The inducible G protein-coupled receptor edg-1 signals via the G(i)/mitogen-activated protein kinase pathway.

The edg-1 gene encodes an inducible G protein-coupled receptor (GPR) homologue that is induced during the in vitro differentiation of human endothelial cells. The aim of this study was to investigate the G protein-coupling and -signaling properties of the edg-1 polypeptide. The third cytosolic loop (i3) of edg-1 associates with G(i) alpha and G(o) alpha polypeptides in a guanosine 5'-O-(thiotriphosphate)-sensitive manner. Immunoprecipitation of the edg-1 polypeptide in transfected cells results in the co-precipitation of G(i) alpha 1 and G(i) alpha 3 polypeptides. These data strongly suggest that edg-1 is capable of coupling to the Gi pathway. Overexpression of the edg-1 GPR in human embryonic kidney 293 cells results in the sustained activation of the MAP kinase activity that is blocked by pertussis toxin treatment. Moreover, NIH3T3 cells permanently transfected with edg-1 exhibit enhanced MAP kinase and phospholipase A2 activities. These data suggest that the G(i)/mitogen-activated protein kinase pathway is a major signaling pathway regulated by the orphan receptor edg-1.

Angiogenesis, new blood vessel formation, is a critical component of many physiological processes such as wound healing and development. Uncontrolled angiogenesis is associated with numerous pathological conditions, including diabetic retinopathy, rheumatoid arthritis, and solid tumor growth (1). The process of angiogenesis is initiated by vascular endothelial cells and involves their orderly migration, proliferation, and differentiation into new capillary channels. Cultured human umbilical vein endothelial cells (HUVEC) 1 exhibit many of the characteristics of endothelium in vivo and is a widely used in vitro model system to study the molecular mechanisms of angiogenesis. For example, HUVEC proliferate in response to angiogenic mitogens, such as fibroblast growth factor-1 (FGF-1) (2) and vascular endothelial growth factor (3), and are growtharrested by cytokines such as tumor necrosis factor-␣, transforming growth factor-␤, as well as phorbol 12-myristate 13-acetate (PMA) (4,5). Moreover, prolonged treatment of HUVEC with PMA in three-dimensional collagen or fibrin gels results in the formation of differentiated capillary-like tubular structures (5)(6)(7).
To study the early transcriptional events in angiogenesis, we have cloned several immediate-early genes induced by PMA in HUVEC (8 -11). One such gene, termed endothelial differentiation gene-1 (edg-1), encodes a G protein-coupled receptor (GPR) homologue (8). Given that PMA causes HUVEC to differentiate phenotypically and since immediate-early genes have been shown to play critical roles in the control of growth and differentiation, we proposed that edg-1 may be functionally involved in endothelial cell differentiation (8). Recent studies have shown that the edg-1 transcript is also induced during endochondral ossification which occurs in the developing skeletal system. 2 Mesenchymal cells that are in the process of differentiating into osteoblasts express high levels of the edg-1 transcript. 2 The expression patterns and the promoter structure of the edg-1 gene is similar to the SPARC/osteonectin gene, which was originally identified from the activated endothelial cells (13). These data suggest that the edg-1 GPR is induced during the differentiation of multiple cell types and suggest that it may regulate signaling events in the developing cardiovascular system and the skeletal system. An important step in the characterization of a GPR is to identify the associated G protein(s). Such information may help reveal the downstream signaling pathways. Three approaches have been used to determine which G protein subtypes can couple to a specific GPR. Kurose et al. (14) reconstituted purified ␣ 2a -and ␣ 2b -adrenergic receptors with recombinant G ␣ subunits in phospholipid vesicles and measured the ability of agonists to stimulate GTPase activity. Second, specific G ␣ subunits have been immunoprecipitated from cellular extracts and the presence of co-precipitated GPR was measured by ligand binding assays (15). Third, co-transfection of receptors and specific G protein subunit cDNAs into receptor negative cells followed by the measurement of ligand-activated second messenger pathways have been used (16). The principal requirement of each of these approaches is the knowledge of agonists for the GPRs. However, in the case of edg-1, the ligands or agonists/antagonists are unknown at present. Thus, edg-1 is an example of a putative GPR, based solely on sequence similarity with the known GPR superfamily. We therefore explored other methodologies for examining the signaling properties of edg-1.
