{"gene":"GPR174","run_date":"2026-06-10T01:55:21","timeline":{"discoveries":[{"year":2012,"finding":"GPR174 expression in CHO cells constitutively elevates intracellular cAMP, and LysoPS stimulation further increases cAMP and phosphorylates Erk; these responses are blocked by NF449, an inhibitor of Gαs protein, establishing GPR174 as a LysoPS receptor that couples to Gαs/adenylyl cyclase signaling.","method":"Stable expression in CHO cells, intracellular cAMP measurement, cholera toxin comparison, lipid/nucleotide ligand screen, Erk phosphorylation assay, pharmacological inhibition with NF449","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional cell-based assay with pharmacological validation (NF449) and multiple readouts (cAMP, Erk), single lab","pmids":["23178570"],"is_preprint":false},{"year":2015,"finding":"GPR174 functions as a LysoPS receptor that constrains regulatory T cell (Treg) generation in the thymus and reduces the fraction of CD103+ peripheral Tregs; LysoPS acting via GPR174 suppresses T cell proliferation and Treg generation in vitro, and GPR174 deficiency reduces susceptibility to experimental autoimmune encephalomyelitis.","method":"Gpr174 knockout mice (X-linked gene deletion), in vitro T cell proliferation assay with LysoPS, flow cytometry of thymic/peripheral Treg populations, in vivo EAE model, genetic complementation with Treg-specific deletion","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal in vitro and in vivo KO experiments with defined cellular phenotypes, multiple orthogonal readouts, replicated across immune contexts","pmids":["26077720"],"is_preprint":false},{"year":2015,"finding":"Structure-activity relationship analysis of synthetic LysoPS analogues identified structural requirements of the fatty acid, glycerol, and L-serine modules for potency and receptor subtype selectivity at GPR174 (LPS3), yielding potent and selective GPR174 agonists.","method":"Synthetic chemistry, SAR analysis of LysoPS analogues in receptor activation assays","journal":"Journal of medicinal chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic SAR with multiple analogues across receptor subtypes, single lab","pmids":["25970039"],"is_preprint":false},{"year":2017,"finding":"GPR174 (LPS3) mediates LysoPS-induced suppression of IL-2 production in CD4+ T cells at both mRNA and protein levels; LysoPS-stimulated suppression is absent in LPS3-deficient splenocytes and CD4+ T cells, and LysoPS with various fatty acids is upregulated upon T cell activation.","method":"Gpr174 (LPS3) knockout mice, anti-CD3/anti-CD28 T cell activation assay, ELISA and RT-PCR for IL-2, lipid profiling of activated T cells","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with defined molecular phenotype (IL-2 suppression) and two orthogonal readouts (mRNA/protein), single lab","pmids":["29017923"],"is_preprint":false},{"year":2018,"finding":"LysoPS/GPR174-mediated suppression of T cell activation requires Gαs proteins; mechanistically, GPR174 signals through Gαs to suppress IL-2 production and limit upregulation of activation markers CD25 and CD69 on naive T cells in vitro and in vivo.","method":"In vivo T cell proliferation models (sublethal irradiation, Treg depletion), Gαs inhibition experiments in vitro, CD25/CD69 flow cytometry, IL-2 measurement, Gpr174 KO comparison","journal":"Immunology and cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — both in vivo and in vitro models, Gαs mechanistic dissection, multiple orthogonal readouts, replicates findings from prior lab with added mechanistic depth","pmids":["29457279"],"is_preprint":false},{"year":2019,"finding":"GPR174, encoded on the X chromosome, acts as a receptor for CCL21 in B cells; CCL21 triggers calcium flux and preferentially induces migration of male B cells via GPR174, which associates more with Gαi protein in male than female B cells. Testosterone drives this sex difference: orchidectomy impairs GPR174-mediated CCL21 migration and testosterone treatment of female B cells confers male-like GPR174-Gαi association and migration. GPR174 suppresses germinal centre formation in males by positioning B cells towards the T-B border rather than follicular centre.","method":"Biochemical fractionation of conditioned media to identify CCL21 ligand, calcium flux assay, B cell migration assay, Co-IP of GPR174 with Gαi, orchidectomy and testosterone treatment models, Gpr174 conditional KO in B cells, germinal centre analysis by flow cytometry and histology, EAE model","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (ligand identification, calcium flux, migration assay, Co-IP, genetic KO, in vivo models), mechanistic dissection of sex-dependent signaling","pmids":["31875850"],"is_preprint":false},{"year":2019,"finding":"GPR174 deficiency in Tregs promotes CTLA-4 and IL-10 expression in Tregs, which drives anti-inflammatory M2 macrophage polarization and protects against sepsis-induced lung damage; Gpr174-deficient Tregs also directly suppress pro-inflammatory cytokines (IL-6, TNF-α) in macrophages in vitro.","method":"Gpr174 KO