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Endogenous agonist–bound S1PR3 structure reveals determinants of G protein–subtype bias

Active S1PR3 structure reveals the unique ligand-binding mode and signaling selectivity switching mechanism.


INTRODUCTION
G protein-coupled receptors (GPCRs) form the largest superfamily of transmembrane proteins that transduce signals to the interior of cells in response to extracellular stimuli (1). The binding of GPCR agonists activates heterotrimeric G protein-mediated downstream signaling dependent on each subunit (G, G, and G) (2)(3)(4). In humans, G subunits are encoded by 16 genes and classified into four major families (G s , G i/o , G q/11 , and G 12/13 ) that trigger distinct signaling cascades. Although many GPCRs couple with multiple G families upon activation, the activated G-mediated signaling pathways are ligand dependent in a process referred to as "biased agonism" (5,6). Although unbiased agonism transduces several intrinsic signaling pathways via its own receptor, biased agonists selectively transduce specific signaling pathways and can enhance therapeutic efficacy or diminish side effects (7). For example, endogenous agonists that activate G i/o rather than G q/11 subunits via chemokine receptor 5 potentially block HIV-1 infection without inducing inflammation (8). Despite recent advances in our understanding of GPCR-G protein coupling (9)(10)(11)(12)(13), it remains elusive how ligands in the ligand-binding pocket change the intracellular conformation of receptors to modulate signaling-pathway selectivity.
Sphingosine-1-phosphate (S1P) is a lysophospholipid characterized by a single alkyl chain and was initially identified as a phospholipid precursor and metabolite (14). S1P is a signaling molecule that acts through five GPCRs (S1PR1 to S1PR5) (15)(16)(17), among which S1PR3 couples to G q/11, G i/o , and G 12/13 upon S1P binding and regulates immune responses including P-selectin-dependent leukocyte rolling via a G q/11 -mediated signaling (18), cellular chemotaxis stimulation via a G i/o -mediated signaling, and cellular chemotaxis inhibition via a G 12/13 -mediated signaling (19,20). A previous study of S1PR3 showed that phosphorylated fingolimod (FTY720-P), a structural analog of S1P characterized by a shorter lipidic tail containing a phenyl acyl group, exerted G i/o -biased agonism of S1PR3 (21). The underlying mechanism on how the alkyl tail length of ligands determines the G protein selectivity of S1PR3 was, however, unclear.
Structural examination of S1PR1 in complex with the sphingosine-mimic antagonist ML056 (22) provides molecular basis of the inactive state of the S1PRs yet does not inform us as to how S1PRs are activated. To gain insight into the S1PR-activation mechanism, here, we report the crystal structure of human S1PR3 in complex with its natural agonist d18:1 S1P. The results show that the ligand-binding pocket forms a long penetrating tunnel not seen in other GPCRs. Furthermore, the structure reveals that amino acid residues involved in formation of the long tunnel play an important role in the receptor activation and biased agonism.
The overall structure of S1PR3 shows the canonical seventransmembrane bundles holding the unbent d18:1 S1P (Fig. 1A). This structure shares common features with a previously determined inactive S1PR1 structure in complex with the sphingosine-mimic antagonist (22), including the extracellular lid composed of the N-terminal helix, extracellular loop 2 (ECL2), and ECL3 (Fig. 1, B and C). The N-terminal region forms a helical cap and contributes to ligand interaction, and disulfide bonds are formed in ECL2 (Cys178-Cys185) and ECL3 (Cys269-Cys274). These extracellular structures block ligand access from the extracellular region and are conserved in both reported S1P receptors. These findings strongly suggest that ligand entry to the binding pocket of S1PRs occurs primarily from the membrane bilayer through the side gap between helical transmembrane domain 1 (TM1) and TM7 (Fig. 1, B