Previous studies have shown that the third intracellular loop (i 3 ) of GPRs are important for G protein interaction and signal transduction (reviewed in Ref. 17). Moreover, structural characteristics of G ␣ contact sites on the receptors have been predicted to form amphipathic ␣-helices (17). The i 3 domain of edg-1 fits this structural model as determined by Helicalwheel analysis (18). Therefore, we constructed a fusion protein of i 3 and glutathione S-transferase (GST-i 3 ) and utilized it as an affinity matrix to study G protein interaction in vitro. In this report, we show that G i␣ and G o␣ associates with the GST-i 3 fusion protein in vitro. Furthermore, we demonstrate that intact edg-1 binds to G i␣ and signals via the G i pathway to regulate cellular MAP kinase activity.
Construction and Isolation of GST-i 3 Fusion Protein-DNA fragment corresponding to the third cystolic loop (i 3 ) of edg-1 was amplified by the polymerase chain reaction (PCR) and subcloned into pGEX-2T (Pharmacia Biotech Inc.). The primers used were 1) 5Ј-ATG GAT CCA GAA TCT ACT CCT TGG TCA GGA CT-3Ј (sense) and 2) 5Ј-TAC CCG GGT TAC TTG AGC AGC GCC AGC GAC TTC TC-3Ј (antisense). The resulting clones were verified by DNA sequencing. The fusion protein construct was introduced into Escherichia coli DH5␣ (Life Technologies, Inc.) and induced with isopropyl-1-thio-␤-D-galactopyranoside. The fusion protein was purified by glutathione-Sepharose 4B (Pharmacia) affinity chromatography using the Sarkosyl extraction method (19).
Cellular Protein Extraction and G ␣ Binding Assay-HUVEC cells were extracted with 1 ml of buffer A (50 mM Tris, pH 7.8, 1 mM EGTA, 5 mM MgCl 2 , 20 mM CHAPS, 20% glycerol, 10 g of both aprotinin and leupeptin, and 20 l of 50 mM phenylmethylsulfonyl fluoride). The sample was extracted on ice for 45 min with constant stirring and centrifuged at 23,000 ϫ g for 15 min at 4°C, the supernatant was collected, and the protein concentration was determined by Bradford method (Bio-Rad).
To perform the in vitro binding assay, 750 g of HUVEC extracts were incubated with GST fusion protein beads (approximately 10 g of GST-i 3 fusion protein bound to 165 l of 75% glutathione-Sepharose slurry) for 2 h at 4°C. Beads were then washed five times with phosphate-buffered saline. Subsequently, bound G ␣ subunits were detected by [ 32 P]ADP-ribosylation with pertussis or cholera toxins. In vitro ADPribosylation reactions were carried out essentially as described (20). The reactions were terminated by the addition of 2 ϫ SDS sample buffer (4.6% (w/v) SDS, 10% (v/v) ␤-mercaptoethanol, 20% (w/v) glycerol, 95.2 mM Tris-HCl, pH 6.8, 0.01% (w/v) bromphenol blue). The [ 32 P]ADPlabeled proteins were then subjected to SDS-PAGE on 10% polyacrylamide gels, and autoradiographed.
The yeast strain Y190 was simultaneously transformed with Gal4-i 3 and Act D-G i␣2 plasmids using the lithium acetate procedure (22). After 3 days of growth, cells were lifted with nylon membranes and assayed for ␤-galactosidase activity as described elsewhere (23). ␤-Galactosidase activity was quantitated using the luminescence-based assay (Tropix Inc.).
The Association of G ␣ with edg-1 in Transiently Transfected HEK293 Cells-Full-length human edg-1 cDNA was subcloned behind the FLAG epitope sequence (24) in the mammalian expression vector pcDNANeo (Invitrogen Inc.). Four micrograms of the cDNA from the resulting clone, pDNFedg-1, either alone or in combination with 4 g of individual G ␣ cDNAs were transiently transfected into subconfluent HEK293 cells with lipofectAMINE (Life Technologies, Inc.) following the manufacturer's recommendations. Two days after transfection, cells were washed three times with ice-cold PBS and extracted as described above. Cellular extracts were then incubated overnight with FLAG epitopespecific monoclonal antibody (M2, Eastman Kodak Co.), which was cross-linked to protein A-Trisacryl GF2000 (Pierce) by the dimethyl pimelimidate method (25). The presence of G ␣ subunits in the anti-M2 immunoprecipitates was determined by (i) immunoblotting with G ␣ antibodies and (ii) [ 32 P]ADP-ribosylation in the presence of pertussis or cholera toxin as described.
