From the Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, Merville House, University College Dublin, Belfield, Dublin 4, Ireland
Received for publication, October 17, 2002, and in revised form, November 27, 2002
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ABSTRACT |
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We have previously established that
isoprenylation of the prostacyclin receptor (IP) is required for its
efficient G protein coupling and effector signaling (Hayes, J. S.,
Lawler, O. A., Walsh, M. T., and Kinsella, B. T. (1999)
J. Biol. Chem. 274, 23707-23718). In the present
study, we sought to investigate whether the IP may actually be subject
to palmitoylation in addition to isoprenylation and to establish the
functional significance thereof. The human (h) IP was efficiently
palmitoylated at Cys308 and Cys311, proximal to
transmembrane domain 7 within its carboxyl-terminal (C)-tail domain,
whereas Cys309 was not palmitoylated. The
isoprenylation-defective hIPSSLC underwent palmitoylation
but did not efficiently couple to Gs or Gq,
confirming that isoprenylation is required for G protein coupling.
Deletion of C-tail sequences distal to Val307 generated
hIP Modification of proteins through the covalent attachment of lipid
groups occurs within a wide variety of cellular proteins and may be
involved in mediating protein-membrane and/or protein-protein interactions (1-3). Three of the most common lipid modifications are
N-myristoylation, isoprenylation and
thio(S)-acylation. In contrast to myristoylation and
isoprenylation, which typically occur either as co-translational or as
immediate post-translational events, S-acylation through
attachment of palmitate to Cys residue(s), via a labile thioester bond,
occurs post-translationally (1). Moreover, whereas the former two
modifications remain attached until protein degradation, palmitoylation
is a reversible, dynamic modification that turns over more rapidly than
the protein itself and thus has the potential to be regulated (2, 3). A
diverse family of cellular proteins are established to be palmitoylated including It has been suggested that palmitoylation is a general feature of
GPCRs; ~80% of all known receptors contain at least one palmitoylable cysteine residue usually located between 10 and 14 amino
acids downstream of transmembrane domain 7, within their intracellular carboxyl-terminal tail (C-tail) region (6). Rhodopsin was
the first of its class to be identified as a target for palmitoylation, occuring within the C-tail at Cys322 and Cys323
(7). Moreover, Ovchinnikov et al. (7) proposed that the hydrophobic palmitate moiety becomes integrated into the lipid membrane
bilayer, resulting in the formation of a putative fourth intracellular
loop. More recently, the use of fluorescent fatty acid analogues with
rhodopsin demonstrated that indeed this is the case (8). Numerous other
GPCRs have been identified as targets for palmitoylation. With the
finding that GPCRs undergo repeated cycles of
palmitoylation/depalmitoylation, it is generally accepted that it may
play a role in the regulation of diverse signaling cascades (1-3). For
example, the Isoprenylation occurs by attachment of either C-15 farnesyl or C-20
geranylgeranyl isoprenoids, derived from the mevalonate/cholesterol biosynthetic pathway, via stable thioether linkages to specific carboxyl-terminal cysteine residues located in distinct
"isoprenylation motifs" of proteins (14). Isoprenylation, an
immediate post-translational modification, occurs in the cytoplasm, but
subsequent proteolysis and carboxyl methylation, should they occur, are
membrane-associated events (14). Palmitoylation of isoprenylated
proteins generally occurs on cellular membranes whereby isoprenylation
precedes palmitoylation (1). Many proteins are modified exclusively
with palmitate, for example G The prostanoid prostacyclin signals through interaction with its
signature GPCR, termed the prostacyclin receptor, mediating the
inhibition of platelet aggregation and vascular smooth muscle relaxation. The prostacyclin receptor, also termed the IP (16), signals
primarily through activation of adenylyl cyclase via Gs (17, 18) but may also couple to other G protein-effector systems including Gq-dependent activation of
phospholipase (PL) C and mobilization of intracellular calcium (18,
19). Recently, it was established that in addition to Gs,
the mouse IP also couples to inhibition of adenylyl cyclase via
Gi and to Gq/PLC activation through a switching
mechanism involving its initial coupling to Gs/adenylyl
cyclase activation and subsequent cAMP-dependent protein kinase A phosphorylation of the mouse IP at Ser357 within
its C-tail region (20). Additionally, IPs are widely reported to
undergo rapid agonist-induced desensitization, internalization, and
down-regulation in human platelets and in other cell types (21-24).
We have recently established that the IP may be unique among GPCRs in
that it is isoprenylated through attachment of a C-15 farnesyl
isoprenoid to a Cys within a highly conserved CAAX motif in
its C-tail domain (18, 25). Inhibition of isoprenylation of both the
mouse and human (h) IP, either through site-directed mutagenesis of the
critical acceptor Cys of the CAAX motif or through the use
of the statin inhibitors of hydroxymethylglutaryl CoA reductase,
established that although isoprenylation is not required for ligand
binding by IP, it is absolutely required for receptor activation of
adenylyl cyclase via G Materials
Cicaprost was obtained from Schering AG (Berlin, Germany).
FURA2/AM was purchased from Calbiochem.
[3H]iP3 (20-40 Ci/mmol) and
[3H]cAMP (15-30 Ci/mmol) were purchased from American
Radiolabeled Chemicals Inc. [3H]Iloprost (15.3Ci/mmol)
was purchased from Amersham Biosciences. [9,10-3H]Palmitic acid (60 Ci/mmol) was obtained from
PerkinElmer Life Sciences. Polyvinylidene difluoride (PVDF) filters,
Expand High Fidelity Taq DNA polymerase, Chemiluminescence
Western blotting kit, and rat monoclonal 3F10 anti-hemagglutinin (HA)
peroxidase-conjugated antibody were purchased from Roche Molecular
Biochemicals. The oligonucleotides were synthesized by Genosys
Biotechnologies. Mouse monoclonal anti-HA 101R antibody was obtained
from BabCO. Anti-G Methods
Deletion- and Site-directed Mutagenesis of Human Prostacyclin
Receptor--
The plasmids pBluescript-hIP and pHM-hIP have been
previously described (20, 26). Deletion of the amino acids carboxyl to
Val307 within the hIP to generate the hIP
All site-directed mutagenesis was performed using the QuikChangeTM
site-directed mutagenesis kit. Conversion Cys308 of the hIP
to Ser308 was performed using pHM-hIP as a template and the
oligonucleotide primers: 5'-CTC AAG CTC TGG GTC AGC TGC CTG
TGC CTC G-3' (sense primer) and 5'-C GAG GCA CAG GCA GCT GAC
CCA GAG CTT GAG-3' (antisense primer; the sequence complimentary to the single mutator codon is in bold type), resulting in the generation of
the plasmid pHM-hIPC308S. Conversion of Cys309
of the hIP to Ser309 was performed using pHM-hIP as a
template and the oligonucleotide primers: 5'-C AAG CTC TGG GTC TGC
AGC CTG TGC CTC GGG CC-3' (sense primer) and 5'-GG CCC GAG
GCA CAG GCT GCA GAC CCA GAG CTT G-3' (antisense primer; the
sequence complimentary to the single mutator codon is highlighted in
bold type), resulting in the generation of the plasmid
pHM-hIPC309S. Conversion of Cys311 of the hIP
to Ser311 was performed using pHM-hIP as a template and the
oligonucleotide primers: 5'-GTC TGC TGC CTG AGC CTC GGG CCT
G-3' (sense primer) and 5'-C AGG CCC GAG GCT CAG GCA GCA
GAC-3' (antisense primer; the sequence complimentary to the single
mutator codon is highlighted in bold type), resulting in the generation
of the plasmid pHM-hIPC311S. Conversion of
Cys308 and Cys309 of the hIP to
Ser308 and Ser309 was performed using
pHM-hIPC309S as a template and the oligonucleotide primers:
5'-CTC AAG CTC TGG GTC AGC AGC CTG TGC CTC GG-3' (sense
primer) and 5'-CC GAG GCA CAG GCT GCT GAC CCA GAG CTT GAG-3'
(antisense primer; the sequence complimentary to the single mutator
codon is highlighted in bold type), resulting in the generation of the
plasmid pHM-hIPC308S,C309S. Conversion of
Cys308 and Cys311 of the hIP to
Ser308 and Ser311 was performed using
pHM-hIPC308S as a template and the oligonucleotide primers:
5'-GTC AGC TGC CTG AGC CTC GGG CCT G-3' (sense primer) and
5'-C AGG CCC GAG GCT CAG GCA GCT GAC-3' (antisense primer;
the sequence complimentary to the single mutator codon is highlighted
in bold type), resulting in the generation of the plasmid
pHM-hIPC308S,C311S. Conversion of Cys309 and
Cys311 of the hIP to Ser309 and
Ser311 was performed using pHM-hIPC309S as a
template and the oligonucleotide primers: 5'-GTC TGC AGC CTG
AGC CTC GGG CCT G-3' (sense primer) and 5'-C AGG CCC GAG
GCT CAG GCT GCA GAC-3' (antisense primer; the sequence complimentary to the single mutator codon is highlighted in bold type),
resulting in the generation of the plasmid
pHM-hIPC309S,C311S. Conversion of Cys308,
Cys309, and Cys311 of the hIP to
Ser308, Ser309, and Ser311 was
performed using pHM-hIPC308S,C309S as a template and the
oligonucleotide primers: 5'-GTC AGC AGC CTG AGC CTC GGG CCT
G-3' (sense primer) and 5'-C AGG CCC GAG GCT CAG GCT GCT
GAC-3' (antisense primer; the sequence complimentary to the single
mutator codon is highlighted in bold type), resulting in the generation
of the plasmid pHM-hIPC308S,C309S,C311S.
All of the resulting plasmids were verified by double-stranded DNA
sequencing and encode HA epitope-tagged forms of hIP Cell Culture and Transfections--
Human embryonic kidney (HEK)
293 cells were obtained from the American Type Culture Collection and
were maintained at 37 °C, 5% CO2. HEK 293 cells were
cultured in minimal essential medium with Earle's salts, 10% fetal
bovine serum. HEK.hIP and HEK.hIPSSLC cells stably
overexpressing the wild type hIP and the isoprenylation-defective hIPSSLC have been previously described (20, 26). The
plasmids pCMV-G
To create the HEK.hIP Western Blot Analysis--
HEK.hIP, HEK.hIP Palmitoylation of the Prostacyclin Receptor in HEK 293 Cells--
Palmitoylation of the prostacyclin receptor was performed
essentially as previously described (28). Briefly, HEK.hIP,
HEK.hIPSSLC, HEK.hIP Radioligand Binding Studies--
For membrane preparation, the
cells were harvested by centrifugation at 500 × g at
4 °C for 5 min followed by washing three times with PBS. The cells
were then resuspended in homogenization buffer (25 mM
Tris-HCl, pH 7.5, 0.25 M sucrose, 10 mM
MgCl2, 1 mM EDTA, 0.1 mM
phenylmethylsulfonyl fluoride), homogenized, and then centrifuged at
100,000 × g for 40 min at 4 °C. The pellet fraction
(P100), representing crude membranes, was resuspended in
MES-KOH buffer (10 mM MES-KOH, pH 6.0, 10 mM
MnCl2, 1 mM EDTA, 10 mM
indomethacin). IP radioligand binding assays were carried out at
30 °C for 1 h, using 100 µg of protein (P100), in
100-µl reactions in the presence of 4 nM
[3H]iloprost (15.3 Ci/mmol) as previously described
(18).
