Palmitoylation of the Human Prostacyclin Receptor

FUNCTIONAL IMPLICATIONS OF PALMITOYLATION AND ISOPRENYLATION*

Sinead M. Miggin, Orlaith A. Lawler, and B. Therese KinsellaDagger

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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 hIPDelta 307 that was neither palmitoylated nor isoprenylated and did not efficiently couple to Gs or to Gq, whereas hIPDelta 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

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 alpha  subunits of the heterotrimeric G protein subunits, for example Galpha s and Galpha 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).

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 beta 2-adrenergic receptor (AR) is palmitoylated at Cys341, and palmitoylation at this site is required for beta 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 alpha 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.

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 Galpha 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).

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 Galpha s coupling and for efficient coupling to Galpha 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

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-Galpha s (K-20) or the Galpha q/11 (C-19) antisera were obtained from Santa Cruz. QuikChangeTM site-directed mutagenesis kit was purchased from Stratagene. Lovastatin was obtained from Merck.

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 hIPDelta 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-hIPDelta 307. Deletion of the amino acids carboxyl to Leu312 within the hIP to generate the hIPDelta 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-hIPDelta 312.

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 hIPDelta 307, hIPDelta 312, hIPC308S, hIPC309S, hIPC311S, hIPC308S,309S, hIPC308S,C311S, hIPC309S,C311S, and hIPC308S,C309S,C311S.

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-Galpha q, pCMV-Galpha 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.

To create the HEK.hIPDelta 307, HEK.hIPDelta 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:hIPDelta 307, pHM6:hIPDelta 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.

Western Blot Analysis-- HEK.hIP, HEK.hIPDelta 307, HEK.hIPDelta 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-Galpha s or pCMV-Galpha 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% beta -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-Galpha s (K-20) (1: 1000) or the Galpha 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.

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.hIPDelta 307, HEK.hIPDelta 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% beta -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.

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 10-12-10-6 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 Galpha s on cAMP generation, HEK 293, HEK.hIPDelta 307, HEK.hIPDelta 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-Galpha 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.

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-Galpha 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.

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 (10-8-10-5 M). To examine the effect of co-transfection of Galpha q on cicaprost-induced [Ca2+]i mobilization, HEK.hIPDelta 307, HEK.hIPDelta 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-Galpha 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 (Delta ) in [Ca2+]i mobilized as a function of time (s) upon ligand stimulation or, alternatively, were calculated as mean changes in [Ca2+]i mobilized (Delta [Ca2+]i ± S.E.; n = 4).

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Galpha s/adenylyl cyclase or efficient coupling to Galpha q/PLC activation (25). In contrast to these findings, another study demonstrated that hIPDelta 312, a variant of hIP lacking a substantial portion of the C-tail including the isoprenylation CAAX motif, exhibited both Galpha s coupling at levels comparable with hIP and Galpha 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 hIPDelta 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.

We initially investigated the above paradigm by comparing signaling by the hIPSSLC and the hIPDelta 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.hIPDelta 312 cells stably overexpressing the hIPDelta 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, hIPDelta 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 hIPDelta 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 hIPDelta 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 hIPDelta 312 and, consistent with our previous reports (25), were not substantially greater than those generated by HEK 293 cells. Although co-transfection of Galpha q significantly augmented cicaprost-induced IP3 generation by both the hIP (p < 0.05) and the hIPDelta 312 (p < 0.05), it did not enhance IP3 generation by the hIPSSLC (Fig. 1D); Western blot analysis confirmed overexpression of Galpha q (Fig. 1E).

                              
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Table I
Radioligand binding assays
Radioligand binding assays were performed on membrane fractions and whole cells in the presence of 4 nM [3H]iloprost at 30 and 4 °C, respectively. The data are presented as the mean ± S.E. (n = 3). In general, it was established that the level of [3H]iloprost bound to the IPs 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.


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Fig. 1.   Analysis of intracellular signaling by hIPDelta 312. A, HEK.hIP (hIP), HEK.hIPSSLC (hIPSSLC), and HEK.hIPDelta 312 (hIPDelta 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.hIPDelta 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 (Delta [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, Delta [Ca2+]i = 145 ± 6.6 nM; HEK.hIPSSLC, Delta [Ca2+]i = 58 ± 4.6; HEK.hIPDelta 312, Delta [Ca2+]i = 151 ± 5.3 nM. C, HEK.hIP (hIP) and HEK.hIPDelta 312 (hIPDelta 312) transiently co-transfected with pCMV-Galpha 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.hIPDelta 312 cells transiently co-transfected with the vector pCMV5 (-) or with pCMV-Galpha q (+) encoding Galpha 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 Galpha q in HEK.hIP cells transiently co-transfected with pCMV-Galpha q (+) or, as a control, with pCMV5 (-). Similar results were obtained for HEK.hIPSSLC and HEK.hIPDelta 312 cells (data not shown). The position of the 46-kDa molecular mass marker is indicated to the right.