Activation of MAP Kinase by edg-1 Overexpression-HEK293 cells transfected with pDNFedg-1 along with 0.2 g of hemagglutinin epitope-tagged extracellular signal regulated kinase-2 (HA-ERK2) plasmid were subjected to immune complex MAP kinase assay (26). Briefly, transfected cells were made quiescent by serum starvation for 12 h. Subsequently, cells were solubilized for 1 h with 0.5 ml of ERK lysis buffer (50 mM ␤-glycerophosphate, 20 mM HEPES (pH 7.4), 0.5% Triton X-100, 2 mM MgCl 2 , 1 mM EGTA, 0.1 mM sodium vanadate, 1 mM dithiothreitol, 2 g/ml leupeptin, 10 g/ml aprotinin, 2 g/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride), sonicated, and clarified by centrifugation at 24,000 ϫ g for 15 min at 4°C. The HA-ERK2 in 100 g of cell extract was immunoprecipitated by incubating the lysate with 1 g of monoclonal HA antibody (12CA5, Boehringer Mannheim) for 1 h. This was followed by the addition of 20 l of protein A-Trisacryl GF2000. The immune complexes were washed twice with ERK lysis buffer and twice with ERK kinase buffer (50 mM ␤-glycerophosphate, 20 mM HEPES (pH 7.4), 10 mM MgCl 2 , 1 mM EGTA, 0.1 mM sodium vanadate, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). The beads were then resuspended in 40 l of ERK kinase buffer containing 100 M [␥-32 P]ATP (5000 cpm/pmol), and the reaction was carried out for 30 min at room temperature using 5 g of myelin basic protein (MBP) as substrate. The reaction was terminated by the addition of 2 ϫ SDS sample buffer. Proteins were resolved on 12% SDS-PAGE and autoradiographed. Alternatively, for the in-gel kinase assay, transfected cell extracts (500 g) were immunoprecipitated with 1 g of anti-ERK-2 antibody (SC-154, Santa Cruz Biotech.) and 20 l of 50% slurry of protein A-Trisacryl GF2000 overnight at 4°C. Immunoprecipitates were separated on a MBP-impregnated 10% SDS-PAGE and ingel kinase assays were performed as described previously (27).
MAP Kinase Activity in Stably Transfected NIH3T3 Cells-NIH3T3 cells were transfected with pMexNeo vector or pMexNeo containing FLAG-edg-1 and neomycin-resistant colonies were cloned. These clones were then assayed for the expression of the human edg-1 transcript by Northern blot analysis (8). Stably transfected NIH3T3 cells (1 ϫ 10 6 ) were serum-starved for 24 h. Subsequently, cells were lysed in 0.5 ml of ERK lysis buffer. Extracts (100 g) were then immunoprecipitated with 1 g of anti-ERK-2 antibody for 1 h. This was followed by the addition of 20 l of protein A-Trisacryl GF2000. The immune complexes were washed, and in vitro kinase assay using MBP as substrate were carried out as described previously (26).
Measurement of Phospholipase A 2 Activity-Assay of phospholipase A 2 activity was carried out by measuring arachidonic acid release from prelabeled cells essentially as described (28). In brief, cells were seeded into 24-well plate (2 ϫ 10 5 cells/well) containing DMEM with 10% FBS. Cultures were labeled with 0.25 Ci/ml of [ 3 H]arachidonic acid (100 Ci/mmol, DuPont NEN) for 24 h at 37°C. Subsequently, cells were washed twice with DMEM containing 10 mM HEPES (pH 7.4) and 0.3% fatty acid-free bovine serum albumin (Sigma), and then re-fed with the same medium to trap the released fatty acid. At indicated times, cells were placed on ice, the medium was collected, and the radioactivity was determined. A23187 (2 M) was used to determine the maximal release.