Alternatively, to confirm cell surface IP expression, whole cells were
harvested by centrifugation at 500 × g at 4 °C for 5 min followed by washing three times with ice-cold PBS. Thereafter, the whole cells were resuspended in MES-KOH buffer (10 mM
MES-KOH, pH 6.0, 10 mM MnCl2, 1 mM
EDTA, 10 mM indomethacin). IP radioligand binding assays
were carried out at 4 °C for 1 h, using 100 µg of whole cell
protein in 100 µl reactions in the presence of 4 nM
[3H]iloprost (15.3 Ci/mmol) essentially as previously
described (18). As a control to determine any
temperature-dependent changes in the ability of the IP to
bind agonist at 4 °C as opposed to 30 °C, the level of
[3H]iloprost binding to membrane fractions
(P100 fractions) was also determined at 4 °C and was
compared with that which occurred at 30 °C. In general, the level of
[3H]iloprost bound to IPs expressed in crude membranes
when assayed at 4 °C was reduced to 72% of that bound at 30 °C
and thereby accounted for the apparent reduced level of radioligand
bound to whole cells, determined at 4 °C, relative to that bound to crude membranes, determined at 30 °C. The protein determinations were carried out using the Bradford assay (32).
Measurement of cAMP--
cAMP assays were carried out as
previously described (18). Briefly, the cells were harvested by
scraping and washed three times in ice-cold PBS; the cells
(~1-2 × 106 cells) were resuspended in 200 µl of
HEPES-buffered saline (HBS; 140 mM NaCl, 4.7 mM
KCl, 2.2 mM CaCl2, 1.2 mM
KH2PO4, 11 mM glucose, 15 mM HEPES-NaOH, pH 7.4) containing 1 mM
3-isobutyl-1-methylxanthine and were preincubated at 37 °C for 10 min. Thereafter, ligands (50 µl) were added, and cells were
stimulated at 37 °C for 10 min in the presence of the ligand (1 µM cicaprost, 10 µM forskolin, or 1 µM cicaprost plus 10 µM forskolin). For
concentration response studies, the cells were stimulated with
10
In each case, the cAMP reactions were terminated by heat inactivation
at 100 °C for 5 min, and the level of cAMP produced was quantified
using the cAMP binding protein assay (33). The levels of cAMP produced
by ligand-treated cells over basal stimulation, determined in the
presence of HBS, were expressed as fold stimulation relative to basal
(fold increase ± S.E.).
Measurement of IP3 Levels--
Intracellular
IP3 levels were measured as previously described (29).
Briefly, the cells were transiently co-transfected with either
pCMV-G Measurement of Intracellular Ca2+
Mobilization--
Measurement of intracellular Ca2+
mobilization ([Ca2+]i) in FURA2/AM-preloaded
cells was carried out essentially as previously described (27).
Cicaprost was diluted in HBSSHB (modified
Ca2+/Mg2+-free Hanks' buffered salt solution
containing 20 mM HEPES, pH 7.67, 0.1% bovine serum albumin
plus 1 mM CaCl2) to the appropriate concentration such that addition of 20 µl of the diluted cicaprost to
2 ml of cells resulted in the correct working concentrations (10 Data Analyses--
Statistical analysis was carried out using
the unpaired Student's t test using the GraphPad Prism V2.0
program (GraphPad Software Inc., San Diego, CA). p values of
less than or equal to 0.05 were considered to indicate a statistically
significant difference. Amino acid sequence alignments were carried out
using the Clustal W software (34), where sequences were aligned to show
maximum homology.
The Role of C-tail Sequences in Signaling by the hIP--
Previous
studies have demonstrated that isoprenylation of the IP within its
C-tail region is required for its efficient intracellular signaling
(18, 25). Specifically, mutation of the CAAX motif of the
hIP from -C383SLC to -S383SLC abolished
isoprenylation and generated hIPSSLC that exhibited
identical ligand binding properties to that of the wild type hIP but
failed to show significant coupling to G
We initially investigated the above paradigm by comparing signaling by
the hIPSSLC and the hIP
To exclude the possibility that the carboxyl-terminal four amino acids
of the truncated hIP
Although the hIP
Thereafter, the ability of hIP
Thus, it appears that although the hIP Palmitoylation of the Human Prostacyclin Receptor--
To
investigate whether the hIP and its variants are indeed palmitoylated,
HEK.hIP, HEK.hIPSSLC, HEK.hIP
Palmitoylation of both the isoprenylation-defective hIPSSLC
and hIP Palmitoylation and Signaling by hIPC308S,
hIPC309S, and hIPC308S,C309S--
Herein, we
have demonstrated that whereas the hIP, hIPSSLC, and
hIP
Initial characterization of HEK.hIPC308S,
HEK.hIPC309S, and HEK.hIPC308S,C309S cells,
recombinant HEK 293 cells stably overexpressing hIPC308S,
hIPC309S, and hIPC308S,C309S by saturation
radioligand-binding confirmed receptor expression at levels comparable
with those observed in HEK.hIP cells and also confirmed that the
mutations per se did not affect the ligand binding
properties of the receptors or their targeting to the plasma membrane
(Table I). To ascertain whether Cys308 and/or
Cys309 residues of the hIP are actually targets for
palmitoylation, HEK.hIP, HEK.hIPC308S,
HEK.hIPC309S, and HEK.hIPC308S,C309S cells were
metabolically labeled with [3H]palmitic acid in the
presence of cicaprost. Palmitoylation of both the hIP and
hIPC309S was observed as evidenced by the detection of a
broad radiolabeled band between 46 and 66 kDa in the immunoprecipitates
from HEK.hIP and HEK.hIPC309S cells (Fig.
6A, lanes 1 and
3, respectively). Additionally, it was noteworthy that the
level of palmitoylation of the hIP (4.23-fold increase) and the
hIPC309S (3.76-fold increase) were comparable, suggesting
that Cys309 may not be a target for palmitoylation. In
contrast, although the hIPC308S and
hIPC308S,C309S did undergo palmitoylation, levels of
metabolic labeling of both receptors were significantly reduced
relative to hIP (Fig. 6A, compare lanes 2 and
4 with lane 1; 2.44- and 2.34-fold increases in
palmitoylation relative to basal levels, respectively). Following fluorography/PhosphorImager analysis, the above PVDF membrane was
subject to secondary screening by Western blot analysis using the
anti-HA 3F10 peroxidase conjugate and confirmed equivalent expression
and the recovery of hIP, hIPC308S, hIPC309S,
and hIPC308S,C309S receptors in their respective
immunoprecipitates (Fig. 6B, lanes 1-4,
respectively).
Thereafter, cicaprost-induced signaling by the
HEK.hIPC308S, HEK.hIPC309S, and
HEK.hIPC308S,C309S cells was examined and was compared with
that of the HEK.hIP and nontransfected HEK 293 cells. Stimulation of
hIPC308S, hIPC309S, and
hIPC308S,C309S each resulted in significant,
concentration-dependent increase in cAMP generation to levels
that were not significantly different from the hIP, throughout the
range of cicaprost concentrations examined (Fig.
7A; p > 0.05). Moreover, co-transfection of cells with G
Whereas stimulation of HEK.hIPC309S cells exhibited
increases in IP3 generation that were not significantly
different from that generated by HEK.hIP cells throughout the range of
cicaprost concentrations examined (Fig.
8A; p = 0.96),
stimulation of HEK.hIPC308S and
HEK.hIPC308S,C309S cells failed to induce measurable
increases in IP3 generation, even at the 1 µM
cicaprost (p < 0.05). Moreover, co-transfection with
G Analysis of Palmitoylation and Signaling Properties of
hIPC308S,C309S,C311S, hIPC311S,
hIPC308S,C311S, and
hIPC309S,C311S--
Analysis of the palmitoylation status
of hIPC308S,C309S (Fig. 6A, lane 4)
demonstrated that whereas a significant reduction in metabolic labeling
of hIPC308S,C309S by [3H]palmitic acid was
evident relative to hIP (p < 0.05), the levels of
metabolic labeling detected were consistently ~2-fold greater than
that detected in control cells, suggesting that
hIPC308S,C309S may be palmitoylated at another Cys residue
distinct from Cys308 and Cys309. It was
postulated that the remaining semi-conserved Cys residue, Cys311, located between amino acids 308 and 312 may be
palmitoylated, accounting for the higher than basal levels of metabolic
labeling of the hIPC308S,C309S. Thus, to investigate
whether Cys311 may indeed be palmitoylated, site-directed
mutagenesis was used to generate hIPC308S,C309S,C311S.
Initial characterization of HEK.hIPC308S,C309S,C311S
cells by saturation radioligand binding confirmed high level receptor
expression at levels comparable with those observed in HEK.hIP cells
(Table I). Metabolic labeling of HEK.hIPC308S,C309S,C311S
cells with [3H]palmitic acid established that, unlike the
hIP (Fig. 9A, lane 1), the hIPC308S,C309S,C311S was not palmitoylated
(Fig. 9A, lane 2). Subsequent screening of the
PVDF membrane by immunoblot analysis using the anti-HA 3F10
peroxidase conjugate followed by chemiluminescence detection confirmed
that both hIPC308S,C309S,C311S and hIP were expressed and
immunoprecipitated at comparable levels (Fig. 9B,
lanes 1 and 2, respectively).
To further confirm that Cys308 and
Cys311, but not Cys309, were subject to
palmitoylation, site-directed mutagenesis was used to generate hIPC311S, hIPC308S,C311S, and
hIPC309S,C311S. Saturation radioligand binding assays again
confirmed high level receptor expression in HEK.hIPC311S,
HEK.hIPC308S,C311S, and HEK.hIPC309S,C311S
cells at levels comparable with those observed in HEK.hIP cells and
confirmed that the mutations per se did not affect the
ligand binding properties of the receptors or their targeting to the plasma membrane (Table I). Metabolic labeling of
HEK.hIPC308S,C311S cells with [3H]palmitic
acid established that, like the hIPC308S,C309S,C311S (Fig.