S1P-binding mode
The ligand-binding pocket of S1PR3 is highly amphipathic, which is ideal for a hydrophobic-zwitterionic ligand such as S1P (Fig. 2, A and B). The phosphate moiety and amine moiety of d18:1 S1P are surrounded by polar residues, whereas the alkyl tail is surrounded by hydrophobic residues (Fig. 2, A and B). The phosphate moiety of d18:1 S1P forms hydrogen bonds with Tyr22 N-term and Thr103 ECL1 and a salt bridge with Arg114 3.28 [superscripts refer to Ballesteros-Weinstein numbering (26)]. The amine moiety forms hydrogen bonds with Glu115 3.29 and Asn95 2.60 . To verify the functional roles of these residues experimentally, we performed mutagenesis analyses using the transforming growth factor- (TGF) shedding assay, which reflects G q/11 and G 12/13 signaling. Alanine substitutions of Tyr22 N-term , Asn95 2.60 , Thr103 ECL1 , Arg114 3.28 , and Glu115 3.29 reduced d18:1 S1P potency (  ; and table S2). As previously reported, these polar residues are important for high-affinity interaction with zwitterionic S1P with reference to the sphingosine-mimic antagonistbound S1PR1 structure (22). Meanwhile, alanine substitutions of almost all residues around the alkyl tail of d18:1 S1P, except for Leu189 ECL2 and Ile284 7.39 , had little effect on d18:1 S1P affinity. These data suggest that polar interactions with the amine and phosphate moieties are more important for S1P binding than hydrophobic interaction with the alkyl tail ( Ligand length effects on G protein signaling As mentioned above, the difference between active S1PR3 and inactive S1PR1 is seen in the lower parts of the ligand-binding pocket that accommodate the alkyl tail of ligands, suggesting that the interaction of the ligands' alkyl tail with receptors decides ligand activity (Fig. 1, D and E). We thus hypothesized that the shorter lipidic tail of FTY720-P relative to d18:1 S1P is a determinant for G proteinsubtype bias (Fig. 3A). To test this, we profiled each G proteindependent S1PR3 signaling pathway using d16:1 S1P, which is an S1P analog with two fewer carbons in the alkyl chain than d18:1 S1P. To evaluate G protein coupling, we performed a GloSensor cyclic adenosine monophosphate (cAMP) accumulation assay to detect G i/o -dependent cAMP inhibition and a TGF shedding assay (27,28) to detect G q/11 -and G 12/13 -dependent signaling ( fig. S4A). The TGF shedding assay showed G q/11 -and G 12/13 -dependent a disintegrin and metalloprotease 17 (ADAM17)-induced~ metallopeptidase domain  Views of residues interacting with d18:1 S1P in the d18:1 S1P-S1PR3-Fab AS55 complex (A) with views rotated 90° counterclockwise (B). The ligand-binding residues in S1PR3 are shown as a licorice model in dark salmon. Hydrogen bonds are shown as black dashed lines. (C) Comparison of pEC 50 values of d18:1 S1P in TGF shedding assay with HEK293 parental cells expressing mutant and wild-type S1PR3. pEC 50 values of mutant S1PR3 N95A, R114A, and E115A were estimated to be very low according to the dose-response curve in fig. S4. ****P < 0.0001, mutant versus wild-type pEC 50 values according to one-way analysis of variance (ANOVA) and Dunnett's post test. (D) Schematic of the interaction between d18:1 S1P and S1PR3. Hydrogen bonds and salt bridges are shown as black dashed lines. Critical residues for d18:1 S1P binding according to mutation analyses are shown in blue boxes.
17-induced ectodomain shedding of alkaline phosphatase-fused TGF (AP-TGF). As expected, both FTY720-P and d16:1 S1P lowered the maximal response of G q/11 signaling by ~40% relative to d18:1 S1P. However, all compounds showed approximately equal maximal response via G 12/13 and G i/o signaling ( Fig. 3B; fig. S4, B to D; and table S3). These data demonstrated that FTY720-P and d16:1 S1P are G i/o -and G 12/13 -biased agonists for S1PR3 (Fig. 3C). A previously determined sphingolipid-mimic antagonist-bound S1PR1 structure and docking simulations revealed that increased pocket-volume requirements for agonists drive the transition from an inactive to active conformation (22). Combined with the results in the present study, these findings suggest that biased signaling by shorter ligands is driven by lower pocket-volume requirements relative to d18:1 S1P.
Numerous S1P receptor modulators have been developed using the sphingosine backbone (29,30). One of these analogs (VPC23019) has a chemical structure similar to that of FTY720-P, except for the position of the alkyl tail (VPC23019 has it in the meta-position of the phenyl ring, whereas FTY720-P has it in the para-position) ( Fig. 3A). Here, VPC23019 exhibited a lower potency in G 12/13 and G i/o signaling and no ability to initiate G q/11 signaling as compared to FTY720-P, suggesting that both length and chemical structure of hydrophobic tail moiety affect ligand activity ( fig. S4, B to D, and table S3).