RESULTS
Pertussis Toxin-sensitive G ␣ Subunits Associate with the GST-i 3 Fusion Protein-In order to characterize the G proteins that are coupled to edg-1, the GST-i 3 fusion protein was constructed and purified by glutathione-Sepharose chromatography (Fig. 1, A and B). The fusion protein was then incubated with HUVEC extracts and the affinity-selected protein complexes were subjected to ADP-ribosylation with pertussis or cholera toxins in the presence of [ 32 P]NAD. As shown in Fig.  1C, a single band of 41 kDa and three bands of 43-, 48-, and 52 kDa from HUVEC extracts were ADP-ribosylated by pertussis and cholera toxins, respectively. The single 41-kDa ADP-ribosylated band may represent G i␣ and G o␣ , whereas the three ADP-ribosylated bands observed in the presence of cholera toxin may represent the G s␣ subtypes. As shown in Fig. 1C, pertussis toxin substrates but not cholera toxin substrates specifically associated with GST-i 3 polypeptide. This result clearly indicates that G i␣ and/or G o␣ are capable of interacting with edg-1-i 3 .
G i␣ and G o␣ Associate with GST-i 3 Fusion Protein in Vitro-We next examined whether G i␣ , G o␣ , or both are capable of interacting with GST-i 3 . This was addressed by immunoblot analysis of the GST-i 3 associated polypeptides with anti-G ␣ antibodies. As shown in Fig. 2A, both anti-G i␣1 /G i␣2 and anti-G i␣3 /G o␣ antibodies react with the 41-kDa protein band from the GST-i 3 affinity isolates. This association was specific because G ␣ subunits did not bind to the GST control polypeptide. We also evaluated the nucleotide dependence of this in vitro interaction. HUVEC extracts were preincubated with the non-hydrolyzable analog of GTP (GTP␥S) and the interaction of G ␣ subunits and GST-i 3 fusion protein was assessed. As shown in Fig. 2B, GTP␥S treatment dramatically reduced GST-i 3 -associated the G ␣ subunits, indicating that the interaction between G ␣ subunits and edg-1-i 3 is dependent on the nucleotide-bound state of the G protein.
To determine the G protein specificity of GST-i 3 , specific G ␣ subunits were expressed in HEK293 cells by transient transfection. Cell extracts were prepared and analyzed for their ability to bind to GST-i 3 . As shown in Fig. 3, immunoblot analysis with subunit-specific antisera show that all three G i␣ subunits as well as the G o␣ subunit associated with the GST-i 3 fusion protein but not with the GST control protein. These data suggest that the i 3 domain of edg-1 is capable of binding to the G i family and G o polypeptides in vitro.
Direct Interaction of edg-1-i 3 Domain with G i␣ Polypeptide in the Two-hybrid Assay-Since the in vitro binding assays described above do not distinguish between direct binding versus association in a multicomponent complex, we utilized the twohybrid interaction system to determine if the i 3 polypeptide is capable of directly binding to the G i␣2 polypeptide. The edg-1-i 3 domain was cloned as a C-terminal fusion with the DNA binding domain of GAL4 (GAL4-i 3 ). In addition, the myristoylation site deleted form of the G i␣2 polypeptide was fused with the activation domain of GAL4 (Act D-G i␣2 ). The resulting transformants were plated on selective media and the colonies were assayed for ␤-galactosidase activity. While the single plasmid transformants did not transactivate the ␤-galactosidase reporter gene, co-expression of GAL4-i 3 and Act D-G i␣2 resulted in strong transactivation, indicating direct interaction between the two proteins (Table I). Co-transformation of the GAL4-i 3

FIG. 2. Association of G i␣ and G o␣ with GST-i 3 in vitro is GTP␥S-sensitive.
A, HUVEC extracts were incubated with glutathione-Sepharose bound GST or GST-i 3 , followed by immunoblotting with anti-G i␣1 /G i␣2 (left panel) or anti-G i␣3 /G o (right panel). The position of molecular weight makers is shown. B, HUVEC extracts were treated with (lanes 2 and 4) or without (lanes 1 and 3) 50 M GTP␥S followed by incubation with glutathione-Sepharose bound GST-i 3 . The G ␣ associated with GST-i 3 was then detected by immunoblotting with anti-G i␣1 / G i␣2 (lanes 1 and 2) or anti-G i␣3 /G o (lanes 3 and 4). Data are representative of three independent experiments. plasmid with irrelevant controls such as FGF-1 fused to the GAL4 activation domain produced colonies which were negative (white) for ␤-galactosidase activity (Table I). This result strongly suggests that the edg-1-i 3 domain interacts directly with the G i␣2 polypeptide.
