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J Biol Chem, Vol. 274, Issue 39, 27351-27358, September 24, 1999


Differential Coupling of the Sphingosine 1-Phosphate Receptors Edg-1, Edg-3, and H218/Edg-5 to the Gi, Gq, and G12 Families of Heterotrimeric G Proteins*

Rolf T. WindhDagger , Menq-Jer Lee§, Timothy Hla§, Songzhu An, Alastair J. BarrDagger parallel , and David R. ManningDagger **

From the Dagger  Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, the § Department of Physiology, University of Connecticut School of Medicine, Farmington, Connecticut 06030, and the  Department of Medicine, University of California at San Francisco, San Francisco, California 94143

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sphingosine 1-phosphate (S1P) is one of several bioactive phospholipids that exert profound mitogenic and morphogenic actions. Originally characterized as a second messenger, S1P is now recognized to achieve many of its effects through cell surface, G protein-coupled receptors. We used a subunit-selective [35S]GTPgamma S binding assay to investigate whether the variety of actions exerted through Edg-1, a recently identified receptor for S1P, might be achieved through multiple G proteins. We found, employing both Sf9 and HEK293 cells, that Edg-1 activates only members of the Gi family, and not Gs, Gq, G12, or G13. We additionally established that Edg-1 activates Gi in response not only to S1P but also sphingosylphosphorylcholine; no effects of lysophosphatidic acid through Edg-1 were evident. Our assays further revealed a receptor(s) for S1P endogenous to HEK293 cells that mediates activation of G13 as well as Gi. Because several of the biological actions of S1P are assumed to proceed through the G12/13 family, we tested whether Edg-3 and H218/Edg-5, two other receptors for S1P, might have a broader coupling profile than Edg-1. Indeed, Edg-3 and H218/Edg-5 communicate not only with Gi but also with Gq and G13. These studies represent the first characterization of S1P receptor activity through G proteins directly and establish fundamental differences in coupling.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sphingolipid metabolites, including sphingosine 1-phosphate (S1P),1 regulate many aspects of cell growth and differentiation. S1P is a mitogen (1-4), opposes ceramide-induced apoptosis (5, 6), inhibits cell motility (2, 7), activates platelets (8), and causes retraction of neurites (9, 10). S1P also elicits diverse biochemical responses, including activation of mitogen-activated protein (MAP) kinases (2, 11), phospholipase C (12-14), phospholipase D (15-17), and Ik(ACh) (18, 19), inhibition of adenylyl cyclase (16, 19), and mobilization of Ca2+ (1, 12, 20-24).

Although postulated to function as an intracellular messenger in some cases (21, 22, 25, 26), S1P exerts many of its effects through cell surface receptors (9, 11, 16, 18, 19). Recently, the former orphan receptor Edg-1 (endothelial differentiation gene 1) was identified as a high affinity receptor for S1P (27-29). In cells overexpressing Edg-1, S1P promotes activation of MAP kinase and inhibition of adenylyl cyclase (28, 30, 31). Additionally, S1P causes a mobilization of calcium in Chinese hamster ovary cells, although not in Sf9, HEK293, or COS-7 cells, overexpressing Edg-1 (29-31). The sensitivity of these S1P/Edg-1-induced responses to a pertussis toxin (PTX) indicates that members of the Gi family of G proteins mediate them (28-32). In addition to the functional coupling of Edg-1 with Gi proteins, physical interaction of Edg-1 with members of this family has been demonstrated. In transfected HEK293 cells, all four PTX-sensitive Galpha subunits, alpha i1, alpha i2, alpha i3, and alpha o, associate with the third intracellular loop of Edg-1 in a GTPgamma S-sensitive manner, and alpha i1 and alpha i3 co-immunoprecipitate with Edg-1 itself (32).

The signaling events mediated by Edg-1 are not uniformly sensitive to PTX, however. Neither the morphological changes nor the expression of P-cadherin induced by S1P in HEK293 cells expressing Edg-1 was inhibited by PTX (28). Instead, both of these responses were attenuated by the C3 exotoxin, which ADP-ribosylates the monomeric G protein Rho. The dependence of morphological changes and P-cadherin expression on Rho is consistent with a role for G12 or G13 as an intermediate in transduction. Constitutively active forms of alpha 12 and alpha 13 activate serum response factor (33, 34) and induce stress fiber formation (35), both in a Rho-dependent manner. Moreover, a guanine nucleotide exchange factor for Rho, p115 RhoGEF, has been identified as a target for alpha 13 (36, 37). The involvement of Rho, however, does not necessarily imply use of G12/13. Recent data implicate Gq, too, as a G protein that can either activate Rho or whose actions in part require already active Rho (34, 38). The lack of closely linked or unique effector read-outs for G12, G13, and Gq makes conclusive demonstration of potential Edg-1-G12/13/q interactions difficult. Thus, the coupling of Edg-1 to G proteins, although partially mapped, is not fully understood.

Several other G protein-coupled receptors related to Edg-1 have also been characterized. Edg-2, or Vzg-1, the first member of the Edg family for which a ligand was identified, serves as a receptor for lysophosphatidic acid (LPA) (39-42). On the basis of sequence homology with Edg-2, three other previously identified genes were added to this family. One receptor, Edg-4, was determined to be a receptor for LPA (42), whereas two other receptors, Edg-3 (43) and Edg-5 (also named H218) (44), were identified as receptors for S1P (45). Edg-3 and Edg-5 are 44% similar to each other and approximately 50% similar in turn to Edg-1. S1P, acting through Edg-3 and Edg-5 but not Edg-1, elicits serum response element (SRE)-driven gene transcription in Jurkat cells and calcium mobilization in Xenopus oocytes, human TAg-Jurkat, and rat HTC4 hepatoma cells (45, 46). As the calcium and SRE responses elicited through these receptors are largely PTX-insensitive, and because Edg-1 does not activate SRE-driven gene transcription in these cells, Edg-3 and Edg-5 appear to use a set of signaling mechanisms, and thus possibly G proteins, partly distinct from that used by Edg-1.

The assumption of involvement of specific G proteins on the basis of changes in second messengers or other downstream read-outs, however, is often specious. Convergence of signaling pathways initiated by different G proteins, e.g. the stimulation of phospholipase C by alpha q and beta gamma dimers released by other alpha  subunits, or initiated by G protein and non-G protein inputs, e.g. the activation of mitogen-activated kinases by G protein beta gamma dimers or by tyrosine kinases, makes confident assignment of G protein identity difficult. This is especially true in model systems in which endogenous receptors and possible autocrine mechanisms further remove the measured function from the ligand binding event. Thus, although differences in signaling through Edg-1, Edg-3, and Edg-5 are recognized, the basis for the differences has only been indirectly approached.