mice, LPS-induced sepsis and CLP models, flow cytometry, ELISA, in vitro Treg–macrophage co-culture assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KO model with cellular mechanistic readout (M2 polarization), in vitro validation, single lab","pmids":["30850582"],"is_preprint":false},{"year":2022,"finding":"GPR174 in Tregs negatively regulates angiogenesis after ischemic injury; GPR174 deficiency upregulates amphiregulin (AREG) expression in Tregs by inhibiting nuclear accumulation of EGR1 via the Gαs/cAMP/PKA signaling pathway, thereby enhancing endothelial cell function and reducing pro-inflammatory macrophage polarization.","method":"Treg-specific Gpr174 KO mice, hindlimb ischemia model, blood flow recovery measurement, AREG overexpression/KD, EGR1 nuclear translocation assay, cAMP/PKA pathway inhibition","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KO with defined cellular mechanism (Gαs/cAMP/PKA/EGR1/AREG axis), multiple pathway readouts, single lab","pmids":["36473866"],"is_preprint":false},{"year":2023,"finding":"Cryo-EM structure of LysoPS-bound human GPR174 in complex with Gs protein reveals: (1) negatively charged LysoPS head group forms extensive polar interactions with ligand binding pocket residues; (2) the L-serine moiety inserts deeply into a positively charged cavity; (3) a partially open pocket on TM4–TM5 allows lateral ligand entry from the membrane; (4) Gs coupling involves deep insertion of the αH5 helix with extensive polar interactions.","method":"Cryo-EM structure determination, structural analysis, functional mutagenesis (implied by ligand binding pocket characterization)","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic-resolution cryo-EM structure with structural-functional analysis of ligand recognition and G protein coupling mode","pmids":["36823105"],"is_preprint":false},{"year":2023,"finding":"Cryo-EM structures of GPR174 without exogenous ligand reveal endogenous LysoPS co-purified with the receptor, conferring high constitutive Gs signaling comparable to fully activated D1R; GPR174 mutants with reduced ligand-binding affinity can be dose-dependently activated by exogenous LysoPS (but not other lipids), confirming LysoPS as the specific endogenous ligand. GPR174 adopts a non-canonical Gs coupling mode.","method":"Cryo-EM structure determination of GPR174-Gs complex without exogenous ligand, cAMP assay comparing GPR174 to D1R, site-directed mutagenesis of ligand-binding residues, LysoPS dose-response in GPR174 mutants, lipid specificity assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure plus mutagenesis plus functional assays in one rigorous study, definitively establishes endogenous ligand and constitutive activation mechanism","pmids":["37737235"],"is_preprint":false},{"year":2023,"finding":"Cryo-EM structures of human GPR34 and GPR174 in complex with LysoPS and G protein elucidate the lipid-binding modes and structural features of these receptors in the active state, providing comparative insights into LysoPS receptor ligand recognition.","method":"Cryo-EM structure determination, structural analysis and comparison, functional studies of ligand binding","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure with functional validation, single lab but orthogonal to other structural studies confirming findings","pmids":["38048360"],"is_preprint":false},{"year":2025,"finding":"The LysoPS/GPR174 axis drives ESCC metastasis via the cAMP-PKA-CREB signaling pathway: LysoPS upregulates GPR174 expression, which activates cAMP-PKA, causing the catalytic subunit of PKA to translocate into the nucleus and phosphorylate CREB, promoting invasion and metastasis in vitro and in vivo.","method":"LC-MS for LysoPS measurement, Western blotting, wound healing and Transwell migration assays, nuclear-cytoplasmic fractionation, CCK-8 proliferation assay, nude mouse metastasis model, GPR174 overexpression","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo mechanistic pathway dissection with nuclear fractionation, single lab, multiple assays","pmids":["40229851"],"is_preprint":false},{"year":2026,"finding":"High-resolution cryo-EM structures (2.0 Å) of LysoPS-activated GPR174 bound to Gs and Gi reveal a continuous hydration-mediated signal transduction network bridging the sodium-binding pocket, NPxxY and DRY motifs, and G protein-binding interface. This water network stabilizes the active state and differentially reshapes the intracellular cavity to enable selective engagement of Gs versus Gi. Molecular dynamics and functional assays confirm the hydration network is essential for activation and G protein selectivity.","method":"Cryo-EM structure determination (2.0 Å GPR174-Gs, 3.4 Å GPR174-Gi), molecular dynamics simulations, functional assays (cAMP/G protein coupling), sequence alignment across class A GPCRs","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structures for both Gs and Gi complexes with MD simulations and functional validation, multiple orthogonal methods","pmids":["42096402"],"is_preprint":false},{"year":2025,"finding":"Molecular