S1PR-activation mechanism
Comparison of the agonist d18:1 S1P in S1PR3 with the antagonist ML056 in S1PR1 indicated that the alkyl tail of ligands strongly affects receptor activation (Fig. 4). The alkyl tail of d18:1 S1P exhibits an unbent conformation in active S1PR3 and is inserted into the gap between TM4 and TM5. In contrast, ML056 exhibits a bent conformation accommodated in the short hydrophobic pocket in S1PR1 (Fig. 4A). To accommodate d18:1 S1P in this conformation, four residues in S1PR3 (Leu122 3.36 , Trp256 6.48 , Phe204 5.47 , and Phe260 6.52 ) are positioned differently from their positions in the inactive S1PR1. The side chains of Leu122 3. 36   (B) Normalized G protein signaling efficacy. E max of each G protein subtype-specific signaling in response to d18:1 S1P was normalized to 100%. Each bar is colored as follows: G q/11 , yellow; G 12/13 , salmon; and G i/o , light blue. ****P < 0.0001, d18:1 S1P versus S1P analogs % G protein signaling according to two-way ANOVA followed by Dunnett's post test. (C) Schematic summary of biased agonism in S1PR3. Major S1P, d18:1 S1P, induces G q/11 -, G 12/13 -, and G i/o -dependent signaling via S1PR3. On the other hand, S1P analogs with a short lipidic tail, such as d16:1 S1P and FTY720-P, induce G 12/13 -and G i/o -biased signaling via S1PR3.
owing to steric hindrance with the flipped side chain of Leu122 3.36 (Fig. 4B). Although a similar structural rearrangement is observed in cannabinoid receptor 1 (CNR1) (11,23), Phe200 3.36 in CNR1 (Leu122 3.36 in S1PR3) is reportedly flipped by hydrophobic interaction with the dimethyl group of the agonist (fig. S5). The side chain of Phe204 5.47 is stabilized by a - interaction with Phe260 6.52 in the active S1PR3 structure, whereas in the inactive S1PR1 structure, Phe 5.47 plugs the gap between TM4 and TM5 (Fig. 4B). The Phe204 5.47 -Phe260 6.52 interaction in S1PR3 opens the long and wide gap between TM4 and TM5 connecting to the membrane, which allows for the accommodation of S1P analogs with various alkyl tail lengths (31) (fig. S6, A and B).
During the conformational change of the four residues upon the activation of receptor, TM6 is displaced outward by ~11 Å, and a partially unwound TM7 shifts inward by ~2.8 Å (Fig. 4C). Rearrangements of conserved activation motifs (P-I-F, sodium-binding site, D/E-R-Y, and NPxxY) within class A GPCRs are also similar to previously reported active GPCR structures ( fig. S6, C to G) (32)(33)(34). Consistent with this model, alanine substitution of these four residues, except for Phe204 5.47 , reduced maximal response to d18:1 S1P relative to that on wild-type S1PR3 (Fig. 4D). As these four residues concertedly alter their side-chain position to form an active conformation for G protein coupling, we referred to them as the "quartet core." The role of the quartet core for G protein selectivity The quartet core is located in proximity to the alkyl tail of d18:1 S1P, suggesting a possible role in lipid length-dependent G proteinsubtype bias. To reveal the residues involved in determining G i/oand G 12/13 -biased signaling, we performed a TGF shedding assay using G q/11 -and G 12/13 -deficient cells (G q/11 and G 12/13 ). Among the four residues of the quartet core, alanine substitution of Leu122 3.36 decreased only G q/11 -signaling efficacy and maintained equivalent G 12/13 -signaling efficacy in response to d18:1 S1P as compared with wild-type S1PR3 (Fig. 5, A to C, and table S4). This effect is similar to that caused by short lipidic agonists (FTY720-P and d16:1 S1P), ig. 4. S1PR-activation mechanism. (A) Superposition of the S1PR3 (dark salmon) and S1PR1 (3V2Y, dark gray) structures. Each molecule is colored as follows: S1PR3, dark salmon; S1PR1, light gray; d18:1 S1P, yellow; and ML056, light blue. (B) Comparison of residues comprising the quartet core in active S1PR3 and inactive S1PR1 structures. Extracellular view of S1PR3 superposed on S1PR1. ECL, extracellular loop. (C) Intracellular view of S1PR3 superposed on S1PR1. ICL, intracellular loop. (D) Comparison of maximal response (E max ) of d18:1 S1P in TGF shedding assay with HEK293 parental cells expressing mutant and wild-type S1PR3. **P < 0.01, ****P < 0.0001, mutant versus wild-type (WT) E max values according to one-way ANOVA and Dunnett's post test. n.s., not significant.
suggesting that the interaction between Leu122 3.36 and agonists is important for G q/11 signaling in S1PR3 (Fig. 3, A to C). In contrast, alanine substitution of both Trp256 6.48 and Phe260 6.52 decreased both G q/11 -and G 12/13 -signaling efficacy in response to d18:1 S1P, whereas alanine substitution of Phe204 5.47 showed little effect on both G q/11 -and G 12/13 -signaling efficacy (Fig. 5, A to C). These data suggest that Trp256 6.48 and Phe260 6.52 are responsible for S1PR3 forming an active conformation capable of coupling with all G q/11 , G i/o , and G 12/13 subfamilies and that Leu122 3.36 specifically plays an important role in G q/11 signaling (Fig. 5, A to C).

