A single amino acid determines preference between phospholipids and reveals length restriction for activation ofthe S1P4 receptor

Background Sphingosine-1-phosphate and lysophosphatidic acid (LPA) are ligands for two related families of G protein-coupled receptors, the S1P and LPA receptors, respectively. The lysophospholipid ligands of these receptors are structurally similar, however recognition of these lipids by these receptors is highly selective. A single residue present within the third transmembrane domain (TM) of S1P receptors is thought to determine ligand selectivity; replacement of the naturally occurring glutamic acid with glutamine (present at this position in the LPA receptors) has previously been shown to be sufficient to change the specificity of S1P1 from S1P to 18:1 LPA. Results We tested whether mutation of this "ligand selectivity" residue to glutamine could confer LPA-responsiveness to the related S1P receptor, S1P4. This mutation severely affected the response of S1P4 to S1P in a [35S]GTPγS binding assay, and imparted sensitivity to LPA species in the order 14:0 LPA > 16:0 LPA > 18:1 LPA. These results indicate a length restriction for activation of this receptor and demonstrate the utility of using LPA-responsive S1P receptor mutants to probe binding pocket length using readily available LPA species. Computational modelling of the interactions between these ligands and both wild type and mutant S1P4 receptors showed excellent agreement with experimental data, therefore confirming the fundamental role of this residue in ligand recognition by S1P receptors. Conclusions Glutamic acid in the third transmembrane domain of the S1P receptors is a general selectivity switch regulating response to S1P over the closely related phospholipids, LPA. Mutation of this residue to glutamine confers LPA responsiveness with preference for short-chain species. The preference for short-chain LPA species indicates a length restriction different from the closely related S1P1 receptor.


Background
Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are phospholipid growth factors which are present in normal serum and plasma. These lipids elicit diverse responses from a wide range of cell types, including enhanced cell survival, cell proliferation, induction of cytoskeletal changes and chemotaxis (reviewed in [1][2][3][4]. Some of these responses reflect activation of G proteincoupled receptors of the endothelial differentiation gene (Edg) family. The Edg receptor family is classified into two clusters based on ligand selectivity: S1P 1/2/3/4/5 (formerly Edg1/5/3/6/8) specifically respond to S1P whilst LPA 1/2/3 (formerly Edg2/4/7) respond to LPA [5]. Members of the S1P receptor family display higher sequence similarity to each other (approximately 40% identity) than to members of the LPA receptor family (approximately 30% identity). These homologies, coupled with observed differences in the structure of S1P and LPA receptor genes, suggest that these receptor families evolved from distinct ancestral genes. The S1P receptors contain a conserved glutamic acid residue present within the third TM that corresponds to glutamine in the LPA receptors. Interaction between distinct functional groups present on S1P and LPA with this residue was shown for the S1P 1 and LPA 1 receptors using computational modelling techniques [6,7] and was demonstrated as the basis for the ligand preference displayed by the receptors. Experimental characterisation confirmed that replacement of glutamic acid with glutamine in S1P 1 changed ligand specificity from S1P to LPA, and the reciprocal mutation in LPA 1 resulted in recognition of both LPA and S1P [7].
In the present study, the role of this residue in determining ligand selectivity for the S1P 4 receptor was examined. Phylogenetic analysis of the Edg family of receptors indicates that S1P 4 is more closely related to other S1P receptors than receptors which respond to LPA. However, S1P 4 lies on the edge of the S1P family cluster and has been shown to bind S1P with lower affinity than other S1P receptors and hence it has been suggested that S1P is not the true endogenous agonist of this receptor [8]. We therefore decided to investigate whether replacement of this residue (E 3.29(122) ) with glutamine conferred LPA-responsiveness to the S1P 4 receptor and hence determine the role of this residue in this lower-affinity S1P receptor. To achieve this, we expressed wild type and E 3.29(122) Q mutant S1P 4 receptors in CHO-K1 cells and studied responses to lysophospholipids using a [ 35 S]GTPγS binding assay. Since CHO-K1 cells respond to LPA, we utilised fusion proteins constructed between the S1P 4 receptor and a pertussis toxin-insensitive Gα i1 (C 351 I) G protein.
Expression of these proteins in CHO-K1 cells followed by treatment with pertussis toxin prior to harvest allowed elimination of any signal due to stimulation of endogenous LPA receptors. Within this study, we also examined how the length of the LPA acyl chain affected potency at the mutant S1P 4 receptor, using a panel of naturally occurring LPA analogues. Computational models of complexes between the wild type or mutant S1P 4 receptor and S1P and LPA species were used to provide a molecular interpretation of the experimental findings.