We have developed an assay for defining interactions among receptors and G proteins using Spodoptera frugiperda (Sf9) cells as a vehicle for biosynthetic reconstitution (47). Receptors and selected mammalian G protein alpha , beta , and gamma  subunits can be co-expressed in intact Sf9 cells through infection with appropriate recombinant baculoviruses. The activation of the G protein in response to an agonist can then be analyzed in subsequently isolated membranes by facilitated exchange of GDP for [35S]GTPgamma S. In distinction from mammalian expression systems, Sf9 cells contain few endogenous G protein-coupled receptors, and the levels of insect G proteins are quite low relative to those that can be expressed heterologously. Using Sf9 cells and GDP/[35S]GTPgamma S exchange, we have characterized the coupling profiles of a variety of receptors working through Gs, Gi, Gq, and G12/13 singly or in combination (47).

S1P can promote [35S]GTPgamma S binding in membranes expressing Edg-1, Edg-3, or Edg-5 (48), but the differential use of G proteins by these receptors has not been directly addressed. The studies presented here were carried out to evaluate whether Edg-1 couples to G proteins beyond Gi and what the specificity of Edg-1 for various bioactive lipids might be. We were particularly interested in the question of whether the requirement for Rho ascertained previously for Edg-1 (28) could be correlated with the activation of Gq, G12, or G13. We also wished to determine whether differences in Edg-1, Edg-3, and Edg-5 signaling might be attributable to differences in the G proteins employed.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- S1P was obtained from both Biomol Research Laboratory (Plymouth Meeting, PA) and Sigma, and sphingosylphosphorylcholine (SPC) was obtained from Biomol Research Laboratory, Sigma, and Matreya Inc. (Pleasant Gap, PA). Other lipids, protein A-Sepharose, aprotinin, TNM-FH, activated charcoal, and normal rabbit serum were also obtained from Sigma. Pansorbin cells and Nonidet P-40 were purchased from Calbiochem (La Jolla, CA). [35S]GTPgamma S (1300 Ci/mmol) was obtained from NEN Life Science Products. Anti-FLAG M2 antibody was obtained from Kodak (New Haven, CT). Pertussis toxin was obtained from Research Biochemicals International (Natick, MA). Phenylmethylsufonyl fluoride and leupeptin were obtained from Roche Molecular Biochemicals. Fetal bovine serum was obtained from Hyclone Laboratories (Logan, UT). Dulbecco's modified Eagle's medium, Sf900-II, and G418 were obtained from Life Technologies, Inc.

Baculoviruses-- Recombinant baculoviruses encoding alpha s-s, alpha i1, alpha q, beta 1, and gamma 2 were kindly provided by Drs. T. Kozasa and A. Gilman at Southwestern Medical Center (Dallas, TX). Those encoding alpha i2, alpha i3, and alpha o1 were the gift of Dr. J. Garrison at the University of Virginia (Charlottesville, VA). Those encoding alpha 12 and alpha 13 were the gift of Dr. N. Dhanasekaran at Temple University (Philadelphia, PA). Baculoviruses for the rat beta 1-adrenoreceptor and human NK-1 receptor were gifts from Drs. E. Ross at Southwestern Medical Center and T. Fong at Merck. The recombinant baculoviruses encoding alpha z and the human 5-HT1A receptor were reported by us previously (49). The human Edg-1 cDNA containing the FLAG epitope (32) was subcloned into the baculovirus vector from Pharmingen (San Diego, CA), and viral propagation was performed using procedures recommended by the manufacturer. The cDNAs encoding full-length human Edg-3 and rat Edg-5/H218 were subcloned from the pEdg-3/EF3 and pEdg-5/EF3, respectively (46), into the pFASTBAC vector. The production of the recombinant baculoviruses was performed according to the instructions for the Bac-To-Bac baculovirus expression system (Life Technologies, Inc.).

Cell Culture and Membrane Preparation-- Sf9 cell culture and membrane preparation were carried out as described (47). Sf9 cells (Invitrogen) were maintained in TNM-FH + 10% charcoal-treated serum and 0.6% pluronic F-68 at 27 °C and ambient oxygen/CO2 tension. For infection with recombinant baculoviruses, cells were subcultured in monolayer and infected with combinations of baculoviruses at a mulitiplicity of infection of one for G protein subunits and two for receptors. The medium was changed to Sf900-II optimized serum-free medium after 18 h, and cells were harvested 30 h thereafter. To make membranes, harvested cells were washed three times in 0.9% saline and then homogenized in ice-cold HE/PI buffer (20 mM HEPES, pH 8.0, 1 mM EDTA, 10 µg/ml leupeptin, 2 µg/ml aprotinin, and 0.1 mM phenylmethylsufonyl fluoride) by 15 strokes through a 26 G needle. The homogenate was centrifuged for 5 min at 110 × g, and the resulting supernatant was centrifuged at 20,800 × g for 30 min to pellet the membranes. The membranes were resuspended at approximately 2 mg/ml protein in HE/PI buffer for storage at -70 °C.

HEK293 cells stably transfected with Edg-1 (HEK/Edg-1 cells) or empty vector (HEK/pcDNA cells) were cultured as described (28). Briefly, cells were maintained in Dulbecco's modified Eagle's medium with 10% charcoal-treated fetal calf serum and 1 mg/ml G418 at 37 °C with 5% CO2. Membranes were made in essentially the same manner as for Sf9 cells.

[35S]GTPgamma S Binding Assay-- [35S]GTPgamma S binding was assayed essentially as described previously (47). Briefly, membranes (20 µg protein/assay point) were incubated with or without ligands (1:30, v/v) for 10 min at 30 °C in the presence of 3 mM Mg2+ and 0.1-30 µM GDP, depending on the G protein. The ligands were dissolved previously in methanol and diluted into 0.2% fatty acid-free bovine serum albumin, yielding final concentrations of 0.6% methanol and 0.06% bovine serum albumin in the assay. [35S]GTPgamma S (final concentration, 1 or 5 nM) was subsequently added, and membranes were incubated an additional 10 min at 30 °C. Following incubation, membrane protein was solubilized with 0.5% Nonidet P-40 under nondenaturing conditions in the presence of 100 µM each of GDP and GTP, and Galpha subunits were immunoprecipitated using subunit-selective antisera, which were generated with C-terminal decapeptides (47, 49). Nonspecific binding was determined by immunoprecipitation with nonimmune sera. Bound radioactivity was quantitated by scintillation spectrometry.