DISCUSSION
Recent advances in lipidomics analysis revealed that S1P analogs (d16:1, d17:1, d18:0, d18:2, and d20:1 in addition to d18:1 S1P) are present in mammalian blood plasma (35). Among these, the level of d16:1 S1P, which is normally present in very small amounts in physiological conditions, is reportedly increased under platinumbased chemotherapy and is associated with chemotherapy-induced peripheral neuropathy (36). The results of the present study revealed d16:1 S1P as a G i/o -and G 12/13 -biased agonist for S1PR3, whereas S1P analogs, which have a lipid tail with 18 or more carbons such as d18:0 S1P and d20:1 S1P, are balanced agonists (Fig. 3 and fig. S7). These findings suggest that modifying signaling bias offers a potential therapeutic strategy. Biased agonism in S1PRs is affected by various factors, including chaperone-dependent stability of S1P (37,38). Although our findings described structural mechanism of liganddependent biased agonism in S1PR3, the mechanism of chaperonerelated biased agonism is still unclear. Further research is required to enhance our understanding of biased agonism of S1PRs in a biological system. Among ~800 GPCRs, at least 36 recognize lipids (39,40). Although these lipid GPCRs regulate important physiological and pharmacological functions, there is little information available on their   WT and mutant S1PR3 were examined for d18:1 S1P-induced AP-TGF shedding responses in the G 12/13 -knockout HEK293 cell line, reflecting G q/11 -mediated signaling. (B) WT and mutant S1PR3 were examined for d18:1 S1P-induced AP-TGF shedding responses in the G q/11 -knockout HEK293 cell line, reflecting G 12/13 -mediated signaling. Symbols and error bars in (A) and (B) represent mean and SEM, respectively, of three to six independent experiments with each performed in triplicate. Each line represents WT and mutant S1PR3 and is colored as follows: WT, gray; Leu122Ala, orange; Phe204Ala, light blue; Trp256Ala, pink; and Phe260Ala, green. (C) Bias plot of mutant and WT S1PR3 of maximal G q/11 -dependent % AP-TGF shedding versus maximal G 12/13 -dependent % AP-TGF shedding. Symbols and error bars represent mean of maximal response and SEM in TGF shedding assay with HEK293 G 12/13 or G q/11 cell lines. Leu122Ala mutant shows G 12/13 -biased activity. Phe204Ala mutant shows similar response to WT in both assays. Phe260Ala and Trp256Ala mutants show lower activity in both assays than WT. (D) Schematic models of S1PR3 activation and G protein-subtype bias. S1PR3 cannot couple to any G proteins in the antagonist-bound state (left). S1PR3 couples only to G 12/13 and G i/o upon binding to an agonist with a shorter lipidic chain, such as d16:1 S1P or FTY720-P (middle). S1PR3 couples with G q/11 , G i/o , and G 12/13 upon binding to the major natural ligand 18:1 S1P (right).
structure as compared with those for amine and peptide GPCRs (41). Previous studies provided structural insights into the activation mechanism of some lipid GPCRs. The agonist-bound structure of prostaglandin E receptor 3 (PTGER3) suggests that direct interaction between the agonist and Trp 6.48 is important for activation (24,25,42). Moreover, in cannabinoid receptors, Phe 3.36 -Trp 6.48 interaction plays a pivotal role in activation (11,12,23). In the present study, the d18:1 S1P-bound S1PR3 structure provided insight into a unique mechanism for lipid GPCR activation. In the active S1PR3 structure, d18:1 S1P dynamically rearranges four residues (termed the quartet core) in the lower part of the ligand-binding pocket, resulting in the formation of a long tunnel obliquely crossing S1PR3. Simultaneously, these rearrangements promote the active conformation, thereby explaining why S1PRs are activated by S1P analogs of varying lengths, even d20:1 S1P (31). There is, however, a caveat that the agonist-induced receptor activation mechanism dependent on the quartet core is specific for S1PR3 since we just compared the active S1PR3 structure and the inactive S1PR1 structure. To assess whether this proposed mechanism is correct and shared with other lipid GPCRs, further structural works on inactive conformations of S1PR3 and active conformations of other lipid GPCRs are required. We also revealed that Leu122 3.36 in S1PR3 plays an important role in G q/11 signaling. Short lipidic agonists lose the ability to interact with Leu122 3.36 , resulting in greater G 12/13 -and G i/o -mediated activation rather than G q/11 . Angiotensin II (AngII) receptors (AGTR1 and AGTR2) show a similar mechanism for weaker G q/11 signaling (43)(44)(45). In the antagonist-bound structure, Leu112 3.36 in AGTR1 (Leu122 in S1PR3) is near TM4, whereas the balanced agonist AngII changes the side-chain orientation of Leu112 3.36 from being near TM4 to TM7 ( fig. S8A). However, the biased agonist TRV026 induces a distinct active conformation, including orientation of the Leu112 3.36 side chain in the proximal TM4 region, similar to that observed in the antagonist-bound structure (fig. S8A). These observations suggest that, in S1PR3, Leu122 3.36 is similarly involved in biased signaling (Fig. 5D). In many G q/11 -coupling class A GPCRs, the residue corresponding to Leu122 3.36 in S1PR3 is a conserved short aliphatic residue, such as Leu, Met, or Val, suggesting that G q/11 -coupling GPCRs share a similar mechanism underlying liganddependent biased signaling ( fig. S8B). The findings in the present study can potentially promote the design of G protein subtypebiased agonists for optimized therapeutics targeting GPCRs.