Results
Human HA-S1P 4 was mutated at position 122 to replace the naturally occurring glutamic acid with glutamine. The mutant and wild type receptors were stably expressed in CHO-K1 cells as in-frame GPCR-G protein fusions with pertussis toxin-insensitive Gα i1 (C 351 I). Western blotting was used to detect expression of these fusion proteins. Membranes from HA-S1P 4 -Gα i1 (C 351 I)-or HA-S1P 4 (E 3.29(122) Q)-Gα i1 (C 351 I)-transfected cells contained a polypeptide with an apparent molecular mass of approximately 110 kDa, which reacted with anti-HA and anti-SG1 antibodies ( Figure 1A and 1B) and was consistent with expression of the GPCR-G protein fusion. Confirmation of comparable cell-surface expression of these proteins was obtained via FACS analysis using an anti-HA antibody directly conjugated with fluorescein ( Figure 1 Panel III).
These results indicate that introduction of the E 3.29(122) Q mutation in the S1P 4 receptor confers LPA-responsiveness, and that a short form of LPA was a more effective agonist than the intermediate and longer forms, when tested at this single concentration. Dose response curves were constructed for ligand-induced activation of the E 3.29(122) Q S1P 4 mutant by the 14:0 and 18:1 forms of LPA as well as S1P ( Figure 3B). An EC 50 could only be determined for the 14:0 form of LPA as S1P and 18:1 LPA caused minimal stimulation at only the highest concentration tested. The EC 50 value for activation of HA-S1P 4 (E 3.29(122) Q)-Gα i1 (C 351 I) was calculated to be 3.8 ± 1.4 µM. However, since a plateau of maximal stimulation Expression of HA-S1P 4 -Gα i1 and HA-S1P 4 (E 122 Q)-Gα i1 (C 351 I) in CHO-K1 cells   was not achieved, interpretation of this EC 50 value needs caution. This result clearly showed that 14:0 LPA was a weak agonist of HA-S1P 4 (E 3.29(122) Q)-Gα i1 (C 351 I) and hence confirmed the involvement of residue 122 in S1P 4 ligand preference. Similar results were obtained using a second CHO-K1 clone expressing this fusion protein (not shown).
Computational modelling of the S1P complex with the wild type S1P 4 receptor identifies the best S1P binding site within the TM with the phosphate group at the extracellular end ( Figure 4A). Ion pairs appear between the phosphate group of S1P and two cationic amino acids, R 3.28(121) and K 5.38(202) . An additional ion pair occurs between the cationic ammonium of S1P and E 3.29(122) . Hydrophobic residues from TM2, TM3, TM5 and TM6 line the binding pocket and surround the alkyl chain of S1P.
The best complex of 14:0 LPA in the E 3.29(122) Q S1P 4 mutant receptor model has striking similarity to the best complex of S1P in the wild type S1P 4 receptor model (Figure 4B). Both models demonstrate ion pairing between the phosphate group and two cationic amino acids, R 3.28(121) and K 5.38(202) . Each ligand interacts with the amino acid at position 3.29(122), S1P by an ion pair with the carboxylate of the wild type glutamate and 14:0 LPA by a hydrogen bond with the mutated glutamine. Multiple hydrophobic residues surround the nonpolar tails of the lipid ligands. The superimposition of the two complexes ( Figure 4B) also demonstrates that the ligands occupy almost identical volumes. Common interactions and overlap volumes are qualitatively consistent with the experimental findings that these ligands give similar 48% and 40% maximal stimulation over basal for S1P at the wild type and 14:0 LPA at the mutant receptor, respectively.
In contrast to the complexes of 14:0 LPA with E 3.29(122) Q S1P 4 and S1P with wild type S1P 4 , the remaining complexes show much less common volume ( Figure 4C). Most complexes exhibit the phosphate interactions described for 14:0 LPA with E 3.29(122) Q S1P 4 and S1P with wild type S1P 4 . Of particular interest is the observation that the best complexes generated by Autodock for the 18:1 LPA species with wild type S1P 4 has a very high positive van der Waals interaction energy, > 3000 kcal/mol, compared to values well under 200 kcal/mol for every other complex studied. In the best complexes found for 16:0 LPA and 18:1 LPA in both constructs, the terminal six to eight carbons of the hydrophobic tails fold into Lshaped conformations quite different from the extended conformations observed in the S1P complex with wild type S1P 4 or the 14:0 LPA complex with the E 3.29(122) Q S1P 4 mutant. The terminal carbons in several complexes curl out of the receptor between TM5 and TM6 ( Figure   4C) due to the restricted length of the binding pocket. These results suggest that the complete lack of S1P 4 activation in response to 18:1 LPA is likely due to failure to form a complex. The strongest complexes formed, S1P with wild type S1P 4 and 14:0 LPA with the E 3.29(122) Q S1P 4 mutant, have complementary interactions with the residue at position 3.29(122). These strong complexes give the most robust activation. Weak complexes are formed for other combinations due to mismatched interactions with position 3.29(122) or excessive length of the hydrophobic tail. The presence of hydrophobic tails of 16:0 or 18:1 LPA between transmembrane domains may additionally impair the conformational change necessary for full agonist responses.