To exclude the possibility that the carboxyl-terminal four amino acids of the truncated hIPDelta 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 hIPDelta 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 hIPDelta 312 (p = 0.717) or by the hIPSSLC (p = 0.93), confirming that neither the hIPDelta 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 beta 2-adrenergic receptor or [Ca2+]i mobilization by the alpha  or beta  isoforms of the human thromboxane A2 receptors (data not shown).

                              
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Table II
Effect of lovastatin on radioligand binding
The cells were preincubated with 10 µM lovastatin (+) or with its vehicle (-) 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.

Although the hIPDelta 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 hIPDelta 307, a variant of the hIP devoid of amino acids 308-386 and differing from the hIPDelta 312 in that residues 308-312 were removed. Initial characterization of HEK.hIPDelta 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 hIPDelta 307 (Table I). Although stimulation of hIPDelta 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 Galpha s resulted in a 1.81-fold augmentation of cicaprost-induced cAMP generation in control HEK.hIP cells, co-transfection of Galpha s did not significantly alter cAMP generation in HEK.hIPDelta 307 cells (Fig. 3B; p < 0.11). Overexpression of Galpha s in HEK.hIPDelta 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.


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Fig. 3.   Analysis of cAMP generation by HEK.hIPDelta 307 cells. HEK.hIPDelta 307 (hIPDelta 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.hIPDelta 307 and HEK.hIP cells that had been transiently co-transfected with the vector pCMV5 (-) or with pCMV-Galpha s(+), encoding Galpha 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 Galpha s in HEK.hIPDelta 307 cells transiently co-transfected with pCMV-Galpha s (+) or, as a control, with pCMV5 (-). The position of the 46-kDa molecular mass marker is indicated to the right.

Thereafter, the ability of hIPDelta 307 to couple to Galpha q and to PLC activation was investigated. Levels of cicaprost-induced [Ca2+]i mobilization by the hIPDelta 307 were significantly impaired relative to the hIP (Fig. 4A; Delta [Ca2+]i by hIP = 145 ± 6.59 nM; Delta [Ca2+]i by hIPDelta 307 = 56 ± 2.08 nM, p < 0.0001). Moreover, in concentration response experiments, levels of [Ca2+]i mobilization by the hIPDelta 307 were significantly reduced at all concentrations employed relative to the hIP (Fig. 4B). Transient overexpression of Galpha q resulted in a 1.45-fold augmentation of intracellular [Ca2+]i mobilization in control HEK.hIP cells (Fig. 4C; Delta [Ca2+]i = 210 ± 5.18 nM by hIP + Galpha q, p < 0.0001) but failed to augment [Ca2+]i mobilization in HEK.hIPDelta 307 cells (Fig. 4C; Delta [Ca2+]i = 58 ± 6.06 nM by hIPSSLC + Galpha q; p = 0.699). Additionally, stimulation of hIPDelta 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 Galpha q did not augment IP3 generation in HEK.hIPDelta 307 cells compared with cells transfected with the vector pCMV5 (Fig. 4E; p < 0.20). Overexpression of Galpha q in HEK.hIPDelta 307 cells was confirmed by Western blot analysis (Fig. 4F).


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Fig. 4.   Analysis of Galpha q coupling by HEK.hIPDelta 307 cells. A-C, HEK.hIPDelta 307 (hIPDelta 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.hIPDelta 307 (hIPDelta 307) and HEK.hIP (hIP) cells that had been transiently co-transfected with either the vector pCMV5 (-) or with pCMV-Galpha q (+), encoding Galpha q, were stimulated with 1 µM cicaprost (C). The data were calculated as changes in intracellular calcium mobilized (Delta [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 (Delta [Ca2+]i, nM ± S.E., n = 4). D, HEK.hIPDelta 307 (hIPDelta 307) and HEK.hIP (hIP) cells transiently co-transfected with pCMV-Galpha q were stimulated with cicaprost (10-12-10-6 M). E, alternatively, HEK.hIPDelta 307 and HEK.hIP cells transiently co-transfected with pCMV5 (-) or with pCMV-Galpha 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 Galpha q in HEK.hIPDelta 307 cells transiently co-transfected with pCMV-Galpha q (+) or, as a control, with pCMV5 (-). The position of the 46-kDa molecular mass marker is indicated to the right.

Thus, it appears that although the hIPDelta 312 exhibits near identical coupling to Gs and Gq to that of the hIP (p > 0.08), signaling by the hIPDelta 307 is significantly impaired (p < 0.05) and is not substantially different from that of the hIPSSLC (p > 0.18).