Expression construct amino acid sequence
The sequence cloned into pFastBac1 vector (Thermo Fisher Scientific) encoded Homo sapiens S1PR3 residues 2 to 315 of the total 378 residues (UniProt accession: Q99500), which are underlined in the sequence provided below. Hemagglutinin (HA) signal sequences and the FLAG epitope sequence on the N terminus, and enhanced green fluorescent protein sequence and 8 × poly-histidine tag on the C terminus of the receptor were retained after the tobacco etch virus (TEV) cleavage site. For removal of glycosylation, the Asn15Gln mutation was induced, which is in italics in the sequence provided below. Details of the cloning procedure are included in the following section. KTIIALSYIFCLVFADYKDDDDKENLYFQGTSATALP-PRLQPVRGQETLREHYQYVGKLAGRLKEASEGSTLTTVLFL-

Fab light chain
The sequence of the Fab AS55 fragment light chain is provided below. The complementarity-determining regions (CDRs) CDR-L1, CDR-L2, and CDR-L3 are underlined. The residues interacting with S1PR3 are shown in bold ( fig. S2).

Fab heavy chain
The sequence of the Fab AS55 fragment heavy chain is provided below. CDR-H1, CDR-H2, and CDR-H3 are underlined. The residues interacting with S1PR3 are shown in bold ( fig. S2).

Preparation of monoclonal antibody AS55
All animal experiments described in this study conformed to the guidelines outlined in the Guide for the Care and Use of Laboratory Animals of Japan and were approved by the Kyoto University Animal Experimentation Committee (approval no. Medkyo18068). S1PR3 (2-330) was used to prepare the monoclonal anti-S1PR3 antibody (AS series). Construct design, expression, and purification were performed according to the protocol described above. Purified S1PR3 was reconstructed in liposomes with a ratio of 20:1 egg phosphatidylcholine (Sigma-Aldrich) to monophosphoryl lipid A (Sigma-Aldrich). MRL/lpr mice were immunized with liposomal S1PR3. Spleen cells were collected from the immunized mice and fused with NS-1 myeloma cells. Hybridoma cell-produced antibodies were screened using liposome enzyme-linked immunosorbent assay, followed by fluorescence SEC and flow cytometry analysis. Monoclonal hit clones were obtained by limiting dilution. Last, we isolated four clones of S1PR3-selective antibodies, including the crystallizing agent AS55. For the crystallization trial, a large number of monoclonal antibodies were prepared from the ascites of nude mice. Monoclonal antibodies were initially purified with protein G resin, eluted with glycine buffer, digested with papain, immobilized on N-hydroxysuccinimide-Sepharose, and incubated for 3 hours at 37°C. Then, the digested immunoglobulin G (IgG) samples were subjected to SEC (Hiload 16/600 Superdex 200 pg). The fraction of Fab fragments was incubated with protein A to remove either the Fc fragment or the residual IgG.