Discussion
Parental CHO-K1 cells respond to LPA in functional assays, reflecting expression of endogenous LPA 1 (G. Holdsworth, et al., manuscript in preparation). For this reason, fusion proteins between wild type or mutant HA-S1P 4 and the pertussis toxin-insensitive Gα i1 (C 351 I) G protein were used in these studies. Expression of these proteins in CHO-K1 cells followed by treatment with pertussis toxin prior to harvest allowed elimination of any signal due to stimulation of endogenous LPA receptors.
McAllister et al. [11] (.(adopted a similar approach for studies of the LPA 1 receptor. We examined the role of residue E 3.29(122) in controlling S1P 4 ligand selectivity using functional and computational methods. This residue, which is conserved throughout the S1P receptors, has been shown to control ligand specificity for the related S1P 1 receptor [7]. Introduction of the E 3.29(122) Q mutation severely affected the response of S1P 4 to S1P: in dose-response experiments S1P caused minimal stimulation at only the highest concentration of ligand used. This is in agreement with published observations for activation of the equivalent S1P 1 mutant [7]. 14:0 LPA was able to induce dose-dependent stimulation of S1P 4 (E 3.29(122) Q) with an EC 50 of approximately 3.8 µM but only promoted minimal stimulation of the wild type S1P 4 receptor. The modelled complexes of 14:0 LPA with E 3.29(122) Q S1P 4 and S1P with wild type S1P 4 demonstrate nearly identical volumes occupied by the two ligands and very similar interactions between these ligands and their respective receptors. Of particular importance are amino acid residues at positions 3.28(121), 3.29(122) and 5.38(202), which either ion pair with the phosphate or interact with the 2-amino or 2-hydroxyl group in S1P and 14:0 LPA, respectively. The importance of interactions with amino acids at positions 3.28 and 3.29 has been previously noted for the S1P 1 [6,7] and LPA 1,2,3 [7,12] receptors.
Computational models of wild type S1P 4 and its E3.29(122)Q mutant with S1P and LPA species Figure 4 Computational models of wild type S1P 4 and its E3.29(122)Q mutant with S1P and LPA species. Computational models of the complexes between the wild type S1P 4 or its E 3.29(122) Q mutant with S1P or various LPA species generated by Autodock 3.0 and minimised using the MMFF94 forcefield in the MOE program. Complexes in each panel are shown from the same viewpoint with the extracellular end of the receptors oriented to the top of the figure. Standard element color codes are used with grey, white red, blue and magenta representing carbon, hydrogen, oxygen, nitrogen and phosphorous. Ribbons are shaded from red at the amino-terminus to blue at the carboxy-terminus. (A) Model of the complex between S1P (spacefilling) and the wild type S1P 4 receptor. Residues in the receptor involved in ion pairs with S1P are shown as stick models and labelled.   The S1P 4 receptor exhibits marked constitutive (agonistindependent) activity ( Figure 5) which was unaffected by the introduction of the E 3.29(122) Q mutation (data not shown). This indicates that the mutation perturbs S1P recognition without affecting the ability of the receptor to spontaneously adopt an active conformation. Similar observations have been reported for the β 2 AR, where a mutation in the sixth transmembrane domain abolished agonist activation but not constitutive activity [13].