9A, lane 2), the hIPC308S,C311S was
not palmitoylated (Fig. 9A, lane 4). Although the
hIPC311S and hIPC309S,C311S did undergo
palmitoylation, the levels of metabolic labeling of both receptors were
significantly reduced relative to hIP (Fig. 9A,
compare lanes 3 and 5 with lane 1;
2.11- and 2.43-fold increases in palmitoylation relative to basal
levels, respectively). Subsequent secondary screening of the PVDF
membrane by immunoblot analysis using the anti-HA 3F10 peroxidase
conjugate followed by chemiluminescence detection confirmed that hIP,
hIPC311S, hIPC308S,C311S, and
hIPC309S,C311S were expressed and immunoprecipitated at
comparable levels (Fig. 9B, lanes 1 and
3-5, respectively). Taken together, these data confirm
that, similar to Cys308, Cys311 is also subject
to palmitoylation. Moreover, these data also demonstrate that
Cys309 is not a site of palmitoylation, because no
palmitate was incorporated into hIPC308S,C311S.
Thereafter, cicaprost-induced signaling by hIPC311S,
hIPC308S,C311S, hIPC309S,C311S, and
hIPC308S,C309S,C311S was examined and compared with
signaling by the hIP. Stimulation of hIPC311S and
hIPC309S,C311S each resulted in significant,
concentration-dependent increase in cAMP generation to levels
that were not significantly different from the hIP throughout the range
of cicaprost concentrations examined (Fig.
10A; p > 0.05). On the other hand, levels of cAMP generation by
hIPC308S,C311S and hIPC308S,C309S,C311S were
significantly lower than those levels generated by the hIP throughout
the range of cicaprost concentrations examined (Fig. 10A;
p < 0.05) and, in fact, were not significantly
different from those levels generated by the hIPSSLC
(p > 0.88; 25) or the hIP
Stimulation of hIPC311S and hIPC309S,C311S with
cicaprost yielded concentration-dependent increases in
[Ca2+]i mobilization to levels that were not
significantly different from those generated by the hIP (Fig.
10C; p = 0.83). However, levels of
[Ca2+]i mobilization by the
hIPC308S,C311S (p = 0.0034) and the
hIPC308S,C309S,C311S (p = 0.003) were
significantly reduced relative to the hIP throughout the
range of cicaprost concentrations (Fig. 10C) and were not
significantly different from those levels generated by the
hIPSSLC (p > 0.05) (25) or the
hIP In the current study, through a combination of deletion and
site-directed mutagenesis, in vivo metabolic labeling
studies, and the use of pharmacologic inhibitors of protein
isoprenylation, we investigated the requirement for palmitoylation and
isoprenylation for IP function and intracellular signaling. To this
end, we initially compared signaling by the wild type hIP, the
isoprenylation-defective hIPSSLC, and the deletion mutant
hIP A number of independent studies have demonstrated that impairment of
receptor palmitoylation, such as in the case of the CCR5 receptor, may
result in accumulation of GPCRs in intracellular stores and in the
lowering of cell surface receptor expression (39). In the present
study, for the hIP and its various mutant forms, we have established
that all stable cell lines under study express equivalent cell surface
receptor numbers, thereby confirming that neither palmitoylation nor,
indeed, isoprenylation per se alter the ability of the hIP
to reach the plasma membrane. Thus, differences in IP signaling by the
palmitoylation-defective mutants may not be attributed to
altered/reduced cell surface receptor expression but rather are most
likely due to direct alteration in their palmitoylation status.
Although our metabolic labeling studies clearly demonstrate that the
hIP is palmitoylated at Cys308 and Cys311 and
our intracellular signaling studies have highlighted the importance of
those palmitoyl moieties in facilitating G protein coupling/effector
signaling, in the absence of an appropriate pharmacologic inhibitor of
palmitoylation, our studies cannot exclude the possibility that it is
the presence of Cys residues at those sites, as opposed to the
palmitoyl-Cys residues, that are critical in mediating that signaling.
Thus, through our previous studies (18, 25, 26) and those outlined in
the present study involving the hIP, it is evident that the IP is
certainly unique among GPCRs thus far characterized in that it is
isoprenylated and palmitoylated, and together these dual lipidations
modulate IP coupling to Gs- and Gq-regulated effector systems but do not affect agonist binding. In attempting to
put forward a model (Fig. 11) to
describe the relative involvements of isoprenylation and palmitoylation
in regulating IP signaling, we have drawn from the prototypic Ha-Ras in
assuming that isoprenylation precedes palmitoylation (14) and that both
types of lipid modifications mediate protein-membrane rather than
protein-protein interactions. However, we preface our model by stating
that this may not strictly be accurate because in the case of the
hIPSSLC, for example, palmitoylation occurs in the absence
of isoprenylation, and further experiments are required to precisely
define or identify the nature of the individual lipid interactants.
However, despite these limitations, we now propose a model, as outlined
in Fig. 11, to explain the role of isoprenylation and palmitoylation in mediating hIP coupling to Gs/adenylyl cyclase and
Gq/PLC effector signaling. Additionally, through the model
outlined below, we propose to explain the apparent paradox as to why
the hIP307 that was neither palmitoylated nor isoprenylated
and did not efficiently couple to Gs or to Gq,
whereas hIP
312 was palmitoylated and ably coupled to
both effector systems. Conversion of Cys308,
Cys309, Cys311, Cys308,309, or
Cys309,311 to corresponding Ser residues, while leaving the
isoprenylation CAAX motif intact, did not affect hIP
coupling to Gs signaling, whereas mutation of
Cys308,311 and Cys308,309,311 abolished
signaling, indicating that palmitoylation of either Cys308
or Cys311 is sufficient to maintain functional
Gs coupling. Although mutation of Cys309 and
Cys311 did not affect hIP-mediated Gq coupling,
mutation of Cys308 abolished signaling, indicating a
specific requirement for palmitoylation of Cys308 for
Gq coupling. Consistent with this, neither
hIPC308S,C309S, hIPC308S,C311S, nor
hIPC308S,C309S,C311S coupled to Gq. Taken
together, these data confirm that the hIP is isoprenylated and
palmitoylated, and collectively these modifications modulate its G
protein coupling and effector signaling. We propose that through lipid
modification followed by membrane insertion, the C-tail domain of the
IP may contain a double loop structure anchored by the dynamically
regulated palmitoyl groups proximal to transmembrane domain 7 and by a
distal farnesyl isoprenoid permanently attached to its carboxyl terminus.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits of the heterotrimeric G protein subunits, for
example G
s and G
q, Ha- and N-Ras
proteins, A kinase anchoring protein 15 and 18, endothelial
nitric-oxide synthase, adenylyl cyclase, G protein-coupled receptor
kinases 4 and 6, diverse members of the Src family of nonreceptor
tyrosine kinases, in addition to several members of the G
protein-coupled receptor
(GPCR)1 superfamily (1, 2, 4,
5).
2-adrenergic receptor (AR) is palmitoylated
at Cys341, and palmitoylation at this site is required for
2-AR coupling to adenylyl cyclase (9) and is increased
upon exposure of cells to agonist (10). Palmitoylation of the
endothelin (ET)A and ETB receptors is required
for their coupling to the extracellular signal regulated
kinase/mitogen-activated protein kinase cascade (11). Mutation of
Cys442 within the C-tail of the
2A-AR
receptor inhibits receptor down-regulation in response to prolonged
agonist exposure (12). Palmitoylation has also been shown to regulate
ligand-induced receptor internalization. Specifically, abolition of
palmitoylation of the luteinizing hormone receptor results in a 2-fold
increase in receptor internalization when compared with the wild type
receptor. Moreover, intracellular receptor degradation was higher in
nonpalmitoylated receptors (13). Thus, it is apparent from these
studies that a diverse array of GPCR cellular signaling cascades may be
regulated either directly or indirectly by palmitoylation.
s, whereas others are
subject to dual lipidation (1). For example, Ha-Ras and N-Ras are
farnesylated and palmitoylated. Dual lipidation may serve to further
increase protein hydrophobicity, resulting in the protein becoming a
permanent resident of the membrane. However, in cases where
palmitoylation is the second signal, the first signal may serve to
allow transient interaction with membranes, thereby facilitating
palmitoylation by a membrane-bound palmitoyl-transferase (1, 15).
s coupling and for efficient
coupling to G
q/PLC (18, 25, 26). Moreover, it was also
established that isoprenylation of the IP is required for efficient
agonist-mediated receptor internalization (25). In the present study,
we sought to investigate whether the human IP is subject to
palmitoylation in addition to isoprenylation and thereafter to
characterize the role of palmitoylation in receptor-G protein coupling.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
s (K-20) or the G
q/11
(C-19) antisera were obtained from Santa Cruz. QuikChangeTM
site-directed mutagenesis kit was purchased from Stratagene. Lovastatin
was obtained from Merck.
307
was achieved by conversion of Cys308 codon to a
Stop308 codon (Cys308 to Stop308,
TGC to TAG). Deletion mutagenesis of hIP was performed using pBluescript-hIP as a PCR template, employing Expand High Fidelity Taq DNA polymerase and the oligonucleotide primers: 5'-G AGA
AGC TTG ATG GCG GAT TCG TGC AGG-3' (sense primer;
nucleotides +1 to +18 of hIP sequences are underlined) and 5'-ATA TGA
ATT CTA GAC CCA GAG CTT GAG TCG C-3' (antisense
primer; the sequences complimentary to nucleotides + 902 to + 920 of
hIP are underlined, and the mutator in-frame stop codon is in bold
type). The resulting PCR-amplified cDNA was subcloned into the
HindIII-EcoRI site of pHM6 (Roche) to generate the plasmid pHM-hIP
307. Deletion of the amino acids
carboxyl to Leu312 within the hIP to generate the
hIP
312 was achieved by conversion of Gly313
codon to a Stop313 codon (Gly313 to
Stop313, GGG to TAG). Deletion mutagenesis of hIP was
performed using pBluescript-hIP as a template and the oligonucleotide
primers: 5'-G AGA AGC TTG ATG GCG GAT TCG TGC AGG-3' (sense
primer; nucleotides +1 to +18 of hIP sequences are underlined) and
5'-ATA TGA ATT CTA GAG GCA CAG GCA GCA GAC-3'
(antisense primer; the sequences complimentary to nucleotides + 918 to + 935 of hIP are underlined, and the mutator in-frame stop codon is in
bold type). The resulting PCR-amplified cDNA was subcloned into the
HindIII-EcoRI site of pHM6 to generate the
plasmid pHM-hIP
312.