Liposome enzyme-linked immunosorbent assay
Purified S1PR3 (2-330) was reconstituted into liposomes containing biotinylated phosphoethanolamine (no. 860562, Avanti Polar Lipids Inc.) and immobilized onto streptavidin-coated plates (no. 436014, Thermo Fisher Scientific). The supernatant of hybridoma cells was added to the plates and mixed with a microplate mixed at 4°C for 1 hour. After washing with PBS containing 1% bovine serum albumin (PBS-B) three times, horseradish peroxidase-conjugated goat antimouse IgG, Fc fragment-specific antibody (no. 115-035-071, Jackson Immune Research, West Grove, PA, USA) was added and mixed with a microplate mixer at 4°C for 1 hour. After washing with PBS-B three times, KPL SureBlue TMB (3,3′,5,5′-tetramethylbenzidine) microwell peroxidase substrate was added. After incubation at room temperature, the reaction was stopped with 2 M H 2 SO 4 . Each well was evaluated by reading the absorbance at 450 nm. To eliminate antibodies recognizing flexible loops, N and C termini, or unstructured regions of S1PR3, we performed the same experiments using S1PR3 (2-330) denatured with 1% sodium dodecyl sulfate.

Crystallization
The protein complex was prepared by incubating S1PR3 with Fab AS55 at a molar ratio of 1:2 for 1.5 hours at 4°C. The protein solution was subjected to SEC (Superdex 200 Increase 10/300 GL, GE Healthcare). Two peak fractions containing the S1PR3-Fab AS55 complex and monomeric Fab AS55 were obtained and mixed again; the final ratio of S1PR3 to Fab AS55 was 1:3. This step was required for better crystal quality for sufficient diffraction. The mixed complex was concentrated to 20 mg/ml by ultrafiltration (Amicon Ultra-0.5 50 K, Millipore), and d18:1 S1P was added to a final concentration of 400 M. Complex and host lipids (monoolein and cholesterol) were mixed at a protein-to-lipid ratio of 2:3 using a mixer that consisted of two 100-l gas-tight syringes (Hamilton Company). The syringe containing the reconstituted LCP was loaded onto an automated crystallization robot (NT-8-LCP, Formulatrix), and 50 nl was dispensed onto 96-well glass sandwich crystallization plates (LD11-50, Molecular Dimensions), which were subsequently overlaid with 800 nl of reservoir solution. Diffraction-quality crystals of S1PR3-Fab AS55 were obtained under conditions of 36% polyethylene glycol (PEG) 400, 0.1 M tris-HCl (pH 8.0), and 80 mM lithium citrate tribasic. Crystals were collected within 3 weeks using mesh grid loops (MiTeGen) and flash-cooled in liquid nitrogen.