Other Immunological Procedures-- Immunotransfer blotting of G protein alpha  subunits and Edg-1 were performed as before (47, 49), using an anti-FLAG M2 monoclonal antibody to recognize the epitope-tagged Edg-1.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We focused in the present study initially on signals conveyed by Edg-1 and specifically on the activation of G proteins achieved through this receptor. Because most mammalian cells express a variety of receptors for bioactive lipids, we began with insect Sf9 cells as a reconstitution system. Sf9 cells provide an essentially null background for the expression of most mammalian receptors (50), which is also true for those recognizing S1P (see below). Coupling of Edg-1 and G proteins was evaluated by a [35S]GTPgamma S binding assay (47), which measures GDP-GTP exchange on the Galpha subunit. Membranes from Sf9 cells co-infected with combinations of baculoviruses encoding mammalian G protein subunits (alpha  with beta 1 and gamma 2 in all cases) and Edg-1 were incubated with or without agonist in the presence of [35S]GTPgamma S, and C-terminal peptide-directed antisera were used to immunoprecipitate selected Galpha subunits from subsequently prepared detergent extracts. The amount of [35S]GTPgamma S in the resulting immunoprecipitate was counted and served as a measure of G protein activation.

As shown in Fig. 1 (upper panel), only a small amount of [35S]GTPgamma S binding was evident for Gi2 (i.e. alpha i2, beta 1, and gamma 2) when expressed alone in Sf9 cells, regardless of S1P. [35S]GTPgamma S binding was easily discerned, however, when Edg-1 was additionally introduced. Binding of [35S]GTPgamma S occurred to a moderate extent without S1P in this latter instance and was increased further with S1P. The agonist-independent activity was not attributable to the vehicle itself (vehicle, H2O, and no addition were equivalent) but was probably due instead to constitutive receptor activity (see "Discussion"). Similar results were observed in membranes from cells co-expressing Edg-1 with other members of the Gi family (Gi1, Gi3, Go, and Gz; not shown). Use of nonimmune sera for immunoprecipitation, which measures nonspecific binding of [35S]GTPgamma S, demonstrated minimal background. Activation by S1P of Gi2 through Edg-1 proceeded in a concentration-dependent manner, with an EC50 of approximately 100 nM (Fig. 1, lower panel). The extent of activation of Gi2 by Edg-1 was comparable with that achieved by the 5-HT1A receptor, a prototypic Gi family-coupled receptor (51-54), assessed in these assays as a positive control and previously (47, 55).


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  		Fig. 1.   Edg-1 confers enhanced [35S]GTPgamma S binding to Gi2 in Sf9 membranes. Membranes from Sf9 cells expressing Gi2 alone (upper panel, left-hand set of columns) or with Edg-1 (right-hand set of columns and lower panel) were assayed for [35S]GTPgamma S binding to alpha i2 in the absence and presence of S1P (10 µM, upper panel; the indicated concentrations, lower panel). Immunoprecipitation was performed with the alpha i-directed antiserum or nonimmune serum. Upper panel, data are from a single experiment performed in duplicate, which was repeated two additional times with equivalent results. Lower panel, S1P-promoted [35S]GTPgamma S binding to alpha i2 was expressed as a percentage of the maximum achieved (10 µM S1P), subtracting agonist-independent binding. Data represent the means ± S.E. of three experiments, each performed in duplicate. The curve was fit using the mean of each concentration.

Whereas Edg-1 activated all members of the Gi family, it had no effect on G proteins from the Gq, G12, and Gs families when co-expressed with these G proteins, either without (not shown) or with S1P (Fig. 2). The data are expressed as a percentage of vehicle to normalize variations among G proteins in [35S]GTPgamma S binding unrelated to receptor expression (47). Expression of Edg-1, as determined by Western analysis using an anti-FLAG antibody, was robust in membranes co-expressing the various G proteins (Fig. 2, inset). Western analysis with subunit-specific antisera also confirmed that G protein alpha  subunits were abundantly expressed (not shown).


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  		Fig. 2.   S1P promotes activation of Gi alone through Edg-1 in Sf9 cell membranes. Membranes from Sf9 cells expressing Edg-1 and mammalian beta 1, gamma 2, and alpha  subunits corresponding to the indicated G proteins were incubated with [35S]GTPgamma S in the absence or presence of S1P (10 µM). The membranes were then extracted, and the alpha  subunits were immunoprecipitated with subunit-specific antibodies. Nonimmune serum was used as a control. S1P-induced [35S]GTPgamma S binding is expressed as a percentage of vehicle to normalize for differences in intrinsic G protein activity and receptor constitutive activity (no constitutive activity was evident for Gs, Gq, G12, or G13). Data represent the means ± S.E. of 3-7 experiments. Conditions used were those found to be optimal for other receptors serving as positive controls, i.e. NK-1 (Gq, G12, and G13), beta 1-adrenoreceptor (Gs), and the 5-HT1A receptor (Gi2) (not shown; see also Ref. 47) and were as follows: 5 nM [35S]GTPgamma S and 0.1 µM GDP for membranes expressing Gq, G12, and G13; 5 nM [35S]GTPgamma S and 1 µM GDP for membranes expressing Gs; and 1 nM [35S]GTPgamma S and 30 µM GDP for membranes expressing Gi2. The asterisk indicates significantly different from vehicle by one-way ANOVA (p < 0.05). Inset, membranes from uninfected Sf9 cells (U.I.) or Sf9 cells expressing Edg-1 with Gi2, Gq, G13, or Gs (from left to right, respectively; 20 µg of protein/lane) were analyzed for Edg-1 by immunotransfer blotting, using an anti-FLAG monoclonal antibody and ECL detection.

The specificity of Edg-1 for S1P among other lipids was assessed in Sf9 membranes co-expressing Edg-1 and Gi2. Of the lipids tested, S1P activated Gi2 to the greatest extent (Fig. 3). SPC also induced significant activation of Gi2, although it was considerably less efficacious than S1P at these concentrations (10 µM each). None of the lipids had any effect on Gi2 when expressed without Edg-1 (not shown). LPA, in concentrations up of 50 µM, failed to activate Gi2 with or without Edg-1, as did phosphatidic acid (PA) and platelet-activating factor (PAF).