Interaction of Intact edg-1 with G ␣ Subunits in HEK 293 Cells-In addition to the third intracellular loop, other intracellular domains have been documented to be involved in the regulation of receptor/G-protein coupling (17). Furthermore, chimeric studies have indicated that all three intracellular loops and the C-terminal tail cooperate to determine specificity in G protein coupling of GPRs (30 -32). While all three G i␣ as well as G o␣ bind to the i 3 domain, other intracellular loops may further discriminate the coupling of edg-1 to G proteins. We therefore examined the ability of G i␣ and G o␣ to interact with intact edg-1 GPR in transfected cells. HEK293 cells were transiently transfected with the epitope-tagged edg-1 in combination with individual G ␣ subunits. Subsequently, edg-1 was immunoprecipitated with the anti-M2 monoclonal antibody which recognizes the FLAG epitope. The G i␣ and G o␣ polypeptides present in the immunoprecipitates were then detected by immunoblot analysis. The level of expression of epitope-tagged edg-1 was similar between transfectants (Fig. 4A). In addition, high level expression of respective G ␣ subunits was achieved by transient transfection. As shown in Fig. 4B, G i␣1 and G i␣3 but not G o␣ and G i␣2 were detected in receptor immunoprecipitates. Consistent with these results, when the anti-M2 complexes were [ 32 P]ADP-ribosylated in vitro in the presence of pertussis toxin, labeled G ␣ polypeptides were only observed in the cells co-transfected with edg-1 and either G i␣1 or G i␣3 (data not shown). No [ 32 P]ADP-labeled G ␣ was detected in the presence of cholera toxin (data not shown), indicating that G s polypeptides are unable to couple to intact edg-1. These data strongly suggest that G i␣1 and G i␣3 interact with intact edg-1 in transfected HEK293 cells.
Activation of MAP Kinase by Edg-1 Overexpression-Recently, Lefkowitz and co-workers (33) proposed a "two-state model" of receptor activation, in which receptors are in dynamic equilibrium between inactive and G-protein coupled, spontaneously active conformations. Therefore, overexpression of receptors results in a significant increase in the population of constitutively active receptors (34). Analogously, overexpression of G q and its cognate receptors induced the basal activity of signaling pathways in the absence of agonists (35). Because G i -coupled receptors are known to modulate signaling pathways such as the MAP kinase pathway (36), we overexpressed edg-1 in HEK293 cells and measured the cellular MAP kinase activity.
Cultured HEK293 cells were transiently transfected with edg-1. MAP kinase activity was assayed by an in-gel kinase assay of extracellular signal regulated kinase-2 (ERK-2) immunoprecipitates. As shown in Fig. 5A, immunoprecipitation of HEK293 cells with the ERK-2 antiserum followed by in- gel   FIG. 3. Interaction of G i␣ and G o␣ with GST-i 3 in vitro. G i␣1 , G i␣2 , G i␣3 , and G o␣ subunits were overexpressed in HEK293 cells by transient transfection. Cell extracts were affinity-isolated with glutathione-Sepharose bound GST-i 3 or GST polypeptides, and protein complexes were immunoblotted with subtype-specific G ␣ antisera. Co-immunoprecipitation of epitope-tagged full-length edg-1 with G i␣1 and G i␣3 . HEK293 cells were transiently transfected with the FLAG epitope-tagged full-length edg-1 and individual G ␣ cDNAs and cellular extracts were prepared as described under "Experimental Procedures." The expression of transfected polypeptides were detected by Western blot analysis with the M2 antibody (FLAG-edg-1), G i␣1 /G i␣2 and G i␣3 /G o␣ antisera. B, cellular extracts from co-transfected HEK293 cells were immunoprecipitated with anti-M2 antibody and the G ␣ subunits present in the immunoprecipitates were detected by Western blot analysis with G i␣1 /G i␣2 or G i␣3 /G o␣ antisera in respective lanes. These results are representative of two independent experiments. kinase assay in MBP-impregnated gels detected a specific band at 42 kDa. The kinase activity of this band is strongly (approximately 6-fold) induced by treatment with PMA for 15 min (data not shown). This result indicates that the 42-kDa band is ERK-2. Transient transfection of the edg-1 expression vector into HEK293 cells, in a dose-dependent manner, induced the ERK-2 kinase activity (Fig. 5B). The ERK-2 MAP kinase activity was induced at 30 -60 h post-transfection and thus appears to be of sustained kinetics. Pertussis toxin treatment blocked the edg-1-dependent MAP kinase activation. The inhibition of ERK-2 activation in edg-1-transfected cells by pertussis toxin is not due to the inhibition of transfected gene expression as indicated in the M2 immunoblots. These data indicate that edg-1 signaling via the G i pathway is responsible for sustained activation of the MAP kinase pathway.