Structure determination
Diffraction data were collected at 100 K at the SPring-8 microfocus beamline BL32XU (Japan) (46), using an Eiger X 9M detector (DECTRIS). A microfocused beam with a size of 15 m by 10 m and a wavelength of 1 Å was used for both raster scan and data collection. A dataset with a total oscillation range of 10° and 0.1° oscillations per frame was collected from each crystal under the absorbed dose of 10 MGy. A total of 481 collected datasets with the automated data collection system ZOO (47) were merged, integrated, and scaled using the KAMO system (48), which exploits BLEND (49), XDS, and XSCALE (50,51). The structure of the S1PR3-Fab AS55 complex was determined by molecular replacement with the program PHASER (52) using the atomic coordinates of Fab 4A03 [Protein Data Bank (PDB) ID 5XLI)] and S1PR1 (PDB ID 3V2Y) as the search model. The model was initially refined using the "jerry body" refinement function implemented in REFMAC5 (53). The reflection data with phase information from molecule replacement undergo the density modification and noncrystallographic symmetry averaging by DM and phenix.density_modification. The structure is rebuilt and refined by phenix.autobuild and Rosetta in the beginning. Simulated annealing is applied in the initial refinement to reduce model bias. Most of the following structure refinement is conducted by phenix. real_space_refine with calculated weighted 2F obs − F calc maps. The sharpened map is produced by phenix.auto_sharpen for guiding manual refinement in COOT. At the tail of the final refinement process, we use riding hydrogen for geometry regularization to reduce clash and outlier. Ligand d18:1 S1P has also been restricted with tight angle and torsion angle restrain to maintain reasonable geometry against relatively weak density map (see table S2). Refined structures were visualized with CueMol: Molecular Visualization Framework (http://cuemol.org/). The C root mean square deviation values of the degree of shifts of helices were calculated by UCSF Chimera (54). C was defined as follows: TM6 as 6.29 to 30 and 32 to 33 and TM7 as 7.49 to 7.53 in Ballesteros-Weinstein numbering (26).

TGF shedding assay
For the S1PR3 receptor activity study, the gene encoding human S1PR3, which was codon-optimized to improve the expression level, was cloned into the pCAGGS expression vector. The signal sequence of influenza HA followed by a FLAG epitope tag was attached to the N terminus of S1PR3. Each point mutation was introduced into S1PR3 by site-directed mutagenesis by whole-plasmid polymerase chain reaction.
The TGF shedding assay was performed as described previously (27). In brief, HEK293 G q/11 , G 12/13 , G q/11/12/13 (28), or parental cells were seeded in a six-well plate at a density of 3 × 10 5 cells per well (for parental and G q/11 ) or 5 × 10 5 cells per well (for G 12/13 and G q/11/12/13 ) and cultured for 24 hours at 37°C with 2 ml of low-glucose DMEM supplemented with 10% charcoal-stripped FBS [(CS-FBS), Invitrogen], penicillin (100 U/ml), and streptomycin (100 g/ml) (Nacalai Tesque). The cells were transfected with a mixture of expression plasmid vectors encoding alkaline phosphatase (AP)-TGF (625 ng per well in six-well plates hereafter, unless otherwise noted) and S1PR3 (250 ng per well), using 2.5 l of Lipofectamine 3000 reagent, 1.75 l of P3000 reagent (Thermo Fisher Scientific), and 250 l of Opti-MEM I reduced serum medium (Thermo Fisher Scientific) according to the manufacturer's instructions. For the negative control, the AP-TGF plasmid and the empty pCAGGS plasmid vector were transfected. After 24 hours of incubation, the cells were dissociated by TrypLE Express Enzyme (Thermo Fisher Scientific), neutralized with 10% CS-FBS containing DMEM, collected in a 15-ml tube, centrifuged, and resuspended in 8 ml of Hanks' balanced salt solution (HBSS, Nacalai Tesque) with Ca 2+ and Mg 2+ containing 5 mM Hepes at pH 7.4. After 10 min of incubation at room temperature to settle the spontaneous AP-TGF release caused by trypsinization, the cells were centrifuged and resuspended in 10 ml of HBSS with Hepes. The cell suspension was seeded in a 96-well culture plate at a volume of 90 l per well. After 30 min of preincubation in a CO 2 incubator, each ligand, diluted in HBSS with Hepes supplemented with 0.01% lipid-free bovine serum albumin (Wako), was added, and the plates were incubated at 37°C in the CO 2 incubator. The plates were centrifuged at 190g for 2 min, and 80 l of supernatant was transferred to an empty 96-well conditioned media (CM) plate. The alkaline phosphatase reaction solution [10 mM p-nitrophenyl phosphate (p-NPP, Wako), 120 mM tris-HCl (pH 9.5), 40 mM NaCl, and 10 mM MgCl 2 ] was dispensed into the cell plates and the CM plates (80 l per well). The absorbance at 405 nm (A 405 ) of the plates was measured using a microplate reader (ARVO X5, PerkinElmer), before and after a 1-hour incubation at room temperature. For each well measurement, the increase in the A 405 over the 1-hour incubation with p-NPP (∆A 405 ) was used to determine alkaline phosphatase activity, and the value from the CM plate was normalized by the total ∆A 405 . AP-TGF release was calculated by subtracting the spontaneous AP-TGF accumulation under the vehicle-treated conditions from that in the compoundstimulated conditions.