307,
hIP
312, hIPC308S, hIPC309S,
hIPC311S, hIPC308S,309S,
hIPC308S,C311S, hIPC309S,C311S, and
hIPC308S,C309S,C311S.
q, pCMV-G
s, and pHM:Ha-Ras
have been previously described (18, 20, 25, 26). HEK 293 cells were
transfected with pADVA (10 µg/10-cm dish) and pCMV- or pHM-based
vectors (25 µg/10-cm dish) using the calcium phosphate/DNA
co-precipitation procedure (27). For transient transfections, the cells
were harvested 48 h post-transfection.
307, HEK.hIP
312,
HEK.hIPC308S, HEK.hIPC309S,
HEK.hIPC311S, HEK.hIPC308S,C309S,
HEK.hIPC308S,C311S, HEK.hIPC309S,C311S, and
HEK.hIPC308S,C309S,C311S stable cell lines, HEK 293 cells
were transfected with ScaI-linearized pADVA (10 µg/10-cm
dish) plus PvuI-linearized pHM6:hIP
307,
pHM6:hIP
312, pHM-hIPC308S, pHM-hIP
C309S, pHM-hIPC311S, pHM-hIP
C308S,C309S pHM-hIPC308S,C311S,
pHM-hIPC309S,C311S, or pHM-hIP
C308S,C309S,C311S (25 µg/10-cm dish), respectively, using
the calcium phosphate/DNA co-precipitation procedure (27). For the
generation of stable cell lines, 48 h post-transfection, G418 (0.8 mg/ml) selection was applied and after ~21 days, G418-resistant
colonies were selected, and individual clonal stable cell isolates were
examined for IP expression by radioligand binding.
307,
HEK.hIP
312, HEK.hIPC308S,
HEK.hIPC309S, HEK.hIPC311S,
HEK.hIPC308S,C309S, HEK.hIPC308S,C311S,
HEK.hIPC309S,C311S, and
HEK.hIPC308S,C309S,C311S stable cell lines were transfected
with 10 µg of pADVA and 25 µg of pCMV-G
s or
pCMV-G
q using the calcium phosphate/DNA co-precipitation procedure (18). The cells were harvested 48 h post-transfection, and aliquots of whole cell protein (75 µg) were resuspended in 1×
solubilization buffer (10%
-mercaptoethanol (v/v), 2% SDS (w/v),
30% glycerol (v/v), 0.025% bromphenol blue (w/v), 50 mM Tris-HCl, pH 6.8; 100 µl) and boiled for 5 min. The samples were then
resolved by SDS-PAGE and were electroblotted onto PVDF membranes (18). Thereafter, the membranes were screened by immunoblot analysis
using either the anti-G
s (K-20) (1: 1000) or the
G
q/11 (C-19) (1:1000) antiserum. Horseradish
peroxidase-conjugated goat anti-rabbit IgG was used as the secondary
antibody in each case (1:2000). The proteins were visualized using the
chemiluminescence detection system, as described by the supplier.
307,
HEK.hIP
312, HEK.hIPC308S,
HEK.hIPC309S, HEK.hIPC311S,
HEK.hIPC308S,C309S, HEK.hIPC308S,C311S,
HEK.hIPC309S,C311S, and
HEK.hIPC308S,C309S,C311S, which express equivalent levels
of IP as determined by radioligand binding assay, and, as controls, HEK
293 cells that had been transiently co-transfected for 48 h with
pHM-Ha-Ras (25 µg/10-cm dish) plus pADVA (10 µg/10-cm dish) or
nontransfected HEK 293 cells were plated to achieve a density of
~3 × 106 cells/10-cm dish (~80% confluency) on
the day of metabolic labeling. The cells were washed once with PBS and
were then metabolically labeled in serum-free minimal essential medium
(1.5 ml) containing 1.5 mCi of [3H]palmitic acid (60 Ci/mmol) in the presence of IP agonist cicaprost (1 µM).
Following incubation at 37 °C for 2 h, the cells were lysed by
the addition of 600 µl of RIPA buffer (20 mM Tris-Cl, pH
8.0, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 1%
deoxycholate, 10 mM EDTA, pH 8.0, 1 mM
phenylmethylsulfonyl fluoride, 2 mM 1,10-phenanthroline, 10 µg/ml aprotinin, 10 µg/ml antipain, 1 µg/ml leupeptin, 10 µg/ml benzamidine). The cells were then harvested followed by disruption of
the lysate by passing through needles of decreasing bore size (gauges
23 and 26). The cell lysates were then subjected to centrifugation at
14, 000 × g for 5 min. The supernatant (600 µg) was
subjected to immunoprecipitation using the anti-HA 101r antibody
(1:300) as previously described (29, 30). Immunoprecipitates were then
resuspended in SDS-PAGE sample buffer (25 mM Tris-HCl, pH 6.8, 1% SDS, 5% glycerol, 0.001% (w/v) bromphenol blue, 2%
-mercaptoethanol), incubated at room temperature for 15 min, and
then resolved by 8% SDS-PAGE followed by electroblotting onto PVDF
membrane. The blots were soaked in Amplify for 30 min followed by
fluorography using Kodak X-Omat XAR film for 60 days at
70 °C. To
test for hydroxylamine-sensitive [3H]palmitate labeling,
SDS page gels were dried, and gels containing replicate samples were
incubated for 4 h in either 1 M Tris-HCl, pH 7.0, serving as a control, or 1 M hydroxylamine, pH 7, as
previously described (4, 31). Thereafter, the blots were subjected to PhosphorImager analysis, and the intensities of the 3H
images relative to basal levels were determined and expressed in
arbitrary units of intensity relative to basal levels. Thereafter, following fluorographic exposure, PVDF membranes were screened by
immunoblot analysis using the anti-HA 3F10 peroxidase conjugate antibody (1:1000) followed by chemiluminescence detection.
12-10
6 M cicaprost. As
controls, the cells were incubated in the presence of 50 µl of HBS in
the absence of ligand. To examine the effect of co-transfection of
G
s on cAMP generation, HEK 293, HEK.hIP
307, HEK.hIP
312,
HEK.hIPC308S, HEK.hIPC309S,
HEK.hIPC311S, HEK.hIPC308S,C309S,
HEK.hIPC308S,C311S, HEK.hIPC309S,C311S, and
HEK.hIPC308S,C309S,C311S cells were transiently
co-transfected with pCMV-G
s (25 µg/10-cm dish) plus
pADVA (10 µg/10-cm dish) and were harvested and assayed 48 h
post-transfection. To investigate the effect of lovastatin on
cicaprost-induced cAMP generation, the cells were preincubated with 10 µM lovastatin for 16 h prior to harvesting for cAMP assays.
q (25 µg/10-cm dish) plus pADVA (10 µg/10-cm dish) or, as controls, with the vector pCMV5 (25 µg/10-cm dish) plus
pADVA (10 µg/10-cm dish). After 48 h, the cells were harvested, washed twice in ice-cold PBS, and then resuspended at ~5 × 106 cells/ml in HBS containing 10 mM LiCl. The
cells (200 µl) were then preincubated at 37 °C for 10 min.
Thereafter, the cells were stimulated for 2 min at 37 °C in the
presence of cicaprost (1 µM) or, for concentration
response studies, were stimulated with cicaprost
10
6-10
12 M. To determine basal
IP3 levels, the cells were incubated in the presence of an
equivalent volume (50 µl) of the vehicle HBS. The IP3
levels produced were determined using the IP3 binding protein assay (29). The levels of IP3 produced by
ligand-stimulated cells over basal stimulation, in the presence of HBS,
were expressed in pmol of IP3/mg cell protein ± standard error of the mean (pmol/mg ± S.E.), and the results are
presented as fold stimulation over basal (fold increase ± S.E.).
The data presented are representative of four independent experiments,
each performed in duplicate.
8-10
5 M). To examine the
effect of co-transfection of G
q on cicaprost-induced [Ca2+]i mobilization,
HEK.hIP
307, HEK.hIP
312,
HEK.hIPC308S, HEK.hIPC309S,
HEK.hIPC311S, HEK.hIPC308S,C309S,
HEK.hIPC308S,C311S, HEK.hIPC309S,C311S, and
HEK.hIPC308S,C309S,C311S cells were transiently
co-transfected with pCMV-G
q (25 µg/10-cm dish) plus
pADVA (10 µg/10-cm dish). For each [Ca2+]i
measurement, calibration of the fluorescence signal was performed in
0.2% Triton X-100 to obtain the maximal fluorescence (Rmax) and 1 mM EGTA to obtain the
minimal fluorescence (Rmin). The ratio of the
fluorescence at 340 and 380 nm is a measure of [Ca2+]i, assuming a Kd of 225 nM Ca2+ for FURA2/AM. The results presented in
the figures are representative data from at least four independent
experiments and are plotted as changes (
) in
[Ca2+]i mobilized as a function of time (s) upon
ligand stimulation or, alternatively, were calculated as mean
changes in [Ca2+]i mobilized
(
[Ca2+]i ± S.E.; n = 4).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
s/adenylyl cyclase or efficient coupling to G
q/PLC activation (25).
In contrast to these findings, another study demonstrated that
hIP
312, a variant of hIP lacking a substantial portion
of the C-tail including the isoprenylation CAAX motif,
exhibited both G
s coupling at levels comparable with hIP
and G
q coupling, albeit at somewhat reduced efficacy
(19). Taken together, these studies suggest that additional elements
within the C-tail, other than its requirement for isoprenylation, may
influence IP-G protein coupling and may ultimately explain the observed
functional differences between the hIPSSLC and the
hIP
312 (19, 25). Hence, in the present study, we sought
to establish whether the hIP may actually undergo dual lipid
modification, namely palmitoylation in addition to its established
isoprenylation and to investigate whether together these lipid
modifications may be involved in regulating signaling by the hIP.