Unlike S1P, which exists as a single species in vivo, the term LPA actually refers to a family of molecules that take the general form 1-o-acyl-2-hydroxy-sn-glyceryl-3-phosphate. Naturally occurring forms of LPA contain acyl chains of differing lengths, with differing degrees of saturation. Investigations into the effect of the length and degree of saturation of the acyl chain of LPA have been undertaken for the LPA receptors [14,15], but limited SAR information is available for S1P receptors (22). The LPAresponsive E 3.29(122) Q S1P 4 mutant facilitates structure activity relationship (SAR) studies due to the greater avail-ability of LPA analogs relative to S1P analogs. Comparison of space-filling models of the structures of S1P and three analogues of LPA ( Figure 4D)  corresponds to the smaller valine in S1P 2 and S1P 3 and the much smaller alanine in LPA 2 . These findings provide a molecular explanation for a similar SAR observed using para-alkyl amide analogs of S1P [9]. SAR obtained with the S1P 4 mutant are in contrast to that shown by LPA Constitutive activity of HA-S1P 4 and HA-S1P 4 -Gα i1 (C 351 I) Figure 5 Constitutive activity of HA-S1P 4 and HA-S1P 4 -Gα i1 (C 351 I). Comparison of basal, vehicle and stimulated (10 µM S1P) GTPγS binding for membranes prepared from CHO cells transfected to express HA-S1P 4 or HA-S1P 4 fused to PTx insensitive Gα i1 . Where indicated, cells were treated with 100ng/ml PTx for 24 hours prior to harvesting. PTx treatment of HA-S1P 4transfected membranes prevented activation by S1P and also caused a dramatic reduction in basal signalling, indicative of constitutive activity. In contrast, when the HA-S1P 4 -Gα i1 fusion-expressing membranes were treated with PTx, there was only a slight reduction in basal signalling and the receptor still responded to exogenous S1P, indicating that the receptor signalled via the tethered, PTx-insensitive G protein. Basal signalling in PTx-treated HA-S1P 4 -Gα i1 -expressing membranes exceeded that seen with membranes from untransfected CHO cells. receptors, which exhibit the general trend of 18:1 ≥ 16:0 > 14:0 for potency and maximal stimulation [15].
Since mutation of residue 122 in the S1P 4 receptor from the naturally occurring glutamic acid to glutamine conferred responsiveness to 14:0 LPA and severely affected responses to S1P, our observations support the hypothesis that this conserved residue in the third transmembrane domain of the S1P receptors is involved in ligand recognition. This is in contrast to a recent paper describing models of several GPCRs, including S1P 4 , which had been generated using novel first principle methods [16]. In this model of S1P 4 , interactions between S1P and residues T 7.34(127) and W 7.37(291) and E 7.30(284) were observed. Interaction of E 7.30(284) with the ammonium group of S1P appeared to control ligand selectivity since the other residues appeared to interact with the phosphate group, which is present on both LPA and S1P. It is therefore surprising that none of these residues are conserved throughout the S1P or LPA receptor families. The data presented here support the assertion that glutamic acid residue 3.29 present in the third transmembrane domain of the S1P receptors controls ligand selectivity and suggest that the S1P 4 model described by Vaidehi et al. [16] is inaccurate.
The current study provides new information for the development of more selective S1P receptor agonists. In particular, an S1P analog with its hydrophobic chain extended by either 2 or 4 carbons would be a very poor agonist of the S1P 4 receptor. On the other hand, the activation of the S1P 1 -E 3.29(121) Q mutant by 18:1 LPA [7] indicates that a chain-extended S1P analog should retain agonist activity at the S1P 1 receptor. S1P receptor agonists with differing selectivity profiles will be useful tools to more completely map the physiological and pathophysiological roles of these receptors.