To further confirm the in-gel kinase assay, the edg-1 expression vector and the hemagglutinin epitope (HA)-tagged ERK-2 cDNA were co-transfected into HEK293 cells, and MAP kinase activity of the HA immunoprecipitates was assayed. As shown in Fig. 6, edg-1 transfection activated the MAP kinase activity. In addition, the edg-1-induced activation of the HA-ERK-2 was suppressed by pertussis toxin. These data indicate that edg-1 signals via the G i pathway to induce the ERK-2/MAP kinase activity.
NIH3T3 Cells Overexpressing edg-1-In order to better characterize the signaling mechanisms regulated by edg-1, NIH3T3 cells were stably transfected with edg-1 expression vector pMexNeo-edg-1 or pMexNeo vector alone and the resulting neomycin-resistant colonies were cloned. Northern blot analysis of the resulting edg-1 clone but not the vector-transfected clone expressed the human edg-1 transcript (Fig. 7A). Immunoprecipitation of ERK-2 polypeptide followed by an immune complex MAP kinase assay indicated that the kinase activity is enhanced 2-fold under steady-state conditions (Fig. 7B). These data suggest that overexpression of edg-1 is associated with the sustained activation of MAP kinase activity in NIH3T3 cells. Since activation of MAP kinase is known to phosphorylate and activate the cellular phospholipase A 2 (37), we measured the phospholipase A 2 activity in edg-1 transfected cells and compared it with the vector-transfected cells. Significant increase (approximately 2-fold) in the phospholipase A 2 activity was observed in edg-1-transfected cells (Fig. 7C). These data suggest that overexpression of edg-1 is associated with enhanced MAP kinase and phospholipase A 2 activities in NIH3T3 cells. DISCUSSION The process of angiogenesis is initiated by vascular endothelial cells and involves their orderly migration, proliferation and differentiation into new capillary channels (1). While considerable efforts have been focused on the study of proliferative events, little is known about the nonproliferative aspects of angiogenesis. Since (i) HUVEC exhibit many of the characteristics of endothelium in vivo and (ii) prolonged treatment of HUVEC grown on collagen or fibrin gels with PMA results in growth arrest and the formation of differentiated capillary-like tubular structures (5, 6), PMA-induced early response genes may play critical roles in the nonproliferative aspects of angiogenesis. The edg-1 transcript was isolated as a PMA-inducible early response gene from HUVEC (8). The cDNA encodes a polypeptide with 382 amino acids that possess many structural features of a GPR (8). Sequence alignment of edg-1 with other GPRs indicate that it belongs to the GPR family group I (38). It is most closely related to other orphan receptors AGR-16 (39) and rat orphan GPR H218 (40), which were isolated by homology cloning of receptors expressed in the cardiovascular system and the brain, respectively. This subfamily of orphan receptors are closely related in structure to the cannabinoid and melanocortin receptor subfamilies (41,42). However, the ligands that are capable of interacting with the edg-1 GPR are un-known at present. The cloning of edg-1, however, raises several questions, such as (i) what is the ligand for edg-1, (ii) what signaling pathways are regulated by edg-1, and (iii) how the induction of edg-1 expression relates to angiogenesis.
Determination of the function of orphan receptors has been onerous due to the intrinsic difficulty in the identification of ligands. For example, the mas oncogene, which was isolated due to its transforming capacity, is still uncharacterized with respect to ligands and signaling properties (43). Due to the advent of homology cloning by PCR, many orphan receptor sequences have become available. However, ligands for only a few of these orphan receptors have been found to date, for example, the cannabinoid and orphanin receptors (42,44). In this report, we present a novel approach to define the signaling properties of the orphan receptor edg-1.