GloSensor cAMP assay
The plasmid encoding human S1PR3 was the same as in TGF shedding assay. pGloSensor 22F plasmid DNA and cAMP reagent were purchased from Promega.
HEK293 cells were seeded in a 100-mm tissue culture dish at a density of 1.5 × 10 6 cells per dish and cultured for 24 hours at 37°C with 10 ml of low-glucose DMEM supplemented with 10% CS-FBS (Invitrogen), penicillin (100 U/ml), and streptomycin (100 g/ml) (Nacalai Tesque). The cells were transfected with a mixture of pGloSensor cAMP 22F plasmid (8 g per dish) and expression plasmid vectors encoding S1PR3 (2 g per dish) using 30 l of Fugene HD transfection kit (Promega) and 650 l of Opti-MEM I reduced serum medium (Thermo Fisher Scientific) according to the manufacturer's instructions. The negative control, the pGloSensor cAMP 22F plasmid, and the empty pCAGGS plasmid vector were transfected. After 24 hours of incubation, the cells were dissociated by TrypLE Express Enzyme (Thermo Fisher Scientific), neutralized with 1% CS-FBS containing CO 2 -independent medium (Invitrogen), collected in a 15-ml tube, centrifuged, and washed with 5 ml PBS (−). Then, cells were resuspended in 1% CS-FBS and 2% GloSensor cAMP reagent containing CO 2 -independent medium at 5 × 10 5 cells/ml and rotated at room temperature for 2 hours. Cell suspension was seeded to LUMITRAC 96-well white plate (Greiner) at a volume of 80 l per well; 10× agonist diluted in HBSS with Hepes supplemented with 0.01% lipid-free bovine serum albumin (Wako) was added, and the plates were incubated at room temperature for 10 min. Ten microliters of 100 M forskolin diluted in HBSS with Hepes supplemented with 0.01% lipid-free bovine serum albumin (Wako) was added to each well (final, 10 M), and plates were incubated for 30 min at room temperature. The luminescence of the plates was measured using a microplate reader (ARVO X5, PerkinElmer). The count of each well was normalized to that of wells treated with forskolin alone as 100%.

Statistical analysis
Concentration-response curves expressed in the TGF shedding assay were fit to a nonlinear regression (four-parameter) model in Prism (v.8.30, GraphPad Software Inc.). For functional analysis of wild-type and S1PR3 mutants, pEC 50 and E max values were calculated from nonlinear regression (four-parameter) analysis of mean data from independent experiments performed in triplicate. Error bars denote SEM.

SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/7/24/eabf5325/DC1 View/request a protocol for this paper from Bio-protocol.