Palmitoylation of the Human Prostacyclin Receptor-- To investigate whether the hIP and its variants are indeed palmitoylated, HEK.hIP, HEK.hIPSSLC, HEK.hIPDelta 312, and HEK.hIPDelta 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.hIPDelta 307, and HEK.hIPDelta 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.hIPDelta 312 (lane 3), HEK.hIPDelta 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, hIPDelta 312, and hIPDelta 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, hIPDelta 312, and hIPDelta 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; hIPDelta 312, 2.28-fold; and hIPDelta 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.

Palmitoylation of both the isoprenylation-defective hIPSSLC and hIPDelta 312 was also observed (Fig. 5C, lanes 2 and 3, respectively). However, although the hIPDelta 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 hIPDelta 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 hIPDelta 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.hIPDelta 312, and HEK.hIPDelta 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, hIPDelta 312, and hIPDelta 307 receptors (Fig. 5B). The chemical identity of the 3H-labeled moiety attached to the hIP, hIPDelta 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).

Palmitoylation and Signaling by hIPC308S, hIPC309S, and hIPC308S,C309S-- Herein, we have demonstrated that whereas the hIP, hIPSSLC, and hIPDelta 312 are palmitoylated, hIPDelta 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.

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).


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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).

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 Galpha 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 Galpha 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-Galpha s(+), encoding Galpha 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 Galpha s in HEK.hIPC308S cells transiently co-transfected with pCMV-Galpha s (+) or, as a control, with pCMV5 (-). Similarly, overexpression of Galpha 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.

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 Galpha 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 Galpha 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 Galpha 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 Galpha 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-Galpha 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-Galpha 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-Galpha q (+) were stimulated with 1 µM cicaprost (D). The data were calculated as mean changes in intracellular calcium mobilized (Delta [Ca2+]i, nM± S.E., n = 4). E, typical Western blot (75 µg of total cellular protein analyzed) confirming overexpression of Galpha q in HEK.hIPC308S cells transiently co-transfected with pCMV-Galpha q (+) or, as a control, with pCMV5 (-). Similarly, overexpression of Galpha 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.

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).


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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.

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 hIPDelta 307 cells (p > 0.18; Fig. 3A). Moreover, co-transfection of cells with Galpha 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 Galpha 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-Galpha s(+), encoding Galpha 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-Galpha q (+) were stimulated with 1 µM cicaprost (D). The data were calculated as the mean changes in intracellular calcium mobilized (Delta [Ca2+]i, nM ± S.E., n = 4). E and F, typical Western blot (75 µg of total cellular protein analyzed) confirming overexpression of Galpha s and Galpha q, respectively, in HEK.hIPC308S,C309S,C311S cells transiently co-transfected with pCMV-Galpha s (+Galpha s), pCMV-Galpha q (+Galpha q) or, as a control, with pCMV5 (-Galpha s/-Galpha q). Similar data was obtained for overexpression of Galpha s and Galpha 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.

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 hIPDelta 307 (p > 0.05; Fig. 4B). Moreover, co-transfection of cells with Galpha 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 Galpha q (Fig. 10F).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 hIPDelta 312. Similar to the hIP, the hIPDelta 312 efficiently coupled to Galpha s and Galpha 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 hIPDelta 312, confirming that the truncated C-tail of the hIPDelta 312 (with the carboxyl sequence -CCLC) is not actually modified by isoprenylation. Similar to that of the hIPSSLC, the hIPDelta 307 exhibited significantly impaired coupling to Galpha s and Galpha 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 Gbeta gamma geranylgeranylation, the fact that lovastatin had no affect on signaling by the hIPDelta 312 (Table II) confirms that lovastatin is not indirectly functioning through inhibition of Gbeta gamma 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.

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 hIPDelta 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 (up-arrow ) 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 hIPDelta 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 hIPDelta 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 Galpha s coupling, there is a strict requirement for palmitoylation of Cys308, but not of Cys311, for efficient Galpha 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 hIPDelta 312, 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 hIPDelta 312. Thus, from studies with hIPDelta 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 hIPDelta 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 Galpha s or Galpha 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 hIPDelta 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 Galpha s-mediated adenylyl cyclase activation but cannot couple to Galpha 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 beta 2-AR, as well as for the ETA and ETB receptors (7, 9, 41). Functional characterization of the nonpalmitoylated beta 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 Galpha q-regulated but not to Galpha 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 beta 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 beta 2-AR (43) and the alpha 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 beta 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 beta 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.

    FOOTNOTES

* 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.

Dagger 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

    ABBREVIATIONS

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).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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