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  		Fig. 3.   SPC but not LPA can activate Gi2 through Edg-1 in Sf9 cell membranes. Membranes from Sf9 cells expressing Edg-1 with Gi2 were incubated with [35S]GTPgamma S in the absence or presence of the lipids indicated (10 µM), and alpha i2 was immunoprecipitated for analysis as described previously. The data are expressed as a percentage of vehicle and represent the means ± S.E. of three experiments, each performed in duplicate. The asterisk indicates significantly different from vehicle by one-way ANOVA (p < 0.05).

The selective coupling of Edg-1 to members of the Gi family alone contrasts with the apparent PTX-insensitive mediation of the morphological changes and P-cadherin expression induced by S1P in HEK293 cells stably expressing Edg-1. Although the fidelity of G protein coupling for other receptors as determined in the Sf9 expression system has been excellent (47), we decided to extend our studies to HEK293 cells directly. The protocol was much the same, with two differences: Edg-1 was introduced by transfection, with cells expressing the receptor subsequently selected (28), and G proteins endogenous to the cells were used as read-outs for [35S]GTPgamma S binding. Edg-1 cannot be detected in HEK293 cells prior to transfection (28).

For membranes from HEK293 cells not expressing Edg-1 (HEK/pcDNA cells), S1P induced an approximately 2-fold increase in [35S]GTPgamma S binding to alpha i (Fig. 4, top and middle panels; the antiserum does not distinguish between alpha i1 and alpha i2 and also recognizes alpha i3 to some extent). S1P also induced a 2-fold increase in [35S]GTPgamma S binding to alpha 13. No effects of S1P on alpha s or alpha q were observed. HEK293 cells express only very low levels of alpha 12, for which no activation could be discerned. Thus, HEK/pcDNA cells appear to express endogenous S1P receptors that couple to both Gi and G13.


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  		Fig. 4.   Edg-1 mediates the activation of Gi alone in HEK293 cell membranes. Membranes from HEK293 cells transfected with vector (HEK/pcDNA cells) or Edg-1 (HEK/Edg-1 cells) were incubated with [35S]GTPgamma S in the absence or presence of S1P (10 µM), and endogenous alpha  subunits were immunoprecipitated subsequently. Top panel, comparison of S1P-induced activation of Gi and G13 in HEK/pcDNA and HEK/Edg-1 cell membranes. Data are from a single experiment performed in duplicate, which was repeated two additional times with equivalent results. Middle panel, activation of G proteins by S1P in HEK/pcDNA cell membranes, normalized to vehicle. Bottom panel, stimulation of G protein activity by S1P in HEK/Edg-1 cell membranes, normalized to vehicle. Data in middle and bottom panels are the means ± S.E. from 3-4 experiments, each performed in duplicate. [35S]GTPgamma S and GDP concentrations are as specified in the legend to Fig. 2, except that 10 µM GDP was used in the assay of Gi. The asterisk indicates significantly different from vehicle by one-way ANOVA (p < 0.05).

For membranes from cells expressing Edg-1 (HEK/Edg-1 cells), [35S]GTPgamma S binding to alpha i in the absence of added agonist was significantly higher than observed under similar conditions for HEK/pcDNA cells (Fig. 4, top panel). [35S]GTPgamma S binding induced by S1P was also higher, as S1P elicited an approximately 4.5-fold increase in [35S]GTPgamma S binding to alpha i in membranes from HEK/Edg-1 cells, as compared with the 2-fold observed in HEK/pcDNA cell membranes (top and bottom panels). In contrast, no activation of G13 beyond that noted in HEK/pcDNA was observed. Gs and Gq were not activated by S1P in HEK/Edg-1 cell membranes. Edg-1 expression in these cells was previously determined to be 650 fmol/105 cells (28), which was similar to the expression in Sf9 cells as determined by Western analysis of membranes (not shown). Expression of G proteins in HEK/pcDNA and HEK/Edg-1 cells was equivalent.

To determine whether a weak interaction between Edg-1 and G13 might be masked by the coupling of Edg-1 to Gi, we treated HEK/Edg-1 cells with PTX. Treatment with PTX eliminated both the agonist-independent and -promoted incorporation of [35S]GTPgamma S into alpha i (Fig. 5). The alpha 13 response was unaltered. PTX treatment similarly eliminated S1P-induced activation of Gi in HEK/pcDNA cell membranes without affecting the G13 response (not shown).


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  		Fig. 5.   Stimulation of Gi, but not G13, by S1P in HEK/Edg-1 membranes is inhibited by pertussis toxin. Membranes prepared from HEK/Edg-1 cells treated with PTX (100 ng/ml, 72 h) were incubated with [35S]GTPgamma S in the absence or presence of S1P (10 µM), and endogenous alpha i or alpha 13 subunits were immunoprecipitated. Data are from a single experiment performed in duplicate, which was repeated two additional times with equivalent results.

We also assessed the activity of other lipids on G protein activation in HEK293 cells. In membranes from HEK/pcDNA cells, both S1P and LPA induced significant activation of Gi, with LPA being slightly more efficacious than S1P at these concentrations (Fig. 6, left set of columns). In membranes from HEK/Edg-1 cells, S1P and SPC stimulated a further increase in [35S]GTPgamma S binding to Gi (Fig. 6, right set of columns). The effects of LPA did not achieve significance.


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  		Fig. 6.   Activation of Gi by SPC is dependent on Edg-1 expression in HEK293 cell membranes. Membranes prepared from HEK/pcDNA and HEK/Edg-1 cells were incubated with [35S]GTPgamma S in the absence or presence of the indicated lipids (10 µM), and endogenous alpha i was immunoprecipitated as described previously. Data are normalized to vehicle and represent the means ± S.E. from three experiments. The asterisk indicates significantly different from vehicle; dagger  indicates significantly different from the response in HEK/pcDNA membranes by two-way ANOVA (p < 0.05 for both).

The work above with HEK293 cells revealed an endogenous receptor(s) for S1P that activates Gi and G13. This receptor is unlikely to be Edg-1, because the data with Sf9 and HEK293 cells with and without Edg-1 indicate that Edg-1 couples only with Gi, and Edg-1 is normally absent in HEK293 cells (28). However, we did find transcripts for Edg-3 and Edg-5, two other recently identified receptors for S1P, in HEK293 cells by Northern analysis of total mRNA (not shown). We therefore returned to Sf9 cells to examine the G protein coupling profiles of both Edg-3 and Edg-5.