312 with that of the
hIP in HEK 293 stably transfected cell lines that were established
under identical conditions, ruling out any possible experimental or
artifactual differences such as differences in levels of receptor
expression, cell type, growth, or assay conditions, for example. Thus,
the effect of the selective IP agonist cicaprost on receptor-mediated
intracellular signaling was investigated in HEK.hIP
312
cells stably overexpressing the hIP
312 and was compared
with signaling in HEK.hIP and HEK.hIPSSLC cells stably
expressing the wild type hIP and isoprenylation-defective hIPSSLC, respectively. Initial radioligand binding assays
established that the levels of hIP, hIP
312, and
hIPSSLC receptor expression in their respective crude
membrane fractions or whole cells were not significantly different from
each other, indicating that the mutations per se did not
affect agonist binding or targeting of the respective IP receptors to
the plasma membrane (Table I). Although
stimulation of the hIP
312 with cicaprost exhibited
efficient increases in cAMP generation comparable with the hIP, levels
of cAMP generation by hIPSSLC were significantly lower
(Fig. 1A) and, at
concentrations less than 10
7 M, were not
significantly different from those generated by control HEK 293 cells
(25). Moreover, stimulation of hIP
312 with cicaprost
also led to efficient increases in [Ca2+]i
mobilization (Fig. 1B) and to
concentration-dependent increases in inositol
1,4,5-trisphosphate (IP3) generation (Fig. 1C)
to levels that were not significantly different from those of the hIP
(Fig. 1, B and C). Levels of
[Ca2+]i mobilization by the hIPSSLC
(Fig. 1B) and IP3 generation (data not shown)
were significantly reduced relative to both the wild hIP and the
hIP
312 and, consistent with our previous reports (25),
were not substantially greater than those generated by HEK 293 cells.
Although co-transfection of G
q significantly augmented
cicaprost-induced IP3 generation by both the hIP
(p < 0.05) and the hIP
312
(p < 0.05), it did not enhance IP3
generation by the hIPSSLC (Fig. 1D); Western
blot analysis confirmed overexpression of G
q (Fig.
1E).
Radioligand binding assays
View larger version (25K):
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Fig. 1.
Analysis of intracellular signaling by
hIP 312. A, HEK.hIP
(hIP), HEK.hIPSSLC (hIPSSLC), and
HEK.hIP
312 (hIP
312) cells were stimulated
with cicaprost (1 µM) for 10 min, with HBS-treated cells
serving as a control. Levels of cAMP produced in ligand-stimulated
cells relative to basal cAMP levels were expressed as fold stimulation
of basal (fold increase in cAMP ± S.E., n = 4).
B, HEK.hIP, HEK.hIPSSLC, and
HEK.hIP
312 cells preloaded with FURA2/AM were stimulated
with cicaprost (1 µM), and changes in
[Ca2+]i mobilization were monitored. The data
presented are representative profiles from at least three independent
experiments and are plotted as changes in intracellular
Ca2+ mobilization (
[Ca2+]i,
nM) as a function of time (s), where cicaprost was added at
the time indicated by the arrow. Actual changes in
[Ca2+]i mobilized by cells were: HEK.hIP,
[Ca2+]i = 145 ± 6.6 nM;
HEK.hIPSSLC,
[Ca2+]i = 58 ± 4.6; HEK.hIP
312,
[Ca2+]i = 151 ± 5.3 nM. C, HEK.hIP (hIP) and
HEK.hIP
312 (hIP
312) transiently
co-transfected with pCMV-G
q were stimulated with
cicaprost (10
12-10
6 M) for 2 min, and the levels of IP3 generation were measured.
D, alternatively, HEK.hIP, HEK.hIPSSLC, and
HEK.hIP
312 cells transiently co-transfected with the
vector pCMV5 (
) or with pCMV-G
q (+) encoding
G
q were stimulated with 1 µM cicaprost. In
each case, basal IP3 levels were determined by exposing the
cells to the vehicle HBS under identical incubation conditions. Levels
of IP3 produced in ligand-stimulated cells relative to
vehicle-treated cells (basal IP3) were expressed as fold
stimulation of basal (fold increase in IP3 ± S.E.,
n = 4). E, typical Western blot (75 µg of
total cellular protein analyzed) confirming overexpression of
G
q in HEK.hIP cells transiently co-transfected with
pCMV-G
q (+) or, as a control, with pCMV5 (
). Similar
results were obtained for HEK.hIPSSLC and
HEK.hIP
312 cells (data not shown). The position of the
46-kDa molecular mass marker is indicated to the
right.
312 mutant, with the sequence -CCLC,
may actually behave as a CAAX motif, facilitating its
isoprenylation within its short C-tail domain and thereby possibly
explaining its efficient signaling relative to the hIPSSLC,
the effect of the hydroxymethylglutaryl CoA reductase inhibitor lovastatin (18) on cicaprost-mediated cAMP generation by
hIP
312 was investigated and was compared with
that which occurred for the hIP. Whereas preincubation of
cells with lovastatin (10 µM, 16 h) significantly
impaired cicaprost-induced cAMP generation by the hIP
(p < 0.005), it had no significant affect on signaling by the hIP
312 (p = 0.717) or by the
hIPSSLC (p = 0.93), confirming that neither
the hIP
312 nor the hIPSSLC are isoprenylated
(Table II). On the other hand, consistent
with our previous reports (26), lovastatin did not impair
agonist-induced cAMP generation by the nonisoprenylated
2-adrenergic receptor or [Ca2+]i
mobilization by the
or
isoforms of the human thromboxane A2 receptors (data not shown).
Effect of lovastatin on radioligand binding
) for 16 h prior to harvesting for cAMP
assays, in response to stimulation with 1 µM cicaprost.
In each case, basal cAMP levels were determined by exposing the cells
to the vehicle HBS under identical incubation conditions. The levels of
cAMP produced in ligand-stimulated cells relative to basal cAMP levels
were expressed as fold stimulation of basal (fold increase in cAMP ± S.E., n = 3). The p values indicate
statistical comparisons of the effect of lovastatin on
cicaprost-induced cAMP generation.
312 is devoid of a substantial portion
of the C-tail domain of the hIP (Fig. 2),
it retains two highly conserved Cys residues, at Cys308 and
Cys309, and a semi-conserved residue at Cys311
that is conserved in the hIP, mouse IP, rat IP but is replaced by a Tyr
within the bovine IP (35-38). Hence, to investigate the potential role
of these conserved Cys residues in hIP signaling, deletion mutagenesis
was used to generate hIP
307, a variant of the hIP devoid
of amino acids 308-386 and differing from the hIP
312 in
that residues 308-312 were removed. Initial characterization of
HEK.hIP
307cells by saturation radioligand binding
confirmed high level receptor expression and established that the
mutation per se did not appreciably affect agonist binding
or plasma membrane targeting by the hIP
307 (Table I).
Although stimulation of hIP
307 with cicaprost did result
in marginal increases in cAMP generation, those levels of cAMP
generation were significantly reduced compared with the hIP throughout
the range of cicaprost concentrations used and, at concentrations less
than 10
7 M cicaprost, were not significantly
greater than those generated in HEK 293 cells in response to cicaprost
(Fig. 3A). Whereas
co-transfection of G
s resulted in a 1.81-fold
augmentation of cicaprost-induced cAMP generation in control HEK.hIP
cells, co-transfection of G
s did not significantly alter
cAMP generation in HEK.hIP
307 cells (Fig. 3B;
p < 0.11). Overexpression of G
s in
HEK.hIP
307 cells was confirmed by Western blot analysis
(Fig. 3C).
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Fig. 2.
Alignment of prostacyclin receptor amino acid
sequences. The deduced amino acid sequences of the C-tail regions
of the human, bovine, mouse, and rat IP aligned to show maximum
homology using the Clustal W software (34) are shown. The putative
isoprenylation motif (-CSLC) is boxed, and downward
arrows indicate the positions of amino acid residues 307 (Val307) and 312 (Leu312) within the hIP
sequence. Throughout the alignment, gaps indicated by
hyphens were inserted to optimize the alignment; identical
amino acids are indicated by asterisks; conservative
substitutions are indicated by colons; and semi-conservative
substitutions are indicated by a periods. Sequences for the
human, mouse, and rat IP are based on the published sequences, whereas
those for the bovine IP were based on those submitted to the
GenBankTM/EMBL Data Bank with accession number
Z93039.
View larger version (9K):
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Fig. 3.
Analysis of cAMP generation by
HEK.hIP 307 cells.
HEK.hIP
307 (hIP
307) and, as controls,
HEK.hIP (hIP) and HEK 293 cells were stimulated with
10
12-10
6 M cicaprost at
37 °C for 10 min (A). Alternatively,
HEK.hIP
307 and HEK.hIP cells that had been transiently
co-transfected with the vector pCMV5 (
) or with
pCMV-G
s(+), encoding G
s, were stimulated
with 1 µM cicaprost (B). In each case, the
basal cAMP levels were determined by exposing the cells to the vehicle
HBS under identical incubation conditions. The levels of cAMP produced
in ligand-stimulated cells relative to basal cAMP levels were expressed
as fold stimulation of basal (fold increase in cAMP ± S.E.,
n = 4). C, typical Western blot (75 µg of
total cellular protein analyzed) confirming overexpression of
G
s in HEK.hIP
307 cells transiently
co-transfected with pCMV-G
s (+) or, as a control, with
pCMV5 (
). The position of the 46-kDa molecular mass marker is
indicated to the right.
307 to couple to
G
q and to PLC activation was investigated. Levels of
cicaprost-induced [Ca2+]i mobilization by the
hIP
307 were significantly impaired relative to the hIP
(Fig. 4A;
[Ca2+]i by hIP = 145 ± 6.59 nM;
[Ca2+]i by
hIP
307 = 56 ± 2.08 nM,
p < 0.0001). Moreover, in concentration response experiments, levels of [Ca2+]i mobilization by
the hIP
307 were significantly reduced at all
concentrations employed relative to the hIP (Fig. 4B).
Transient overexpression of G
q resulted in a 1.45-fold
augmentation of intracellular [Ca2+]i
mobilization in control HEK.hIP cells (Fig. 4C;
[Ca2+]i = 210 ± 5.18 nM by
hIP + G
q, p < 0.0001) but failed to
augment [Ca2+]i mobilization in
HEK.hIP
307 cells (Fig. 4C;
[Ca2+]i = 58 ± 6.06 nM by
hIPSSLC + G
q; p = 0.699).
Additionally, stimulation of hIP
307 did not result in
significant increases in IP3 generation throughout the
range of cicaprost concentrations examined (Fig. 4D;
p < 0.0026), and co-transfection of G
q
did not augment IP3 generation in HEK.hIP
307
cells compared with cells transfected with the vector pCMV5 (Fig. 4E; p < 0.20). Overexpression of
G
q in HEK.hIP
307 cells was confirmed by
Western blot analysis (Fig. 4F).
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Fig. 4.
