylamide gel
electrophoresis.
EE cDNA construct and Gordon Chew for
HA cDNA constructs.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.