Edg-3, but not Edg-5, was found to promote an agonist-independent activation of Gi2, and both Edg-3 and Edg-5 activated Gi2 in response to S1P (Fig. 7, upper left panel and both lower panels). Furthermore, both Edg-3 and Edg-5 activated G13 with S1P (upper right-hand and both lower panels). Agonist-independent activation of G13 was evident only for Edg-5 (upper right-hand panel). Gq was additionally activated by Edg-3 and Edg-5 but only in the presence of S1P (lower panels); no agonist-independent activity was evident for either receptor in this instance. Activation of Gq and G13 by Edg-3 and Edg-5 was comparable with that observed by a substance P analog working through NK-1 receptors used as a positive control in these assays and also thrombin working through the protease-activated receptor-1 (47). Expression of Edg-3 and Edg-5 was not quantified, as the receptors were not epitope-tagged; that co-infection with either of these receptors confers S1P-induced G protein activation demonstrates expression. It is quite clear that although Edg-1 couples strictly to Gi, Edg-3 and Edg-5 couple to Gi, Gq, and G13.


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  		Fig. 7.   Edg-3 and Edg-5 mediate the activation of Gi, Gq, and G13 in Sf9 cell membranes. Membranes prepared from Sf9 cells infected with recombinant baculoviruses encoding Edg-3 or Edg-5 and G protein beta 1, gamma 2, and alpha  subunits corresponding to the indicated G proteins were incubated with [35S]GTPgamma S in the absence or presence of S1P (10 µM), and the alpha  subunits were immunoprecipitated as described previously. Upper panels, direct comparison of agonist-independent and -dependent activities for Gi2 (left-hand panel) and G13 (right-hand panel). Data are from individual experiments performed in duplicate, each consistent with two additional experiments. Lower panels, activation of Gi2, Gq, and G13 in membranes expressing Edg-3 (left-hand panel) and Edg-5 (right-hand panel), normalized to vehicle. Data represent the means ± S.E. of 3-5 experiments. [35S]GTPgamma S and GDP concentrations are those specified in the legend to Fig. 2. The asterisk indicates significantly different from vehicle by one-way ANOVA (p < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our studies constitute the first direct investigation of specific G proteins activated through the Edg-1, Edg-3, and Edg-5 receptors for S1P. We demonstrate here that 1) Edg-1 couples with members of the Gi family but not Gs, Gq, or G12/13; 2) Edg-1 coupling to Gi is stimulated by sphingosylphosphorylcholine in addition to S1P but not by lysophosphatidic acid; 3) Edg-3 and Edg-5 couple not only to Gi but also to Gq and G13; and 4) all three Edg receptors exhibit constitutive activity with respect to G proteins. We have extended, for the first time, the [35S]GTPgamma S assay to mammalian cells using endogenous G proteins as end points. Unlike assays that rely on membrane filtration alone, immunoprecipitation of Galpha subunits makes it possible to assess the activity of individual G proteins in these cells. The specificity noted for Edg-1 in Sf9 cells was found to exist in HEK293 cells. The presence of an endogenous receptor(s) for S1P that resembles to some extent Edg-3 and Edg-5 was also revealed.

Our results with Sf9 cells confirm previous intimations that Edg-1 communicates with Gi (28, 29, 31, 32, 56). Sf9 cells represent an especially good vehicle for assays based on reconstitution. We show here that Sf9 cells contain no functional endogenous G protein-coupled receptor for S1P, and we have demonstrated previously that heterologously expressed mammalian G protein subunits assume dominance in coupling to introduced receptors (47, 49). These two features are especially important, because so many mammalian expression systems, HEK293 cells included (30), respond to S1P in the absence of transfected receptor. Our studies with Sf9 cells also argue that Edg-1 does not activate Gs, Gq, or G12/13. The lack of coupling to these G proteins does not reflect a deficiency in the Sf9 reconstitution system. We have demonstrated previously for several receptors (47), and here for Edg-3 and Edg-5, the utility of the system in monitoring activation of these G proteins.

Our experiments with HEK293 cells corroborate the results obtained with Sf9 cells, i.e. the designation of Gi as the only G protein to which Edg-1 couples. We did not observe in either system the activation of G13 that might have been anticipated based on morphogenic changes (28). The stimulation of G13 by S1P observed here in HEK293 cells was instead attributable to an endogenous receptor(s). Even PTX treatment, used in an attempt to redirect Edg-1 from Gi to other G proteins, did not reveal an activation of G13 through Edg-1. Thus, the data from Sf9 cells, where Edg-1 and G proteins are expressed on an essentially null background, and the data from HEK293 cells, where the activity of endogenous G proteins in their native mileu is measured, are in accord.

Our findings are consistent with the majority of what is known about Edg-1 signaling. First, a physical interaction between Edg-1 and Gi proteins has been demonstrated (32). Second, signals initiated by Edg-1 in a variety of cell types have been almost uniformly PTX-sensitive. For example, S1P-induced activation of extracellular signal-regulated kinases in HEK293 and Chinese hamster ovary cells expressing Edg-1 (28, 29, 31), inhibition of adenylyl cyclase in Sf9 and HEK293 cells (30, 31), and activation of phospholipase C and mobilization of calcium in Chinese hamster ovary cells (29) are all inhibited by PTX. Conversely, in experiments where overexpression of other S1P receptors elicits classically Gq- and G13-mediated signals, Edg-1 fails to mediate these events (45, 56, 57).

Of the reported signals elicited by Edg-1, only the changes in HEK293 morphology and expression of P-cadherin were resistant to PTX (28). Because these effects were sensitive to C3 exotoxin, implying a role for Rho, utilization of G13 had been suggested (28). As we have shown here, however, this is not the case. These results illustrate the difficulties in assigning G protein coupling on the basis of downstream effectors. Direct determination of G protein activation is necessary for confident assignment of G protein coupling.