Analysis of
G q coupling by
HEK.hIP
307 cells. A-C,
HEK.hIP
307 (hIP
307) cells, or as positive
controls, HEK.hIP (hIP) cells were preloaded with FURA2/AM prior to
stimulation with 1 µM cicaprost (A) or with
10
8-10
5 M cicaprost
(B). Alternatively, HEK.hIP
307
(hIP
307) and HEK.hIP (hIP) cells that had been
transiently co-transfected with either the vector pCMV5 (
) or with
pCMV-G
q (+), encoding G
q, were stimulated
with 1 µM cicaprost (C). The data were
calculated as changes in intracellular calcium mobilized
(
[Ca2+]i, nM) as a function of
time (s) following ligand stimulation and, in A, are
presented as representative profiles from at least four independent
experiments. The data presented in B and C were
calculated as the mean changes in intracellular Ca2+
mobilization (
[Ca2+]i, nM ± S.E.,
n = 4). D, HEK.hIP
307
(hIP
307) and HEK.hIP (hIP) cells transiently
co-transfected with pCMV-G
q were stimulated with
cicaprost (10
12-10
6 M).
E, alternatively, HEK.hIP
307 and HEK.hIP
cells transiently co-transfected with pCMV5 (
) or with
pCMV-G
q (+) were stimulated with 1 µM
cicaprost. The basal IP3 levels were determined by exposing
the cells to the vehicle HBS under identical incubation conditions. The
levels of IP3 produced in ligand-stimulated cells relative
to vehicle-treated cells (basal IP3) were expressed as fold
stimulation of basal (fold increase in IP3 ± S.E.,
n = 4). F, typical Western blot (75 µg of
total cellular protein analyzed) confirming overexpression of
G
q in HEK.hIP
307 cells transiently
co-transfected with pCMV-G
q (+) or, as a control, with
pCMV5 (
). The position of the 46-kDa molecular mass marker is
indicated to the right.
312 exhibits near
identical coupling to Gs and Gq to that of the
hIP (p > 0.08), signaling by the hIP
307
is significantly impaired (p < 0.05) and is not
substantially different from that of the hIPSSLC
(p > 0.18).
312, and
HEK.hIP
307 cells were metabolically labeled with
[3H]palmitic acid. As a positive control for the
experimental conditions, metabolic labeling was also investigated in
HEK 293 cells transiently transfected with pHM-Ha-Ras encoding Ha-Ras,
a known substrate for palmitoylation (2, 14). Following metabolic
labeling, HA-tagged IP and Ras proteins were immunoprecipitated and
analyzed by fluorography. Metabolic labeling of Ha-Ras was detected,
consistent with its palmitoylation (Fig.
5, A and B). The
hIP also underwent palmitoylation as evidenced by the detection of a
broad radiolabeled band between 46 and 66 kDa in the immunoprecipitates
from HEK.hIP cells (Fig. 5C, lane 1) but not from
the immunoprecipitates from control HEK 293 cells (Fig. 5C,
lane 5) or indeed from HEK 293 cells transfected with Ha-Ras
(Fig. 5A, lane 1).
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Fig. 5.
Analysis of palmitoylation in HEK.hIP,
HEK.hIPSSLC,
HEK.hIP 307, and
HEK.hIP
312 cells. A and
B, HEK 293 cells transiently transfected with pHM:Ha-Ras
(lane 1) and, as a negative control, nontransfected HEK 293 cells (lane 2) were metabolically labeled with 1.5 mCi of
[3H]palmitic acid for 2 h (A), with
nonmetabolically labeled cells serving as a reference (B).
C and D, HEK.hIP (lane 1),
HEK.hIPSSLC (lane 2), HEK.hIP
312
(lane 3), HEK.hIP
307 (lane 4),
and, as negative controls, HEK 293 cells (lane 5) were
metabolically labeled with 1.5 mCi of [3H]palmitic acid
for 2 h in the presence of 1 µM cicaprost
(C), with nonmetabolically labeled cells serving as a
reference (D). Thereafter, the HA-tagged hIP,
hIPSSLC, hIP
312, and hIP
307
receptors or HA-tagged Ha-Ras were immunoprecipitated using anti-HA
101r antibody, with HEK 293 cells serving as negative control.
Immunoprecipitates were resolved by SDS-PAGE followed by
electroblotting onto PVDF membrane. The blots were then soaked in
Amplify for 30 min followed by autoradiography for 60 days at
70 °C (A and C). Thereafter, blots were
subjected to PhosphorImager analysis, and the intensities of
cicaprost-mediated palmitoylation of hIP, hIPSSLC,
hIP
312, and hIP
307 were determined and
expressed, in arbitrary units, as fold increases in palmitoylation
relative to basal levels detected in HEK 293 cells, as follows: hIP,
4.1-fold; hIPSSLC, 1.44-fold; hIP
312,
2.28-fold; and hIP
307, 1.0-fold. Subsequently, the blots
were screened by immunoblot analysis using the anti-HA 3F10 peroxidase
conjugate followed by chemiluminescent detection (B and
D). The positions of the molecular mass markers (kDa) are
indicated to the left and right of A and
D, respectively. The data presented are representative of
three independent experiments.
312 was also observed (Fig. 5C,
lanes 2 and 3, respectively). However, although
the hIP
312 was palmitoylated to levels comparable with
that of the wild type hIP, the level of palmitoylation of the
hIPSSLC appeared to be somewhat reduced (2.28- and
1.44-fold increase in palmitoylation relative to basal levels observed
in HEK 293 cells, respectively). In contrast, palmitoylation of
hIP
307 was not detected (Fig. 5C, lane
4). To confirm the identities of the palmitoylated proteins to be
those of the hIP and its variants and to ascertain whether failure to
detect palmitoylation of the hIP
307 was not due altered
expression levels, following fluorographic exposure the PVDF membranes
were screened with anti-HA 3F10 peroxidase-conjugated antibody (Fig.
5D). In each case, similar to that observed in the
palmitoylation assays (Fig. 5C), a broad immunoreactive band of 46-66 kDa was observed in the immunoprecipitates from HEK.hIP, HEK.hIPSSLC, HEK.hIP
312, and
HEK.hIP
307 cells but not from the control HEK 293 cells
or from cells transfected with HA-epitope tagged Ha-Ras, thus
confirming recovery of hIP, hIPSSLC, hIP
312,
and hIP
307 receptors (Fig. 5B). The chemical
identity of the 3H-labeled moiety attached to the hIP,
hIP
312, and the hIPSSLC following metabolic
labeling to be a thioester-linked palmitoyl group was confirmed by
treatment of the immunoprecipitates with hydroxylamine (data not shown).
312 are palmitoylated, hIP
307 is not a
substrate for palmitoylation, suggesting that amino acid residues
308-312, inclusively, contain the target site(s) for palmitoylation of
the hIP. The primary sequence within this defined region contains two
identically conserved Cys residues, Cys308 and
Cys309, which may be potential targets for palmitoylation
(Fig. 2). Thus, to investigate the possible involvement of
Cys308 and/or Cys309 in palmitoylation and the
potential implications thereof in the regulation of IP signaling,
site-directed mutagenesis of hIP was used to mutate Cys308
and Cys309 residues individually and collectively to Ser
residues to generate hIPC308S, hIPC309S, and
hIPC308S,C309S.
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Fig. 6.
Analysis of palmitoylation in HEK.hIP,
HEK.hIPC308S, HEK.hIPC309S, and
HEK.hIPC308S,C309S cells. A and B,
HEK.hIP (lane 1), HEK.hIPC308S (lane
2), HEK.hIPC309S (lane 3),
HEK.hIPC308S,C309S (lane 4), and, as a negative
control, HEK 293 cells (lane 5) were metabolically labeled
with 1.5 mCi of [3H]palmitic acid for 2 h in the
presence of 1 µM cicaprost (A). Thereafter,
the HA-tagged hIP, hIPC308S, hIPC309S, and
hIPC308S,C309S receptors were immunoprecipitated using
anti-HA 101r antibody with HEK 293 cells serving as a negative control.
Immunoprecipitates were resolved by SDS-PAGE followed by
electroblotting onto PVDF membrane. The blots were then soaked in
Amplify for 30 min followed by autoradiography for 60 days at
70 °C. The positions of the molecular mass markers (kDa) are
indicated to the left and right of A
and B, respectively. The data presented are representative
of three independent experiments. Thereafter, the blots were subjected
to densitometric analysis, and the intensities of cicaprost-mediated
palmitoylation of hIP, hIPC308S, hIPC309S, and
hIPC308S,309S were determined and expressed, in arbitrary
units, as fold increases in palmitoylation relative to basal levels
detected in HEK 293 cells, as follows: hIP, 4.23-fold;
hIPC308S, 2.44-fold; hIPC309S, 3.76-fold; and
hIPC308S,309S, 2.34-fold. Thereafter, the blots were
screened by immunoblot analysis using the anti-HA 3F10
peroxidase-conjugated antibody followed by chemiluminescent detection
(B).
s
resulted in significant augmentations in cAMP generation in
HEK.hIPC308S, HEK.hIPC309S, and
HEK.hIPC308S,C309S cells to levels comparable with the
control HEK.hIP cells (Fig. 7B). Overexpression of
G
s was confirmed by Western blot analysis (Fig.
7C). Similar to that of the hIP, preincubation of
HEK.hIPC308S, HEK.hIPC309S, and
HEK.hIPC308S,C309S cells with lovastatin significantly
impaired cicaprost-induced cAMP generation, confirming that the
hIPC308S, hIPC309S, and
hIPC308S,C309S are indeed subject to protein isoprenylation
(Table II).
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Fig. 7.
Analysis of cAMP generation by
HEK.hIPC308S, HEK.hIPC309S, and
HEK.hIPC308S,C309S cells. HEK.hIPC308S
(hIPC308S), HEK.hIPC309S
(hIPC309S), HEK.hIPC308S,C309S
(hIPC308S,C309S), and, as positive controls, HEK.hIP (hIP)
cells were stimulated with 10 12-10
6
M cicaprost at 37 °C for 10 min (A).
Alternatively, HEK.hIPC308S HEK.hIPC309S,
HEK.hIPC308S,C309S, and HEK.hIP cells transiently
co-transfected with pCMV5 (
) or with pCMV-G
s(+),
encoding G
s, were stimulated with 1 µM
cicaprost (B). In each case, basal cAMP levels were
determined by exposing the cells to the vehicle HBS under identical
incubation conditions. The levels of cAMP produced in ligand-stimulated
cells relative to basal cAMP levels were expressed as fold stimulation
of basal (fold increase in cAMP ± S.E., n = 4).
C, typical Western blot (75 µg of total cellular protein
analyzed) confirming overexpression of G
s in
HEK.hIPC308S cells transiently co-transfected with
pCMV-G
s (+) or, as a control, with pCMV5 (
).