dynamics simulations of GPR174 bound to an antagonist (mPS, modified LysoPS) versus LysoPS show that mPS inactivates GPR174 by reducing conformational dynamics, disrupting the PIF, DRY, and N/DPxxY conserved activation motifs, and blocking signal transduction to the G protein interface, whereas LysoPS engages these motifs to couple the binding site to Gs. Membrane lipids including PIP2 modulate ligand dynamics and receptor conformational states.","method":"Molecular dynamics simulations in heterogeneous lipid bilayer, network analysis of signaling pathways, protein-lipid interaction analysis","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational study only (MD simulations), preprint, no experimental functional validation reported","pmids":["41000684"],"is_preprint":true}],"current_model":"GPR174 is an X-chromosome-encoded class A GPCR that is constitutively activated by its endogenous lipid ligand lysophosphatidylserine (LysoPS), coupling primarily to Gαs (elevating cAMP via adenylyl cyclase) and also to Gαi; cryo-EM structures reveal LysoPS recognition through polar interactions with a positively charged binding pocket and lateral membrane entry via a TM4–TM5 portal, while a hydration network through the sodium pocket, DRY/NPxxY motifs, and intracellular cavity governs activation and differential Gs/Gi selectivity; in immune cells GPR174 suppresses IL-2 production, constrains regulatory T cell generation, and—in a testosterone-dependent manner—limits germinal centre formation by positioning male B cells toward the T-B border via CCL21-induced, Gαi-associated migration, collectively imparting sexual dimorphism to humoral immunity and modulating autoimmune susceptibility."},"narrative":{"mechanistic_narrative":"GPR174 is an X-chromosome-encoded class A GPCR that functions as a lysophosphatidylserine (LysoPS) receptor governing immune cell activation, with a strong role in adaptive immunity [PMID:26077720, PMID:29017923]. Upon LysoPS engagement it couples to Gαs and elevates intracellular cAMP, and cryo-EM structures show that the negatively charged LysoPS head group makes polar contacts within a positively charged pocket while the receptor accepts ligand laterally through a partially open TM4–TM5 portal in the membrane; co-purified endogenous LysoPS confers high constitutive Gs signaling, and a hydration-mediated network through the sodium pocket and the DRY/NPxxY motifs governs activation and selective engagement of Gs versus Gi [PMID:36823105, PMID:37737235, PMID:42096402]. In CD4+ T cells GPR174 signaling suppresses IL-2 production and limits activation-marker upregulation in a Gαs-dependent manner, constrains regulatory T cell generation, and its deficiency reduces susceptibility to experimental autoimmune encephalomyelitis [PMID:26077720, PMID:29017923, PMID:29457279]. GPR174 also acts as a receptor for CCL21 in B cells, where a testosterone-driven enhancement of GPR174–Gαi association preferentially promotes male B cell migration toward the T–B border and suppresses germinal centre formation, imparting sexual dimorphism to humoral immunity [PMID:31875850]. In regulatory T cells, the Gαs/cAMP/PKA axis links GPR174 to downstream transcriptional control of CTLA-4, IL-10, and amphiregulin, shaping macrophage polarization and tissue responses to injury [PMID:30850582, PMID:36473866].","teleology":[{"year":2012,"claim":"Established the molecular identity and signaling output of GPR174 by showing it is a LysoPS receptor with constitutive and ligand-stimulated cAMP elevation through Gαs.","evidence":"Stable expression in CHO cells with cAMP measurement, ligand screen, Erk phosphorylation, and NF449 Gαs inhibition","pmids":["23178570"],"confidence":"Medium","gaps":["Single cell-based system without structural detail","Physiological cell type and in vivo relevance not addressed"]},{"year":2015,"claim":"Connected GPR174 signaling to an immunological role by showing LysoPS via GPR174 constrains Treg generation and modulates autoimmune susceptibility.","evidence":"Gpr174 knockout mice, in vitro T cell proliferation/Treg assays, and EAE model","pmids":["26077720"],"confidence":"High","gaps":["Downstream transcriptional effectors of Treg suppression not yet defined","G protein dependence of the Treg phenotype not dissected here"]},{"year":2015,"claim":"Defined the structure-activity requirements of LysoPS for GPR174 potency and selectivity, enabling chemical tools.","evidence":"Synthetic chemistry and SAR of LysoPS analogues across receptor subtypes","pmids":["25970039"],"confidence":"Medium","gaps":["Structural basis of selectivity not resolved at this stage","No in vivo validation of agonists"]},{"year":2017,"claim":"Identified IL-2 suppression as a concrete molecular output of GPR174 in CD4+ T cells and linked it to activation-induced LysoPS.","evidence":"Gpr174 KO splenocytes/T cells, anti-CD3/CD28 activation, IL-2 ELISA and RT-PCR, lipid profiling","pmids":["29017923"],"confidence":"Medium","gaps":["G protein coupling responsible for IL-2 suppression not yet pinned down","Single