Several lines of evidence support a mechanism apart from G13 in the Rho-dependent actions of Edg-1. First, a receptor endogenous to HEK293 cells is coupled to both Gi and G13. If the Rho-dependent morphogenic effects of Edg-1 are mediated by G13 or a combination of Gi and G13, the endogenous receptor should mediate an S1P-induced morphogenic change in untransfected HEK293 cells, yet it does not (28). Second, in Jurkat cells transfected with Edg-1, S1P fails to stimulate SRE-driven gene transcription, although in cells transfected with Edg-3 or Edg-5, S1P can (45). C3 exotoxin inhibited the stimulation elcited by S1P through Edg-3 and Edg-5 (45, 58). The question then becomes how Rho might be engaged by S1P in Edg-1-containing HEK293 cells without engagement of any G proteins but Gi by Edg-1. One possibility is that Rho is activated by Edg-1 through a mechanism apart from heterotrimeric G proteins. Mitchell et al. (59), for example, have demonstrated a physical interaction between several G protein-coupled receptors and Rho, and it is quite conceivable that Edg-1 shares this property in a manner possibly conditioned on cell type. Another possibility is that long term overexpression of Edg-1 in some way sensitizes Rho to activation through the endogenous S1P receptors and G13, perhaps through up-regulation of a p115RhoGEF-like molecule or another component whose absence appears to negate a morphogenic response through the endogenous S1P receptor(s). However, it is important to note that an activation of Rho by Edg-1 has never been demonstrated, only a requirement for Rho based on C3 exotoxin sensitivity. Thus, the morphogenic change elicited by Edg-1 may in fact be conditioned on pre-existing Rho activities.

In sharp contrast to the observed coupling of Edg-1 with members of the Gi family alone, both Edg-3 and Edg-5 couple to Gi, Gq, and G13. We have observed this pattern of coupling previously for receptors for thrombin and substance P (NK-1) (47). It is curious that although some receptors, like Edg-1, couple to Gi alone, and others to Gq or Gs alone (e.g. (47, 60)), none have so far been observed to couple selectively to G12 or G13. It is remarkable in relation to the previous point regarding Rho activation by Edg-1 that the differential activation of G proteins by Edg-3 and Edg-5 mirrors functional data obtained in studies of these two receptors. Edg-3 and Edg-5 mobilize Ca2+ in rat hepatoma cells in a largely PTX-insensitive fashion, consistent with utilization of Gq (46), and also in Xenopus oocytes when introduced alone (45, 56) or with mammalian Gq (56). It has recently been reported that Rho-dependent SRE-driven gene transcription can be elicited with GTPase-deficient (constitutively active) forms of Gq and G12 family members (34). Edg-3 and Edg-5, when transfected into Jurkat cells, mediate C3 exotoxin-sensitive activation of SRE-driven gene transcription by S1P (45, 58), consistent with activation of alpha q and/or alpha 12/13. Furthermore, transfection of HEK293 and PC12 cells with Edg-3 and Edg-5 confer S1P-induced rounding (57). Thus, data from studies of the downstream consequences of Edg-3 and Edg-5 activation are consistent with a coupling of these receptors to Gq and G12/13.

All three receptors displayed agonist-independent activation of G proteins. Although Edg-3 and Edg-5 coupled to the same families of G proteins, they could be distinguished in Sf9 cells based on this activity, with Edg-3 preferentially activating Gi2 and Edg-5 activating G13. Without antagonists or inverse agonists, it is difficult to definitively establish this activity as true constitutive activity. However, the data do not support the actions of S1P possibly carried over from the medium or synthesized by the cells, because the apparent constitutive activity was more narrowly restricted to certain G proteins (Edg-3-Gi only; Edg-5-G13 only) than the S1P-stimulated activity through these receptors.

We found that SPC induces the activation of Gi in membranes from Sf9 and HEK293 cells expressing Edg-1 but not those in which Edg-1 is absent, demonstrating that it must interact with Edg-1 in some fashion. This was somewhat surprising, for although SPC agonism at Edg-1 was was recently described (56), SPC was also reported to be unable to displace radiolabeled S1P from Edg-1 in competition binding assays (28, 30). Furthermore, it has recently been reported that 1-O-cis-alk-1'-enyl-2-lyso-sn-glycerol-3-phosphate (alkenyl-GP), present in some commercial preparations of SPC, mediates mitogenesis and activation of mitogen-activated protein kinases in Swiss 3T3 fibroblasts previously attributed to SPC (61). In that study, alkenyl-GP was detected both by bioassay and structural analysis in SPC obtained from Sigma but not in any of the preparations obtained from Matreya, Inc. We used SPC from Sigma, Matreya, and Biomol with identical results, suggesting that SPC itself rather than alkenyl-GP acts as an agonist at Edg-1.

Although we observed activation of Gi proteins by Edg-1 in response to S1P and SPC, LPA failed to activate Gi2 in Sf9 cells co-expressing Gi2 and Edg-1. Similarly, no significant activation of Gi by LPA was observed in membranes from HEK/Edg-1 cells. These observations do not agree with previous studies suggesting that Edg-1 is a low affinity receptor for LPA (62). In those studies, LPA bound to Edg-1-transfected cells with an affinity of 2.3 µM and induced MAP kinase activation, receptor phosphorylation, and cadherin expression. These events occurred in the presence of endogenous LPA receptors, however, and thus LPA may signal through Edg-1 indirectly in intact cell assays by inducing release and subsequent interaction of S1P with Edg-1. Other studies have not observed competition of [32P]S1P binding by LPA (28, 57), and LPA did not function as an agonist for the murine analog of Edg-1, lpB1, when transfected into RH7777 cells (48). Again, by measuring G protein activation, we are able to evaluate agonism more directly than techniques relying on ligand competition or cellular events distal to the primary transduction event.

The use of Sf9 and mammalian expression systems together in the evaluation of receptor-G protein coupling has distinct advantages over the use of either alone. Sf9 cells permit a straightforward analysis of coupling where the receptor and G protein components are unambiguously defined in the absence of endogenous receptors. Mammalian cells provide corroboration for the specificity of coupling in a more realistic setting of multiple receptors and G proteins and permit an easier extension of G protein activation to biological sequels. When the two systems agree, as they do in this report, a relatively firm conclusion regarding coupling can be made. The measurement of G protein activation in mammalian cells can be even more revealing, however. Here, we find activation of a G protein not only by an overexpressed receptor (Edg-1) but of two G proteins by a receptor(s) endogenous to HEK293 cells. The existence of this receptor was unrecognized previously. This finding has an immediate impact on interpretations that any biological action of S1P is exerted through an overexpressed Edg-1, Edg-3 or Edg-5 alone. Rather, the action may instead represent the combination of signaling by multiple receptor isotypes.

    ACKNOWLEDGEMENT

We are grateful to Tara Friebel for technical assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM51196.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.

parallel Present address: Dept. of Medicine, Duke University Medical Center, Durham, NC 27710.

** To whom correspondence should be addressed: Dept. of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia, PA 19104-6084. Tel.: 215-898-1775; Fax: 215-573-2236; E-mail: manning@pharm.med.upenn.edu.