Structure-function analysis of GPRs has defined several features of the GPRs that are essential for specificity in signaling (17). While the structure of the i 3 domain plays a major role in determining the specificity of G protein coupling, other intracellular loops (e.g. the C-terminal portion of intercellular loop 2 and the C-terminal tail) also contribute to the specificity (30,31). For example, the intercellular loop 2, i 3 , and C-terminal domains of rhodopsin are able to interact with transducin independently (32). The i 3 domain of edg-1 is only 34 residues in length and contains the G i -activator motif (BBXXF) as well as other potential regulatory sites (45). We expressed the i 3 domain of edg-1 as a C-terminal GST fusion protein and used it as an affinity matrix to characterize G proteins that interact with it. Toxin labeling studies clearly indicate that the pertussis toxin substrates (G i␣ and G o␣ ) but not the cholera toxin substrates (G s␣ ) are capable of associating with the edg-1-i 3 . While we have not ruled out association of other G proteins (e.g. G q , G z , G 12 , and G 15 ) with i 3 , our data clearly show that the G i␣ and G o␣ family of proteins bind to edg-1-i 3 . The interaction between G ␣ and edg-1-i 3 in this in vitro binding assay is highly specific, since (i) the G ␣ subunits are unable to interact with GST protein and (ii) the interaction was suppressed by GTP␥S. In addition, these data also suggested that the i 3 domain alone in the C-terminal context of the soluble GST fusion polypeptide is of sufficient affinity and specificity to physically associate with the G protein ␣ subunits.
While the in vitro association experiments proved that the GST-i 3 and the G ␣ associates specifically, they do not, however, demonstrate direct interaction. Thus, the yeast two-hybrid system was used to demonstrate direct physical interaction between the edg-1-i 3 domain and the G i␣2 polypeptide. The i 3 domain was expressed as a C-terminal fusion protein with the DNA binding domain of the transcription factor GAL4 (GAL4i 3 ) and the N-terminal deleted form of the rat G i␣2 polypeptide was expressed as a C-terminal fusion with the transactivation domain of the GAL4 protein (Act D-G i ). Interaction of two proteins results in the transactivation of the GAL4-LacZ reporter gene in the host Y190 cells (21). While the GAL4-i 3 alone and Act D-G i alone did not transactivate the LacZ reporter gene, co-expression of both plasmids strongly transactivated the LacZ expression, suggesting that the edg-1-i 3 and the G i polypeptides interact directly. The two-hybrid system has been used previously to demonstrate direct interaction between the G protein subunits and the downstream kinases in the yeast mating pathway (46). To our knowledge, this is the first demonstration of interaction of a signaling domain of the GPR with an ␣ subunit of a G protein. Due to the relative ease of the readout, this system holds promise as a genetic method to delineate sequences involved in the receptor/G protein interaction.
Chimeric receptor studies have indicates that multiple cyto- FIG. 7. Enhanced MAP kinase and phospholipase A 2 activities in NIH3T3 cells stably transfected with edg-1. A, NIH3T3 cell clones transfected with human edg-1 cDNA or vector alone were isolated and the expression of the transfected cDNA was assessed by Northern blot analysis. E, edg-1 clone; M, pMexNeo vector clone. B, activation of MAP kinase in stably transfected NIH3T3 cells. Extracts from serum-starved cells were prepared, immunoprecipitated with anti-ERK-2 antibody, and the immune complex kinase assay using MBP as substrate was conducted. Phosphorylated MBP was qunatitated by densitometry. Values shown represent the mean Ϯ S.E. from two separate experiments. C, activation of phospholipase A 2 activity in stably transfected NIH3T3 cells. Assay of phospholipase A 2 activity was carried out by measuring arachidonic acid release from prelabeled cells as described. Data represent mean Ϯ S.E. of triplicate values from a representative experiment that was repeated three times. solic domains of GPRs cooperate to determine specificity in G protein coupling. For example, when the i 3 domain of the G scoupled ␤ 2 -adrenergic receptor was inserted into the corresponding region of the G q -coupled M1 muscarinic receptor, the resulting chimeric receptor stimulated both G s and G q pathways (47). Thus, the i 3 domain alone is not sufficient to switch the G protein-coupling characteristics. Further chimeric studies have shown that replacement of all the cytosolic loops is sufficient to convert the G protein-coupling specificity (30 -32). While the i 3 domain of edg-1 binds to G i and G o , it is important to establish which G proteins are capable of coupling to intact edg-1 GPR. Thus, the epitope-tagged full-length edg-1 polypeptide was co-expressed with the individual G i and G o polypeptides in HEK293 cells. In this system, the edg-1 polypeptide associated with G i␣1 and G i␣3 . The association of G i␣2 and G o␣ with full-length edg-1 was not observed in transiently transfected HEK293 cells. Thus, multiple domains in the cytosolic loops of edg-1 are likely to be involved in determining the specificity in coupling to G i␣1 and G i␣3 polypeptides. These data demonstrate that edg-1 is a G i -linked receptor.