lab"]},{"year":2018,"claim":"Established that GPR174-mediated T cell suppression operates through Gαs, tying the signaling axis to defined activation phenotypes in vivo.","evidence":"In vivo T cell proliferation models, Gαs inhibition in vitro, CD25/CD69 flow cytometry, Gpr174 KO comparison","pmids":["29457279"],"confidence":"High","gaps":["Did not address Gαi contributions later seen in B cells","Transcriptional targets downstream of cAMP not mapped"]},{"year":2019,"claim":"Revealed a second ligand and a sex-dimorphic mechanism: GPR174 responds to CCL21 and, through testosterone-enhanced Gαi coupling, directs male B cell positioning and suppresses germinal centre formation.","evidence":"Ligand identification by fractionation, calcium flux, migration assays, GPR174–Gαi Co-IP, orchidectomy/testosterone models, conditional B cell KO, germinal centre analysis","pmids":["31875850"],"confidence":"High","gaps":["Reconciliation of CCL21 versus LysoPS ligand usage not fully resolved","Mechanism of testosterone-driven Gαi bias unknown"]},{"year":2019,"claim":"Extended GPR174 function in Tregs to control of CTLA-4/IL-10 expression and macrophage polarization, linking the receptor to inflammatory tissue outcomes.","evidence":"Gpr174 KO mice, LPS sepsis and CLP models, Treg–macrophage co-culture, flow cytometry and ELISA","pmids":["30850582"],"confidence":"Medium","gaps":["Direct signaling pathway linking GPR174 loss to CTLA-4/IL-10 not dissected here","Single lab"]},{"year":2022,"claim":"Mapped a downstream transcriptional axis (Gαs/cAMP/PKA/EGR1/AREG) through which GPR174 in Tregs regulates angiogenesis and macrophage polarization.","evidence":"Treg-specific Gpr174 KO mice, hindlimb ischemia, AREG manipulation, EGR1 nuclear translocation, cAMP/PKA inhibition","pmids":["36473866"],"confidence":"Medium","gaps":["Direct molecular link between PKA and EGR1 nuclear exclusion not fully resolved","Single lab"]},{"year":2023,"claim":"Provided the atomic basis for LysoPS recognition and Gs coupling, including lateral membrane ligand entry and confirmation of LysoPS as the constitutively bound endogenous ligand driving high basal signaling.","evidence":"Cryo-EM of LysoPS-bound and ligand-free GPR174-Gs complexes, mutagenesis, cAMP assays versus D1R, lipid specificity tests","pmids":["36823105","37737235","38048360"],"confidence":"High","gaps":["Gi-bound conformation not resolved in these structures","Structural basis of constitutive activity versus regulated signaling not fully separated"]},{"year":2025,"claim":"Linked the LysoPS/GPR174 cAMP-PKA-CREB axis to a disease setting by showing it drives ESCC invasion and metastasis.","evidence":"LC-MS LysoPS measurement, migration/invasion assays, nuclear-cytoplasmic fractionation, nude mouse metastasis model, GPR174 overexpression","pmids":["40229851"],"confidence":"Medium","gaps":["Causal requirement of endogenous GPR174 (vs overexpression) in patient tumors not established","Single lab"]},{"year":2026,"claim":"Resolved the mechanistic basis of Gs versus Gi selectivity by defining a continuous hydration network bridging the sodium pocket, conserved activation motifs, and the G protein interface.","evidence":"High-resolution cryo-EM of GPR174-Gs and GPR174-Gi complexes, molecular dynamics, and functional coupling assays","pmids":["42096402"],"confidence":"High","gaps":["How physiological cues bias cells toward Gs versus Gi engagement not addressed","Link between hydration-network mutations and immune phenotypes untested"]},{"year":null,"claim":"How GPR174 integrates two ligands (LysoPS and CCL21) and switches between Gαs and Gαi outputs to produce context- and sex-specific immune outcomes remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["Mechanism of ligand-dependent G protein switching in native cells unknown","Molecular basis of testosterone-driven Gαi bias undefined","Relative physiological contribution of constitutive versus ligand-driven signaling unclear"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,8,9]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[8,9]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[5]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[8,9]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,4,12]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,3,5]}],"complexes":[],"partners":["GNAS","GNAI","CCL21"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BXC1","full_name":"Probable G-protein coupled receptor 174","aliases":[],"length_aa":333,"mass_kda":38.5,"function":"G-protein-coupled receptor of lysophosphatidylserine (LysoPS) that plays different roles in immune response (PubMed:36823105). Plays a negative role in regulatory T-cell accumulation and homeostasis. Under inflammatory conditions where LysoPS production increases, contributes to the down-regulation of regulatory T-cell activity to favor effector response. Mediates the suppression of IL-2 production in activated T-lymphocytes leading to inhibition of growth, proliferation and differentiation of T-cells. Mechanistically, acts via G(s)-containing