    ABBREVIATIONS

The abbreviations used are: S1P, sphingosine 1-phosphate; 5-HT1A receptor, the 1A subtype of the 5-hydroxytryptamine (serotonin) receptor; ANOVA, analysis of variance; G protein, GTP-binding regulatory protein; GTPgamma S, guanosine 5'-(3-O-thio)triphosphate; HEK, human embryonic kidney; LPA, lysophosphatidic acid; MAP, mitogen-activated protein; NK-1, neurokinin-1 (substance P); PTX, pertussis toxin; Sf9, Spodoptera frugiperda; SPC, sphingosylphosphorylcholine; SRE, serum response element; alkenyl-GP, 1-O-cis-alk-1'-enyl-2-lyso-sn-glycerol- 3-phosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell Biol. 114, 155-167[Abstract/Free Full Text]
2. Bornfeldt, K. E., Graves, L. M., Raines, E. W., Igarashi, Y., Wayman, G., Yamamura, S., Yatomi, Y., Sidhu, J. S., Krebs, E. G., Hakomori, S., and Ross, R. (1995) J. Cell Biol. 130, 193-206[Abstract/Free Full Text]
3. Gomez-Munoz, A., Waggoner, D. W., O'Brien, L., and Brindley, D. N. (1995) J. Biol. Chem. 270, 26318-26325[Abstract/Free Full Text]
4. Berger, A., Bittman, R., Schmidt, R. R., and Spiegel, S. (1996) Mol. Pharmacol. 50, 451-457[Abstract]
5. Cuvillier, O., Pirianov, G., Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, S., and Spiegel, S. (1996) Nature 381, 800-803[CrossRef][Medline] [Order article via Infotrieve]
6. Edsall, L. C., Pirianov, G. G., and Spiegel, S. (1997) J. Neurosci. 17, 6952-6960[Abstract/Free Full Text]
7. Sadahira, Y., Zheng, M., Ruan, F., Hakomori, S., and Igarashi, Y. (1994) FEBS Lett. 340, 99-103[CrossRef][Medline] [Order article via Infotrieve]
8. Yatomi, Y., Ruan, F., Hakomori, S., and Igarashi, Y. (1995) Blood 86, 193-202[Abstract/Free Full Text]
9. Postma, F. R., Jalink, K., Hengeveld, T., and Moolenaar, W. H. (1996) EMBO J. 15, 2388-2392[Medline] [Order article via Infotrieve]
10. Sato, K., Tomura, H., Igarashi, Y., Ui, M., and Okajima, F. (1997) Biochem. Biophys. Res. Commun. 240, 329-334[CrossRef][Medline] [Order article via Infotrieve]
11. Wu, J., Spiegel, S., and Sturgill, T. W. (1995) J. Biol. Chem. 270, 11484-11488[Abstract/Free Full Text]
12. Okajima, F., Tomura, H., Sho, K., Nochi, H., Tamoto, K., and Kondo, Y. (1996) FEBS Lett. 379, 260-264[CrossRef][Medline] [Order article via Infotrieve]
13. Im, D. S., Fujioka, T., Katada, T., Kondo, Y., Ui, M., and Okajima, F. (1997) Am. J. Physiol. 272, G1091-G1099[Abstract/Free Full Text]
14. Noh, S. J., Kim, M. J., Shim, S., and Han, J. K. (1998) J. Cell. Physiol. 176, 412-423[CrossRef][Medline] [Order article via Infotrieve]
15. Desai, N. N., Zhang, H., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1992) J. Biol. Chem. 267, 23122-23128[Abstract/Free Full Text]
16. Goodemote, K. A., Mattie, M. E., Berger, A., and Spiegel, S. (1995) J. Biol. Chem. 270, 10272-10277[Abstract/Free Full Text]
17. Kiss, Z., Crilly, K. S., and Anderson, W. H. (1997) Biochem. J. 328, 383-391
18. Bünemann, M., Brandts, B., Meyer zu Heringdorf, D., van Koppen, C. J., Jakobs, K. H., and Pott, L. (1995) J. Physiol. 489, 701-777[Medline] [Order article via Infotrieve]
19. van Koppen, C., Meyer zu Heringdorf, D., Laser, K. T., Zhang, C., Jakobs, K. H., Bünemann, M., and Pott, L. (1996) J. Biol. Chem. 271, 2082-2087[Abstract/Free Full Text]
20. Chao, C. P., Laulederkind, S. J., and Ballou, L. R. (1994) J. Biol. Chem. 269, 5849-5856[Abstract/Free Full Text]
21. Ghosh, T. K., Bian, J., and Gill, D. L. (1994) J. Biol. Chem. 269, 22628-22635[Abstract/Free Full Text]
22. Mattie, M., Brooker, G., and Spiegel, S. (1994) J. Biol. Chem. 269, 3181-3188[Abstract/Free Full Text]
23. Meyer zu Heringdorf, D., van Koppen, C. J., Windorfer, B., Himmel, H. M., and Jakobs, K. H. (1996) Naunyn-Schmiedebergs Arch. Pharmacol. 354, 397-403[CrossRef][Medline] [Order article via Infotrieve]
24. Törnquist, K., Saarinen, P., Vainio, M., and Ahlstrom, M. (1997) Endocrinology 138, 4049-4057[Abstract/Free Full Text]
25. Meyer zu Heringdorf, D., Lass, H., Alemany, R., Laser, K. T., Neumann, E., Zhang, C., Schmidt, M., Rauen, U., Jakobs, K. H., and van Koppen, C. J. (1998) EMBO J. 17, 2830-2837[CrossRef][Medline] [Order article via Infotrieve]
26. Alemany, R., Meyer zu Heringdorf, D., van Koppen, C. J., and Jakobs, K. H. (1999) J. Biol. Chem. 274, 3994-3999[Abstract/Free Full Text]
27. Hla, T., and Maciag, T. (1990) J. Biol. Chem. 265, 9308-9313[Abstract/Free Full Text]
28. Lee, M. J., Van Brocklyn, J. R., Thangada, S., Liu, C. H., Hand, A. R., Menzeleev, R., Spiegel, S., and Hla, T. (1998) Science 279, 1552-1555[Abstract/Free Full Text]
29. Okamoto, H., Takuwa, N., Gonda, K., Okazaki, H., Chang, K., Yatomi, Y., Shigematsu, H., and Takuwa, Y. (1998) J. Biol. Chem. 273, 27104-27110[Abstract/Free Full Text]
30. Van Brocklyn, J. R., Lee, M.-J., Menzeleev, R., Olivera, A., Edsall, L., Cuvillier, O., Thomas, D. M., Coopman, P. J. P., Thangada, S., Liu, C., Hla, T., and Spiegel, S. (1998) J. Cell Biol. 142, 229-240[Abstract/Free Full Text]