Recent data have supported the two-state model of receptor function; in this model receptors are in equilibrium between active (R*) and inactive (R) conformations (33). Thus, overexpression of GPRs should increase the concentration of the R* state and thereby lead to constitutive signaling (34,35). Recent studies from in vitro transient transfection studies with orphan receptors (48) and in vivo transgenic studies with the adrenergic receptors (34,35) support this model. Since edg-1 is an inducible receptor, regulation of signaling pathways by modulation of receptor numbers may be of physiological significance. Our data suggest that the fraction of the overexpressed edg-1 receptors that are in the activated (R*) state are capable of coupling to the G i␣1 and G i␣3 polypeptides for productive signaling. The G i family of polypeptides are known to activate a number of cellular signal transduction pathways such as inhibition of the adenylate cyclase, activation of phospholipase A 2 (37,49), induction of MAP kinase activity (36) and the activation of ion channels (50). We thus examined the activity of basal and forskolin-induced cAMP levels and the basal release of [ 3 H]arachidonic acid release (phospholipase A 2 activity) from HEK293 cells transfected with edg-1. Both the cAMP and phospholipase activities were not altered by overexpression of edg-1 with and without the G i and G o polypeptides (data not shown). In contrast, cellular MAP kinase activity was strongly induced by edg-1 transfection and is attenuated by pertussis toxin. It is unclear why the cellular cAMP levels and phospholipase activities are unaltered by these treatments in HEK293 cells. It is possible that edg-1 regulates the MAP kinase activity in a constitutive manner and that regulation of other pathways require the presence of the ligand(s). Alternatively, appropriate effector isoenzymes that respond to the edg-1 signal may not be present in HEK293 cells. Nevertheless, these data do confirm that edg-1-dependent MAP kinase activation occurs via the G i␣ pathway in HEK293 cells. Interestingly, the MAP kinase activity is induced in a sustained manner by edg-1 transfection. In PC-12 phreochromocytoma cells, sustained MAP kinase activation is essential for neurite extension and differentiation (51,52). Because edg-1 expression is associated with differentiation of endothelial cells in vitro (8), these data are consistent with a functional role for edg-1 in cellular differentiation.
In stably transfected NIH3T3 cells, edg-1 overexpression is associated with MAP kinase activation. In contrast to HEK293 cells, the edg-1 expressing NIH3T3 cells also exhibited enhanced phospholipase A 2 activity. Because the cellular phospholipase A 2 enzyme is phosphorylated and activated by MAP kinase (37), these data suggest that edg-1 signaling via the MAP kinase is involved in the induction of phospholipase A 2 activity in NIH3T3 cells.
The MAP kinase pathway is a widely used signaling system that regulates cell growth, differentiation and apoptosis (51)(52)(53)(54)(55)(56)(57). Both GPRs as well as tyrosine kinase receptors induce the MAP kinase activity in a number of cell types (36,49,(53)(54)(55)(56)(57)(58)(59). In fibroblasts and neuronal cells, MAP kinase activation is known to induce cell cycle traverse and differentiation, respectively (51)(52)(53)(54)(55)(56)(57)59). For example, lysophosphatidic acid and thrombin are known to induce fibroblast proliferation via G icoupled receptors (36,60,61). In PC-12 cells, the activation of the MAP kinase pathway regulates differentiation by inducing neurite outgrowth (51,52). Recent studies in early Xenopus development have indicated that MAP kinase activation by the FGF receptor can account completely for the mesoderm-inducing capacity of the FGF polypeptides (12). These studies highlight the importance of the MAP kinase pathway in the control of cell growth and differentiation. The edg-1 polypeptide was originally isolated because it was induced during the in vitro differentiation and growth arrest of endothelial cells (8). While the physiological function of edg-1 is unknown at present, data presented in this report indicate that overexpression of edg-1 results in constitutive activation of the MAP kinase activity by the G i pathway. Whether such a mechanism plays a functional role in endothelial cell differentiation awaits further experimentation.
In conclusion, our data indicate that (i) the edg-1-i 3 domain associates with G i␣ and G o␣ polypeptides, (ii) the edg-1 receptor associates with G i␣1 and G i␣3 , and (iii) overexpression of edg-1 induces MAP kinase activity in HEK293 and NIH3T3 cells and (iv) edg-1 overexpression is associated with enhanced phospholipase A 2 activity in NIH3T3 cells. These data provide a basis for further understanding of the function of the inducible orphan receptor edg-1.