heterotrimeric G proteins to trigger elevated cyclic AMP levels and protein kinase A/PKA activity, which may in turn act to antagonize proximal TCR signaling. Plays an important role in the initial period of sepsis through the regulation of macrophage polarization and pro- and anti-inflammatory cytokine secretions. Upon testosterone treatment, acts as a receptor for CCL21 and subsequently triggers through G(q)-alpha and G(12)/G(13) proteins a calcium flux leading to chemotactic effects on activated B-cells. Signals via GNA13 and PKA to promote CD86 up-regulation by follicular B-cells","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q9BXC1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/GPR174","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/GPR174","total_profiled":1310},"omim":[{"mim_id":"602737","title":"CHEMOKINE, CC MOTIF, LIGAND 21; CCL21","url":"https://www.omim.org/entry/602737"},{"mim_id":"300903","title":"G PROTEIN-COUPLED RECEPTOR 174; GPR174","url":"https://www.omim.org/entry/300903"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Vesicles","reliability":"Additional"},{"location":"Centriolar satellite","reliability":"Additional"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"lymphoid tissue","ntpm":15.7}],"url":"https://www.proteinatlas.org/search/GPR174"},"hgnc":{"alias_symbol":["FKSG79","LPS3"],"prev_symbol":[]},"alphafold":{"accession":"Q9BXC1","domains":[{"cath_id":"1.20.1070.10","chopping":"12-237_283-302","consensus_level":"high","plddt":87.3057,"start":12,"end":302}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BXC1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BXC1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BXC1-F1-predicted_aligned_error_v6.png","plddt_mean":84.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=GPR174","jax_strain_url":"https://www.jax.org/strain/search?query=GPR174"},"sequence":{"accession":"Q9BXC1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BXC1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BXC1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BXC1"}},"corpus_meta":[{"pmid":"31875850","id":"PMC_31875850","title":"A GPR174-CCL21 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medicine","url":"https://pubmed.ncbi.nlm.nih.gov/40229851","citation_count":2,"is_preprint":false},{"pmid":"41000684","id":"PMC_41000684","title":"GPR174 Antagonism: Structure, Function, and Dynamics.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/41000684","citation_count":0,"is_preprint":false},{"pmid":"42096402","id":"PMC_42096402","title":"Structured water molecules drive activation and G protein selectivity in the GPR174 receptor.","date":"2026","source":"PLoS biology","url":"https://pubmed.ncbi.nlm.nih.gov/42096402","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.10.06.680656","title":"Hydration Network Drives Activation and G Protein Selectivity in GPR174","date":"2025-10-06","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.06.680656","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":14705,"output_tokens":3916,"usd":0.051428,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11801,"output_tokens":3783,"usd":0.07679,"stage2_stop_reason":"end_turn"},"total_usd":0.128218,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2012,\n      \"finding\": \"GPR174 expression in CHO cells constitutively elevates intracellular cAMP, and LysoPS stimulation further increases cAMP and phosphorylates Erk; these responses are blocked by NF449, an inhibitor of Gαs protein, establishing GPR174 as a LysoPS receptor that couples to Gαs/adenylyl cyclase signaling.\",\n      \"method\": \"Stable expression in CHO cells, intracellular cAMP measurement, cholera toxin comparison, lipid/nucleotide ligand screen, Erk phosphorylation assay, pharmacological inhibition with NF449\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional cell-based assay with pharmacological validation (NF449) and multiple readouts (cAMP, Erk), single lab\",\n      \"pmids\": [\"23178570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"GPR174 functions as a LysoPS receptor that constrains regulatory T cell (Treg) generation in the thymus and reduces the fraction of CD103+ peripheral Tregs; LysoPS acting via GPR174 suppresses T cell proliferation and Treg generation in vitro, and GPR174 deficiency reduces susceptibility to experimental autoimmune encephalomyelitis.\",\n      \"method\": \"Gpr174 knockout mice (X-linked gene deletion), in vitro T cell proliferation assay with LysoPS, flow cytometry of thymic/peripheral Treg populations, in vivo EAE model, genetic complementation with Treg-specific deletion\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal in vitro and in vivo KO experiments with defined cellular phenotypes, multiple orthogonal readouts, replicated across immune contexts\",\n      \"pmids\": [\"26077720\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Structure-activity relationship analysis of synthetic LysoPS analogues identified structural requirements of the fatty acid, glycerol, and L-serine modules for potency and receptor subtype selectivity at GPR174 (LPS3), yielding potent and selective GPR174 agonists.