Conclusions
These studies confirm that glutamic acid residue 3.29, present in the third transmembrane domain of the S1P receptors is important for the selective recognition of S1P, versus the closely related lipid, LPA. Mutation of E3.29 to glutamine diminished response to S1P and allowed structure activity studies using the diverse available LPA species. The mutant S1P 4 receptor is stimulated most strongly by LPA 14:0 and is not activated by the longer LPA 18:1, in contrast with a previous report on the analogous S1P 1 receptor mutant that responded to LPA 18:1. Thus the S1P 4 receptor ligand binding pocket is shorter in length than the S1P 1 ligand binding pocket.

Residue nomenclature
Amino acids within the TM of S1P 4 can be assigned index positions to facilitate comparison between receptors with different numbers of amino acids, as described by Weinstein and coworkers [17]. An index position is in the format x.xx. The first number denotes the TM in which the residue appears. The second number indicates the position of that residue relative to the most highly conserved residue in that TM which is arbitrarily assigned position 50. E3.29, then, indicates the relative position of glutamate 122 in TM3 relative to the highly conserved arginine 143 in the E(D)RY motif which is assigned index position 3.50 [17]. . The SG1 antiserum was produced previously [18]. All other chemicals were from Sigma Aldrich Company Ltd., (Gillingham, Dorset, U.K.) or BDH Ltd., (Poole, Dorset, U.K.) unless stated otherwise.

Construction of receptor expression plasmids
The S1P 4 coding sequence was cloned from a human PBMC cDNA library using the sense primer 5'-GAGA-GAGCGGCCGCCACCATGTATCCATATGATGTTCCA-GATTATGCTAACGCCACGGGGACCCCGGTG-3', which contains a NotI restriction site (bold) and the haemagglutinin HA epitope tag (YPYDVPVYA, underlined) immediately after the initiator methionine, and the antisense primer 5'-GAGAGAGAATTCGGCGATGCTCCGCACGCT-GGAGATG-3', which contains an EcoRI restriction site (bold) and changes the S1P 4 stop codon to alanine (underlined). A C 351 I mutant of the Gα i1 G protein (previously produced, [19]) was amplified using PCR with the sense cloning primer 5'-GAGAGAGAATTCGCCA CCAT-GGGCTGCACACTGAGCG-3', which contains the EcoRI restriction site (bold), and the antisense cloning primer 5'-GAGAGAGGATCCTTAGAAGAGACCGATGTCTTTTA G-3', which contains a BamHI restriction site (bold). After digestion of each PCR product with the appropriate restriction enzymes, fragments were ligated into the pIRE-Spuro mammalian expression vector (Invitrogen Ltd.) to generate an in-frame fusion between HA-S1P 4 and Gα i1 (C 351 I).
The E 3.29(122) Q mutation was introduced into the S1P 4 sequence in parallel PCR reactions. Complementary oligonucleotides were designed across the residue which was to be mutated such that each primer contained the necessary base change to mutate residue 122 to glutamine (underlined in each primer): sense mutational primer: 5'-CAGTGGTTCCTACGGCAGGGCCTGCTCTTCAC-3'; antisense mutational primer: 5'-GTGAAGAGCAG-GCCCTGCCGTAGGAACCACTG-3'. Mutational sense or antisense primers were used in parallel PCR reactions with the appropriate antisense or sense cloning primer, with HA-S1P 4 plasmid DNA as template. Equimolar amounts of each purified PCR product were mixed and amplified in a further reaction, using the cloning primers described above. The resultant product was digested with the appropriate restriction enzymes and ligated with the Gα i1 sequence in the pIRESpuro expression vector to generate an in-frame fusion between HA-S1P 4 (E 3.29(122) Q) and Gα i1 (C 351 I).
Cell culture and transfection CHO-K1 cells were maintained at 37°C with 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% foetal bovine serum (FBS), 2 mM Lglutamine and non-essential amino acids. Sub-confluent cell monolayers were stably transfected to express either HA-S1P 4 -Gα i1 (C 351 I) or HA-S1P 4 (E 3.29(122) Q)-Gα i1 (C 351 I) fusion proteins using Lipofectamine reagent (Invitrogen). 72 hours post-transfection, cells were seeded in media supplemented with 7.5 µg/mL puromycin and the resultant clones examined for expression of cell surface receptor using FACS analysis. Clonal cell lines were expanded in complete DMEM containing 7.5 µg/mL puromycin and were transferred to serum free DMEM approximately 24 hours prior to harvesting. Where indicated, 100 ng/mL pertussis toxin was included in the serum free medium.
It should be noted that we initially expressed S1P 4 in RH7777 cells, which are unresponsive to S1P and LPA and have been commonly used for studies of Edg family receptors [20]. Unfortunately, our attempts to detect activation of S1P 4 expressed in these cells using a variety of functional assays were unsuccessful. Therefore, we used CHO-K1 cells as an alternative host in these studies; expression of functional S1P 4 in CHO-K1 cells has also been reported by Mandala et al. [21].