Similarly, overexpression of G
s in
HEK.hIPC309S and HEK.hIPC308S,C309S cells was
also confirmed (data not shown). The position of the 46-kDa molecular
mass marker is indicated to the right.
q significantly augmented IP3 generation in
HEK.hIPC309S (p < 0.05) and HEK.hIP
(p < 0.05) cells but did not affect IP3 generation by HEK.hIPC308S (p = 0.65) or
HEK.hIPC308S,C309S (p < 0.78) cells (Fig.
8B). Similarly, stimulation of hIPC309S with
cicaprost yielded concentration-dependent increases in
[Ca2+]i mobilization to levels that were not
significantly different from those generated by the hIP (Fig.
8C; p = 0.68). However, levels of
[Ca2+]i mobilization by the hIPC308S
(p = 0.0048) and the hIPC308S,C309S
(p = 0.0039) were significantly reduced relative to the
hIP throughout the range of cicaprost concentration (Fig.
8C). Moreover, co-transfection of cells with
G
q resulted in a significant augmentation of
[Ca2+]i mobilization in HEK.hIP
(p = 0.0001) and HEK.hIPC309S
(p = 0.0001) cells, but not in HEK.hIPC308S
(p = 0.39) and HEK.hIPC308S,C309S
(p = 0.41) cells (Fig. 8D). Overexpression
of G
q was confirmed by Western blot analysis (Fig.
8E). Thus, taken together, as the metabolic labeling of
hIPC308S and hIPC308S,C309S by
[3H]palmitic acid is reduced relative to hIP and
hIPC309S, it is proposed that Cys308, but not
Cys309, is a target site for palmitoylation and that
palmitoylation at Cys308 is required for efficient hIP
coupling to Gq/PLC activation but may not be required for
Gs/adenylyl cyclase activation.
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Fig. 8.
Analysis of
G q coupling by
HEK.hIPC308S, HEK.hIPC309S, and
HEK.hIPC308S,C309S cells. A and B,
HEK.hIPC308S (hIPC308S),
HEK.hIPC309S (hIPC309S),
HEK.hIPC308S,C309S (hIPC308,C309S), and, as
positive controls HEK.hIP (hIP) cells that had been transiently
co-transfected with pCMV-G
q were stimulated with
cicaprost (10
12-10
6 M) for 2 min at 37 °C (A). Alternatively, cells that had been
transiently co-transfected with either the control vector pCMV5 (
) or
with pCMV-G
q (+) were stimulated with 1 µM
cicaprost (B). In each case, basal IP3 levels
were determined by exposing the cells to the vehicle HBS under
identical incubation conditions. Levels of IP3 produced in
ligand-stimulated cells relative to vehicle-treated cells (basal
IP3) were expressed as fold stimulation of basal (fold
increase in IP3 ± S.E., n = 4).
C and D, HEK.hIPC308S
(hIPC308S), HEK.hIPC309S
(hIPC309S), HEK.hIPC308S,C309S
(hIPC308S,C30s9S), and, as positive controls HEK.hIP (hIP)
cells were preloaded with FURA2/AM prior to stimulation with
10
8-10
5 M cicaprost
(C). Alternatively, cells that had been transiently
co-transfected with either the control vector pCMV5 (
) or with
pCMV-G
q (+) were stimulated with 1 µM
cicaprost (D). The data were calculated as mean changes in
intracellular calcium mobilized (
[Ca2+]i,
nM± S.E., n = 4). E, typical
Western blot (75 µg of total cellular protein analyzed) confirming
overexpression of G
q in HEK.hIPC308S cells
transiently co-transfected with pCMV-G
q (+) or, as a
control, with pCMV5 (
). Similarly, overexpression of
G
q in HEK.hIPC309S and
HEK.hIPC308S,C309S cells was also confirmed (data not
shown). The position of the 46-kDa molecular mass marker is indicated
to the right.
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Fig. 9.
Analysis of palmitoylation in
HEK.hIPC308S,C309S,C311S, HEK.hIPC311S,
HEK.hIPC308S,C311S, and HEK.hIPC309S,C311S
cells. A and B, HEK.hIP (lane 1),
HEK.hIPC308S,C309S,C311S (lane 2),
HEK.hIPC311S (lane 3),
HEK.hIPC308S,C311S (lane 4),
HEK.hIPC309S,C311S (lane 5), and, as a negative
control, HEK 293 cells (lane 6) were metabolically labeled
with 1.5 mCi of [3H]palmitic acid for 2 h in the
presence of 1 µM cicaprost (A). Thereafter,
the HA-tagged hIP, hIPC308,308,311S, hIPC311S,
hIPC308S,C311S, and hIPC309S,C311S receptors
were immunoprecipitated using anti-HA 101r antibody with HEK 293 cells
serving as a negative control. The immunoprecipitates were resolved by
SDS-PAGE followed by electroblotting onto PVDF membrane; the blots were
then soaked in Amplify for 30 min followed by fluorography for 60 days
at 70 °C. Thereafter, the blots were subjected to densitometric
analysis, and the intensities of cicaprost-mediated palmitoylation of
hIP, hIPC308,308,311S, hIPC311S,
hIPC308S,C311S, and hIPC309S,C311S were
determined and expressed, in arbitrary units as fold increases in
palmitoylation relative to basal levels detected in HEK 293 cells, as
follows: hIP, 4.23-fold; hIPC308S,C309S,C311S, 1.03-fold;
hIPC311S, 2.11-fold, hIPC308S,311S, 1.08-fold;
and hIPC309S,311S, 2.43-fold. Subsequently, the blots were
screened by immunoblot analysis using the anti-HA 3F10 peroxidase
conjugate followed by chemiluminescent detection (B). The
positions of the molecular mass markers (kDa) are indicated to the
left and right of A and B,
respectively. The data presented are representative of three
independent experiments.
307 cells
(p > 0.18; Fig. 3A). Moreover,
co-transfection of cells with G
s significantly augmented
cAMP generation by the hIPC311S and the
hIPC309S,C311S to levels comparable with the hIP (Fig.
10B) but failed to significantly augment cAMP generation by
hIPC308S,C311S or by the hIPC308S,C309S,C311S
(Fig. 10B). Positive overexpression of G
s was
confirmed by Western blot analysis (Fig. 10E). Similar to
that of the hIP, preincubation of HEK.hIPC311S,
HEK.hIPC309S,C311S cells with lovastatin significantly
impaired cicaprost-induced cAMP generation, confirming that the
hIPC308S, hIPC311S, and
hIPC308S,C311S are indeed subject to protein isoprenylation
(Table II).
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Fig. 10.
Analysis of intracellular signaling by
HEK.hIPC308,308,311S, HEK.hIPC311S,
HEK.hIPC308S,C311S, and HEK.hIPC309S,C311S
cells. HEK.hIPC308S,C309S,C311S
(hIPC308S,C309S,C311S), HEK.hIPC311S
(hIPC311S), HEK.hIPC308S,C311S
(hIPC308S,C311S), HEK.hIPC309S,C311S
(hIPC309S,C311S), and, as positive controls HEK.hIP (hIP)
cells were stimulated with 10 12-10
6
M cicaprost at 37 °C for 10 min (A).
Alternatively, HEK.hIPC308,308,311S,
HEK.hIPC311S, HEK.hIPC308S,C311S,
HEK.hIPC309S,C311S, and HEK.hIP cells transiently
co-transfected with pCMV5 (
) or with pCMV-G
s(+),
encoding G
s, were stimulated with 1 µM
cicaprost (B). In each case, basal cAMP levels were
determined by exposing the cells to the vehicle HBS under identical
incubation conditions. The levels of cAMP produced in ligand-stimulated
cells relative to basal cAMP levels were expressed as fold stimulation
of basal (fold increase in cAMP ± S.E., n = 4).
C and D, HEK.hIPC308S,C309S,C311S
(hIPC308S,C309S,C311S), HEK.hIPC311S
(hIPC311S), HEK.hIPC308S,C311S
(hIPC308S,C311S), HEK.hIPC309S,C311S
(hIPC309S,C311S), and, as positive controls HEK.hIP (hIP)
cells were preloaded with FURA2/AM prior to stimulation with
10
8-10
5 M cicaprost
(C). Alternatively, the cells that had been transiently
co-transfected with either the control vector pCMV5 (
) or with
pCMV-G
q (+) were stimulated with 1 µM
cicaprost (D). The data were calculated as the mean changes
in intracellular calcium mobilized (
[Ca2+]i,
nM ± S.E., n = 4). E and
F, typical Western blot (75 µg of total cellular protein
analyzed) confirming overexpression of G
s and
G
q, respectively, in
HEK.hIPC308S,C309S,C311S cells transiently co-transfected
with pCMV-G
s (+G
s),
pCMV-G
q (+G
q) or, as a control, with
pCMV5 (
G
s/
G
q). Similar data was
obtained for overexpression of G
s and G
q
in HEK.hIPC311S, HEK.hIPC308S,C311S, and
HEK.hIPC309S,C311S cells (data not shown). The position of
the 46-kDa molecular mass marker is indicated to the right
of E and F.
307 (p > 0.05; Fig. 4B).
Moreover, co-transfection of cells with G
q significantly
augmented of cicaprost-induced [Ca2+]i
mobilization by the hIPC311S (p = 0.0003)
and the hIPC309S,C311S (p = 0.0008) but
failed to significantly augment signaling by the
hIPC308S,C311S (p = 0.58) or by the
hIPC308S,C309S,C311S (p = 0.69). Western
blot analysis confirmed overexpression of G
q (Fig.
10F).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
312. Similar to the hIP, the hIP
312
efficiently coupled to G
s and G
q (Fig.
1). In contrast, consistent with our previous findings (25), signaling
by the hIPSSLC was not substantially different from that of
control HEK 293 cells (Fig. 1). Although the hydroxymethylglutaryl CoA
reductase inhibitor lovastatin, an effective inhibitor of
isoprenylation (18), impaired signaling by the hIP, it did not affect
signaling by the hIP
312, confirming that the truncated
C-tail of the hIP
312 (with the carboxyl sequence -CCLC)
is not actually modified by isoprenylation. Similar to that of the
hIPSSLC, the hIP
307 exhibited significantly
impaired coupling to G
s and G
q (Fig. 3).