\",\n      \"method\": \"Synthetic chemistry, SAR analysis of LysoPS analogues in receptor activation assays\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic SAR with multiple analogues across receptor subtypes, single lab\",\n      \"pmids\": [\"25970039\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"GPR174 (LPS3) mediates LysoPS-induced suppression of IL-2 production in CD4+ T cells at both mRNA and protein levels; LysoPS-stimulated suppression is absent in LPS3-deficient splenocytes and CD4+ T cells, and LysoPS with various fatty acids is upregulated upon T cell activation.\",\n      \"method\": \"Gpr174 (LPS3) knockout mice, anti-CD3/anti-CD28 T cell activation assay, ELISA and RT-PCR for IL-2, lipid profiling of activated T cells\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with defined molecular phenotype (IL-2 suppression) and two orthogonal readouts (mRNA/protein), single lab\",\n      \"pmids\": [\"29017923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"LysoPS/GPR174-mediated suppression of T cell activation requires Gαs proteins; mechanistically, GPR174 signals through Gαs to suppress IL-2 production and limit upregulation of activation markers CD25 and CD69 on naive T cells in vitro and in vivo.\",\n      \"method\": \"In vivo T cell proliferation models (sublethal irradiation, Treg depletion), Gαs inhibition experiments in vitro, CD25/CD69 flow cytometry, IL-2 measurement, Gpr174 KO comparison\",\n      \"journal\": \"Immunology and cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — both in vivo and in vitro models, Gαs mechanistic dissection, multiple orthogonal readouts, replicates findings from prior lab with added mechanistic depth\",\n      \"pmids\": [\"29457279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"GPR174, encoded on the X chromosome, acts as a receptor for CCL21 in B cells; CCL21 triggers calcium flux and preferentially induces migration of male B cells via GPR174, which associates more with Gαi protein in male than female B cells. Testosterone drives this sex difference: orchidectomy impairs GPR174-mediated CCL21 migration and testosterone treatment of female B cells confers male-like GPR174-Gαi association and migration. GPR174 suppresses germinal centre formation in males by positioning B cells towards the T-B border rather than follicular centre.\",\n      \"method\": \"Biochemical fractionation of conditioned media to identify CCL21 ligand, calcium flux assay, B cell migration assay, Co-IP of GPR174 with Gαi, orchidectomy and testosterone treatment models, Gpr174 conditional KO in B cells, germinal centre analysis by flow cytometry and histology, EAE model\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (ligand identification, calcium flux, migration assay, Co-IP, genetic KO, in vivo models), mechanistic dissection of sex-dependent signaling\",\n      \"pmids\": [\"31875850\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"GPR174 deficiency in Tregs promotes CTLA-4 and IL-10 expression in Tregs, which drives anti-inflammatory M2 macrophage polarization and protects against sepsis-induced lung damage; Gpr174-deficient Tregs also directly suppress pro-inflammatory cytokines (IL-6, TNF-α) in macrophages in vitro.\",\n      \"method\": \"Gpr174 KO mice, LPS-induced sepsis and CLP models, flow cytometry, ELISA, in vitro Treg–macrophage co-culture assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO model with cellular mechanistic readout (M2 polarization), in vitro validation, single lab\",\n      \"pmids\": [\"30850582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"GPR174 in Tregs negatively regulates angiogenesis after ischemic injury; GPR174 deficiency upregulates amphiregulin (AREG) expression in Tregs by inhibiting nuclear accumulation of EGR1 via the Gαs/cAMP/PKA signaling pathway, thereby enhancing endothelial cell function and reducing pro-inflammatory macrophage polarization.\",\n      \"method\": \"Treg-specific Gpr174 KO mice, hindlimb ischemia model, blood flow recovery measurement, AREG overexpression/KD, EGR1 nuclear translocation assay, cAMP/PKA pathway inhibition\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO with defined cellular mechanism (Gαs/cAMP/PKA/EGR1/AREG axis), multiple pathway readouts, single lab\",\n      \"pmids\": [\"36473866\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cryo-EM structure of LysoPS-bound human GPR174 in complex with Gs protein reveals: (1) negatively charged LysoPS head group forms extensive polar interactions with ligand binding pocket residues; (2) the L-serine moiety inserts deeply into a positively charged cavity; (3) a partially open pocket on TM4–TM5 allows lateral ligand entry from the membrane; (4) Gs coupling involves deep insertion of the αH5 helix with extensive polar interactions.