FACS analysis
The amino-terminal HA-epitope tag was detected using a fluorescein conjugate of the anti-HA antibody, clone 3F10 (Roche Molecular Biochemicals Ltd., Lewes, U.K.). Cells were harvested non-enzymatically and washed with FACS buffer (PBS containing 3% FBS and 0.1% NaN 3 ) then stained with the 3F10 antibody (or an isotype matched control) for 40 minutes at 4°C in the dark. After washing with FACS buffer, cells were analysed using a FACScalibur flow cytometer (BD Biosciences, Oxon., U.K.).

Preparation of cell membranes
Cells were harvested non-enzymatically, washed with PBS and resuspended in "assay buffer" (20 mM Hepes, pH 7.4, 3 mM MgCl 2 , 100 mM NaCl), supplemented with "com-plete" protease inhibitors (Roche Molecular Biochemicals Ltd.). Cells were homogenised in a nitrogen cavitation chamber (500 psi for 15 minutes). Unbroken cells and nuclei were pelleted by centrifugation (500 × g, 10 minutes, 4°C) and the supernatant fraction was centrifuged at 45,000 × g for 45 minutes at 4°C. Membrane pellets were resuspended in assay buffer, titurated through a fine gauge needle and stored at -80°C until required.

[ 35 S]GTPγS binding assay
[ 35 S]GTPγS binding experiments were performed essentially as described previously [22]. Briefly, membranes were incubated with or without the indicated ligand for 30 minutes at 30°C in assay buffer containing [ 35 S]GTPγS (100 nCi/point), saponin (20 µg/point) and 0.1 µM GDP. 18:1 LPA was prepared as a 2 mM DMSO stock whilst 16:0 and 14:0 LPA were prepared as 2 mM stock solutions in 1:1 ethanol:water per supplier recommendation due to their poor solubility in DMSO. S1P had previously been dispensed as thin film aliquots (dissolved in MeOH and the solvent evaporated under nitrogen) in brown glass vials and stored at -70°C prior to use. Lipids (S1P or LPA forms) were diluted in assay buffer containing 1% fatty acid free BSA, such that the final concentration of BSA in the assay was 0.1%. Following incubation, membrane protein was solubilised with 1.25% NP-40 and 0.4% SDS and after pre-clearance using non-immune serum, Gα i1/2 subunits were immunoprecipitated with SG1 antiserum, used at a dilution of 1 in 200. Non-specific binding was determined by the addition of 100 µM GTPγS. Bound radioactivity was measured using liquid scintillation counting.

Experimental data analysis
Numerical data are expressed as means ± standard error, shown as error bars in the appropriate figures. Statistical comparisons were made using one-way ANOVA with Dunnett's multiple comparison post test.