Whole cell (in vivo) metabolic labeling studies established that the hIP is indeed palmitoylated, and through a combination of
site-directed and deletion mutagenesis, we have confirmed that this
occurs at both Cys308 and Cys311 (Figs. 5, 6,
and 9). Although palmitoylation of the hIP was not required for its
ligand binding (Table I), palmitoylation at either Cys308
or Cys311 is sufficient to maintain hIP coupling to
Gs/adenylyl cyclase, whereas mutation of both
palmitoylation sites completely abolishes that signaling (Figs. 7 and
10). On the other hand, palmitoylation of Cys308 is
specifically required for Gq coupling and PLC activation as indicated by the failure of the hIPC308S to mediate
increases in IP3 generation or
[Ca2+]i mobilization (Fig. 8). Moreover, similar
to that of the hIP, signaling by the hIPC308S,
hIPC309S, hIPC311S, hIPC308S,C309S,
and hIPC309S,C311S was significantly inhibited by
lovastatin (Table II), confirming that these receptors are indeed
isoprenylated at Cys383 within the CAAX motif;
on the other hand, the fact that lovastatin did not affect signaling by
the hIPC308S,C311S and hIPC308S,C309S,C311S was
reflective of the fact that these two palmitoylation-defective receptors did not mediate substantial increases in cAMP generation, even in the absence of lovastatin. Whereas conceptually lovastatin may
inhibit hIP signaling indirectly through inhibition of G
geranylgeranylation, the fact that lovastatin had no affect on signaling by the hIP
312 (Table II) confirms that
lovastatin is not indirectly functioning through inhibition of G
isoprenylation and function but rather is specifically impairing IP
isoprenylation and thereby directly inhibiting IP (hIP,
hIPC308S, hIPC309S, hIPC311S,
hIPC308S,C309S, and hIPC309S,C311S) signaling.
312 but not the hIPSSLC mediates
efficient signaling to both Gs/adenylyl cyclase and Gq/PLC effector systems.
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Fig. 11.
Model of lipid modification of the hIP.
The newly translated, seven transmembrane domain (I-VII)
integral plasma membrane (PM)-bound hIP contains a
CAAX motif within its C-tail domain with the sequence
-C383SLC (CSLC). Isoprenylation of the hIP
through attachment of a farnesyl isoprenoid (jagged tail
symbol) to Cys383 followed by membrane
insertion results in the formation of fourth intracellular loop within
its C-tail domain (IC4C-tail). Subsequent palmitoylation
( ) at Cys308 and Cys311 within its
IC4C-tail followed by membrane insertion results in the
formation of a double loop structure, referred to as loop A and loop B,
within the C-tail domain, which is required for efficient hIP coupling
to Gs and Gq and for effector signaling. The
hIP
312 retains loop A, but not loop B, within its C-tail
domain and exhibits efficient coupling to Gs and
Gq. Neither the deletion mutant hIP
307 nor
hIPC308S,C311S contain loop A or loop B sequences and hence
cannot efficiently couple to Gs- or Gq-mediated
effector signaling. The hIPC308S, although not undergoing
palmitoylation at Ser308 (S), does undergo
palmitoylation at Cys311 and hence contains a modified loop
A and loop B within its C-tail domain. The hIPC311S,
although not undergoing palmitoylation at Ser311, does
undergo palmitoylation at Cys308 and hence contains loop A
and a modified loop B within its C-tail domain (not shown). Although
palmitoylation at either Cys308 or Cys311 alone
is sufficient to maintain G
s coupling, there is a strict
requirement for palmitoylation of Cys308, but not of
Cys311, for efficient G
q coupling. The
hIPSSLC, although not undergoing isoprenylation, does
undergo palmitoylation at Cys308 and Cys311
which following membrane insertion, results in the formation of loop A,
but not loop B, within its C-tail domain; the presence of loop B
sequences not involved in loop B formation either (i) physically
impairs Gs and Gq interaction or (ii)
destabilizes loop A, thereby impairing Gs and
Gq coupling and effector signaling.
In this model (Fig. 11), we propose that the newly translated hIP
undergoes isoprenylation through attachment of a farnesyl isoprenoid to
Cys383 within its CAAX motif. Insertion of the
farnesyl group into the lipid bilayer results in the formation of a
fourth intracellular loop (IC4C-tail) within the C-tail
domain of the hIP and also positions Cys308 and
Cys311 in the proximity of the membrane, making them
available for palmitoylation by the membrane-bound palmitoyl
transferase (15). Palmitoylation of Cys308 and
Cys311 residues, which may be enhanced on agonist binding,
and subsequent membrane insertion of those palmitoyl groups results in
the formation of a double loop structure (loop A and loop B, amino- and
carboxyl-terminal to the palmitoylated Cys308 and
Cys311, respectively) within the IC4C-tail and
is required for hIP coupling to both Gs- and
Gq-mediated effector signaling. The hIP312,
although not undergoing isoprenylation, does undergo palmitoylation at
Cys308 and Cys311. We suggest that following
membrane insertion of the palmitoyl groups, loop A, but not loop B, is
formed within the truncated C-tail domain of the hIP
312.
Thus, from studies with hIP
312, it appears that loop A
is critical for both Gs and Gq coupling. Paradoxically, the hIPSSLC, although not undergoing
isoprenylation, undergoes palmitoylation at Cys308 and
Cys311 and, following membrane insertion, results in the
formation of loop A, and not loop B; however, unlike the
hIP
312, the hIPSSLC does contain those
constituent amino acid residues of loop B (312-386), which we propose
may either (i) physically impair hIPSSLC:Gs and
Gq interaction or (ii) destabilize or shield loop A within the hIPSSLC, thereby inhibiting signaling by the
hIPSSLC. Through co-immunoprecipitation studies, we have
previously established that the hIPSSLC does not physically
associate with either G
s or G
q (25), thereby suggesting that indeed amino acid residues 312-386 within the
hIPSSLC may serve to impair receptor-G protein interaction.
Neither the palmitoylation-defective deletion mutant
hIP
307 nor the site-directed mutants
hIPC308S,C311S and hIPC308S,C309S,C311S contain
either loop A or loop B within their C-tail domains and hence cannot
mediate efficient coupling to Gs or Gq effector
signaling. The hIPC308S, which is predicted to contain a
modified loop A and a loop B, can couple to G
s-mediated
adenylyl cyclase activation but cannot couple to G
q/PLC
activation, suggesting that there is a strict requirement for
palmitoylation at Cys308 for Gq, but not
Gs, coupling.
While recognizing that the IP is somewhat unparalleled among well characterized GPCRs, the proposed double loop A and loop B structure within the C-tail domain of the hIP is not unlike that which has recently been proposed to occur for the 5-hydroxytryptamine (4A) (5-HT4(a)) receptor (40). In their study, Ponimaskin et al. (40) established that, like many other GPCRs, the 5-HT4(a) receptor is subject to palmitoylation at two adjacent Cys residues (Cys328 and Cys329) located proximal to transmembrane domain 7 but is also palmitoylated at Cys386, which is located one amino acid residue from the C terminus. Membrane insertion of the palmitates at the proximal Cys328 and Cys329 and distal Cys386 would generate a dynamic (agonist-regulated) double loop structure within the C-tail of the 5-HT4(a) receptor. Although palmitoylation-deficient mutants of 5-HT4(a) receptor were indistinguishable from the wild type receptor in mediating G protein/effector signaling, mutation of the proximal residues converted the inactive receptor (R) into the active (R*) form and thereby promoted agonist-independent or constitutive activation of the 5-HT4(a) receptor.
The finding that modulation of the palmitoylation status of the hIP
significantly affects its G protein coupling characteristics is indeed
consistent with data generated for several GPCRs including rhodopsin,
the 2-AR, as well as for the ETA and
ETB receptors (7, 9, 41). Functional characterization of
the nonpalmitoylated
2-AR and the ETB
receptor revealed that palmitoylation is essential for
agonist-stimulated coupling to Gs and to both
Gq and Gi proteins, respectively (9, 41).
However, analysis of the nonpalmitoylated ETA receptor
mutant revealed that agonist-mediated Gs coupling was
unaffected, whereas signaling through Gq was abolished
(42). Similar to that of the ETA receptor, we have
established that mutation of Cys308 significantly impaired
hIPC308S coupling to G
q-regulated but not to
G
s-regulated effector signaling, providing further
evidence that palmitoylation may differentially regulate distinct
signal transduction pathways.
As stated, various studies have indeed indicated that the functional
implications of palmitoylation are both individual/specific to the
given GPCR and may also be diverse (1-3). Although agonist stimulation
has been reported to enhance palmitoylation of the 2-AR
(10) and of the 5-HT4(a) receptor (40), it is also widely reported to enhance the rate of the palmitate turnover relative to
protein turnover, such as in the case of the
2-AR (43)
and the
2A AR (44). Further experiments are required to
investigate whether there is enhanced agonist-induced turnover of
palmitate, relative to protein turnover, in the case of the hIP. In
addition to its widely documented role in modulating G protein coupling (1-3), palmitoylation has been shown to influence the phosphorylation, desensitization, and/or internalization status of various GPCRs (12,
13, 39, 45, 46). For example, it has been suggested that the location
of the palmitoylated Cys residue(s) in GPCRs may be strategic, being
juxtaposed between the G protein-binding site and amino acids that may
be phosphorylated and play a role in desensitization (3). Additionally,
mutation of Cys341 of the
2-AR significantly
impairs coupling to Gs/adenylyl cyclase but generated a
receptor that is highly phosphorylated in its basal state, suggesting
that palmitoylation of Cys341 plays a central role in
maintaining the
2-AR in a state capable of interacting
with the G protein and, hence, with its effector system (9, 40, 45).
Whether an analogous regulatory system applies to the hIP remains to be
investigated; however, it is noteworthy that the hIP is subject to
agonist-mediated phosphorylation and desensitization through a protein
kinase C-dependent mechanism whereby Ser328,
within the putative loop B (Fig. 11), has been identified as the target
residue for phosphorylation (19, 30). Additionally, we have established
that isoprenylation of the hIP is required for its efficient
agonist-induced internalization (25), and hence it is indeed likely
that, either in combination with isoprenylation or alone,
palmitoylation may also regulate the processes that contribute to
agonist-induced internalization and desensitization.
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FOOTNOTES |
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* This work was supported by grants from the Wellcome Trust, the Health Research Board of Ireland, Enterprise Ireland, and the Irish Heart Foundation (to B. T. K.).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 nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) Z93039.
To whom correspondence should be addressed. Tel.:
353-1-7161507; Fax: 353-1-2837211; E-mail:
Therese.Kinsella@UCD.IE.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M210637200
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ABBREVIATIONS |
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The abbreviations used are: GPCR, G protein-coupled receptor; AR, adrenergic receptor; C-tail, carboxyl-terminal tail; HA, hemagglutinin; HEK, human embryonic kidney; IP, prostacyclin receptor; IP3, inositol 1, 4, 5-trisphosphate; PL, phospholipase; HBS, HEPES-buffered saline; ET, endothelin; hIP, human IP; PVDF, polyvinylidene difluoride; PBS, phosphate-buffered saline; MES, 4-morpholineethanesulfonic acid; 5-HT4(a), 5-hydroxytryptamine (4A).
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