\",\n      \"method\": \"Cryo-EM structure determination, structural analysis, functional mutagenesis (implied by ligand binding pocket characterization)\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic-resolution cryo-EM structure with structural-functional analysis of ligand recognition and G protein coupling mode\",\n      \"pmids\": [\"36823105\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cryo-EM structures of GPR174 without exogenous ligand reveal endogenous LysoPS co-purified with the receptor, conferring high constitutive Gs signaling comparable to fully activated D1R; GPR174 mutants with reduced ligand-binding affinity can be dose-dependently activated by exogenous LysoPS (but not other lipids), confirming LysoPS as the specific endogenous ligand. GPR174 adopts a non-canonical Gs coupling mode.\",\n      \"method\": \"Cryo-EM structure determination of GPR174-Gs complex without exogenous ligand, cAMP assay comparing GPR174 to D1R, site-directed mutagenesis of ligand-binding residues, LysoPS dose-response in GPR174 mutants, lipid specificity assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure plus mutagenesis plus functional assays in one rigorous study, definitively establishes endogenous ligand and constitutive activation mechanism\",\n      \"pmids\": [\"37737235\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cryo-EM structures of human GPR34 and GPR174 in complex with LysoPS and G protein elucidate the lipid-binding modes and structural features of these receptors in the active state, providing comparative insights into LysoPS receptor ligand recognition.\",\n      \"method\": \"Cryo-EM structure determination, structural analysis and comparison, functional studies of ligand binding\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure with functional validation, single lab but orthogonal to other structural studies confirming findings\",\n      \"pmids\": [\"38048360\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The LysoPS/GPR174 axis drives ESCC metastasis via the cAMP-PKA-CREB signaling pathway: LysoPS upregulates GPR174 expression, which activates cAMP-PKA, causing the catalytic subunit of PKA to translocate into the nucleus and phosphorylate CREB, promoting invasion and metastasis in vitro and in vivo.\",\n      \"method\": \"LC-MS for LysoPS measurement, Western blotting, wound healing and Transwell migration assays, nuclear-cytoplasmic fractionation, CCK-8 proliferation assay, nude mouse metastasis model, GPR174 overexpression\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo mechanistic pathway dissection with nuclear fractionation, single lab, multiple assays\",\n      \"pmids\": [\"40229851\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"High-resolution cryo-EM structures (2.0 Å) of LysoPS-activated GPR174 bound to Gs and Gi reveal a continuous hydration-mediated signal transduction network bridging the sodium-binding pocket, NPxxY and DRY motifs, and G protein-binding interface. This water network stabilizes the active state and differentially reshapes the intracellular cavity to enable selective engagement of Gs versus Gi. Molecular dynamics and functional assays confirm the hydration network is essential for activation and G protein selectivity.\",\n      \"method\": \"Cryo-EM structure determination (2.0 Å GPR174-Gs, 3.4 Å GPR174-Gi), molecular dynamics simulations, functional assays (cAMP/G protein coupling), sequence alignment across class A GPCRs\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structures for both Gs and Gi complexes with MD simulations and functional validation, multiple orthogonal methods\",\n      \"pmids\": [\"42096402\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Molecular dynamics simulations of GPR174 bound to an antagonist (mPS, modified LysoPS) versus LysoPS show that mPS inactivates GPR174 by reducing conformational dynamics, disrupting the PIF, DRY, and N/DPxxY conserved activation motifs, and blocking signal transduction to the G protein interface, whereas LysoPS engages these motifs to couple the binding site to Gs. Membrane lipids including PIP2 modulate ligand dynamics and receptor conformational states.\",\n      \"method\": \"Molecular dynamics simulations in heterogeneous lipid bilayer, network analysis of signaling pathways, protein-lipid interaction analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational study only (MD simulations), preprint, no experimental functional validation reported\",\n      \"pmids\": [\"41000684\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"GPR174 is an X-chromosome-encoded class A GPCR that is constitutively activated by its endogenous lipid ligand lysophosphatidylserine (LysoPS), coupling primarily to Gαs (elevating cAMP via adenylyl cyclase) and also to Gαi; cryo-EM structures reveal LysoPS recognition through polar interactions with a positively charged binding pocket and lateral membrane entry via a TM4–TM5 portal, while a hydration network through the sodium pocket, DRY/NPxxY motifs, and intracellular cavity governs activation and differential Gs/Gi selectivity; in immune cells GPR174 suppresses IL-2 production, constrains regulatory T cell generation, and—in a testosterone-dependent manner—limits germinal centre formation by positioning male B cells toward the T-B border via CCL21-induced, Gαi-associated migration, collectively imparting sexual dimorphism to humoral immunity and modulating autoimmune susceptibility.