31. Zondag, G. C., Postma, F. R., Etten, I. V., Verlaan, I., and Moolenaar, W. H. (1998) Biochem. J. 330, 605-609
32. Lee, M.-J., Evans, M., and Hla, T. (1996) J. Biol. Chem. 271, 11272-11279[Abstract/Free Full Text]
33. Fromm, C., Coso, O. A., Montaner, S., Xu, N., and Gutkind, J. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10098-10103[Abstract/Free Full Text]
34. Mao, J., Yuan, H., Xie, W., and Wu, D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12973-12976[Abstract/Free Full Text]
35. Buhl, A. M., Johnson, N. L., Dhanasekaran, N., and Johnson, G. L. (1995) J. Biol. Chem. 270, 24631-24634[Abstract/Free Full Text]
36. Hart, M. J., Jiang, X., Kozasa, T., Roscoe, W., Singer, W. D., Gilman, A. G., Sternweis, P. C., and Bollag, G. (1998) Science 280, 2112-2114[Abstract/Free Full Text]
37. Kozasa, T., Jiang, X., Hart, M. J., Sternweis, P. M., Singer, W. D., Gilman, A. G., Bollag, G., and Sternweis, P. C. (1998) Science 280, 2109-2111[Abstract/Free Full Text]
38. Katoh, H., Aoki, J., Yamaguchi, Y., Kitano, Y., Ichikawa, A., and Negishi, M. (1998) J. Biol. Chem. 273, 28700-28707[Abstract/Free Full Text]
39. Hecht, J. H., Weiner, J. A., Post, S. R., and Chun, J. (1996) J. Cell Biol. 135, 1071-1083[Abstract/Free Full Text]
40. An, S., Dickens, M. A., Bleu, T., Hallmark, O. G., and Goetzl, E. J. (1997) Biochem. Biophys. Res. Commun. 231, 619-622[CrossRef][Medline] [Order article via Infotrieve]
41. Fukushima, N., Kimura, Y., and Chun, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 6151-6156[Abstract/Free Full Text]
42. An, S., Bleu, T., Hallmark, O. G., and Goetzl, E. J. (1998) J. Biol. Chem. 273, 7906-7910[Abstract/Free Full Text]
43. Yamaguchi, F., Tokuda, M., Hatase, O., and Brenner, S. (1996) Biochem. Biophys. Res. Commun. 227, 608-614[CrossRef][Medline] [Order article via Infotrieve]
44. MacLennan, A. J., Browe, C. S., Gaskin, A. A., Lado, D. C., and Shaw, G. (1994) Mol. Cell. Neurosci. 5, 201-209[CrossRef][Medline] [Order article via Infotrieve]
45. An, S., Bleu, T., Huang, W., Hallmark, O. G., Coughlin, S. R., and Goetzl, E. J. (1997) FEBS Lett. 417, 279-282[CrossRef][Medline] [Order article via Infotrieve]
46. An, S., Bleu, T., and Zheng, Y. (1999) Mol. Pharmacol. 55, 787-794[Abstract/Free Full Text]
47. Barr, A. J., Brass, L. F., and Manning, D. R. (1997) J. Biol. Chem. 272, 2223-2229[Abstract/Free Full Text]
48. Zhang, G., Contos, J. J. A., Weiner, J. A., Fukushima, N., and Chun, J. (1999) Gene (Amst.) 227, 89-99[CrossRef][Medline] [Order article via Infotrieve]
49. Butkerait, P., Zheng, Y., Hallak, H., Graham, T. E., Miller, H. A., Burris, K. D., Molinoff, P. B., and Manning, D. R. (1995) J. Biol. Chem. 270, 18691-18699[Abstract/Free Full Text]
50. Windh, R. T., Barr, A. J., and Manning, D. R. (1999) in Handbook of Experimental Pharmacology: The Pharmacology of Functional, Biochemical, and Recombinant Receptor Systems (Kennakin, T. , and Angus, J. A., eds) , Springer-Verlag, Berlin, in press
51. Innis, R. B., and Aghajanian, G. K. (1987) Eur. J. Pharmacol. 143, 195-204[CrossRef][Medline] [Order article via Infotrieve]
52. Fargin, A., Raymond, J. R., Regan, J. W., Cotecchia, S., Lefkowitz, R. J., and Caron, M. G. (1989) J. Biol. Chem. 264, 14848-14852[Abstract/Free Full Text]
53. Gettys, T. W., Fields, T. A., and Raymond, J. R. (1994) Biochemistry 33, 4283-4290[CrossRef][Medline] [Order article via Infotrieve]
54. Mulheron, J. G., Casañas, S. J., Arthur, J. M., Garnovskaya, M. N., Gettys, T. W., and Raymond, J. R. (1994) J. Biol. Chem. 269, 12954-12962[Abstract/Free Full Text]
55. Barr, A. J., and Manning, D. R. (1997) J. Biol. Chem. 272, 32979-32987[Abstract/Free Full Text]
56. Ancellin, N., and Hla, T. (1999) J. Biol. Chem. 274, 18997-19002[Abstract/Free Full Text]
57. Van Brocklyn, J. R., Tu, Z., Edsall, L. C., Schmidt, R. R., and Spiegel, S. (1999) J. Biol. Chem. 274, 4626-2632[Abstract/Free Full Text]
58. An, S., Goetzl, E. J., and Lee, H. (1998) J. Cell. Biochem. Suppl. 30/31, 147-157
59. Mitchell, R., McCulloch, D., Lutz, E., Johnson, M., MacKenzie, C., Fennel, M., Fink, G., Zhou, W., and Sealfon, S. C. (1998) Nature 392, 411-414[CrossRef][Medline] [Order article via Infotrieve]
60. Hellmich, M. R., Battey, J. F., and Northup, J. K. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 751-756[Abstract/Free Full Text]
61. Liliom, K., Fischer, D. J., Virddager g, T., Sun, G., Miller, D. D., Tseng, J.-L., Desiderio, D. M., Seidel, M. C., Erickson, J. R., and Tigyi, G. (1998) J. Biol. Chem. 273, 13461-13468[Abstract/Free Full Text]
62. Lee, M.-J., Thangada, S., Liu, C. H., Thompson, B. D., and Hla, T. (1998) J. Biol. Chem. 273, 22105-22112[Abstract/Free Full Text]

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