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GPR174 is an X-chromosome-encoded class A GPCR that functions as a lysophosphatidylserine (LysoPS) receptor governing immune cell activation, with a strong role in adaptive immunity [#1, #3]. Upon LysoPS engagement it couples to G\\u03b1s and elevates intracellular cAMP, and cryo-EM structures show that the negatively charged LysoPS head group makes polar contacts within a positively charged pocket while the receptor accepts ligand laterally through a partially open TM4\\u2013TM5 portal in the membrane; co-purified endogenous LysoPS confers high constitutive Gs signaling, and a hydration-mediated network through the sodium pocket and the DRY/NPxxY motifs governs activation and selective engagement of Gs versus Gi [#8, #9, #12]. In CD4+ T cells GPR174 signaling suppresses IL-2 production and limits activation-marker upregulation in a G\\u03b1s-dependent manner, constrains regulatory T cell generation, and its deficiency reduces susceptibility to experimental autoimmune encephalomyelitis [#1, #3, #4]. GPR174 also acts as a receptor for CCL21 in B cells, where a testosterone-driven enhancement of GPR174\\u2013G\\u03b1i association preferentially promotes male B cell migration toward the T\\u2013B border and suppresses germinal centre formation, imparting sexual dimorphism to humoral immunity [#5]. In regulatory T cells, the G\\u03b1s/cAMP/PKA axis links GPR174 to downstream transcriptional control of CTLA-4, IL-10, and amphiregulin, shaping macrophage polarization and tissue responses to injury [#6, #7].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"Established the molecular identity and signaling output of GPR174 by showing it is a LysoPS receptor with constitutive and ligand-stimulated cAMP elevation through G\\u03b1s.\",\n      \"evidence\": \"Stable expression in CHO cells with cAMP measurement, ligand screen, Erk phosphorylation, and NF449 G\\u03b1s inhibition\",\n      \"pmids\": [\"23178570\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Single cell-based system without structural detail\", \"Physiological cell type and in vivo relevance not addressed\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Connected GPR174 signaling to an immunological role by showing LysoPS via GPR174 constrains Treg generation and modulates autoimmune susceptibility.\",\n      \"evidence\": \"Gpr174 knockout mice, in vitro T cell proliferation/Treg assays, and EAE model\",\n      \"pmids\": [\"26077720\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Downstream transcriptional effectors of Treg suppression not yet defined\", \"G protein dependence of the Treg phenotype not dissected here\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined the structure-activity requirements of LysoPS for GPR174 potency and selectivity, enabling chemical tools.\",\n      \"evidence\": \"Synthetic chemistry and SAR of LysoPS analogues across receptor subtypes\",\n      \"pmids\": [\"25970039\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Structural basis of selectivity not resolved at this stage\", \"No in vivo validation of agonists\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified IL-2 suppression as a concrete molecular output of GPR174 in CD4+ T cells and linked it to activation-induced LysoPS.\",\n      \"evidence\": \"Gpr174 KO splenocytes/T cells, anti-CD3/CD28 activation, IL-2 ELISA and RT-PCR, lipid profiling\",\n      \"pmids\": [\"29017923\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"G protein coupling responsible for IL-2 suppression not yet pinned down\", \"Single lab\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established that GPR174-mediated T cell suppression operates through G\\u03b1s, tying the signaling axis to defined activation phenotypes in vivo.\",\n      \"evidence\": \"In vivo T cell proliferation models, G\\u03b1s inhibition in vitro, CD25/CD69 flow cytometry, Gpr174 KO comparison\",\n      \"pmids\": [\"29457279\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Did not address G\\u03b1i contributions later seen in B cells\", \"Transcriptional targets downstream of cAMP not mapped\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed a second ligand and a sex-dimorphic mechanism: GPR174 responds to CCL21 and, through testosterone-enhanced G\\u03b1i coupling, directs male B cell positioning and suppresses germinal centre formation.\",\n      \"evidence\": \"Ligand identification by fractionation, calcium flux, migration assays, GPR174\\u2013G\\u03b1i Co-IP, orchidectomy/testosterone models, conditional B cell KO, germinal centre analysis\",\n      \"pmids\": [\"31875850\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Reconciliation of CCL21 versus LysoPS ligand usage not fully resolved\", \"Mechanism of testosterone-driven G\\u03b1i bias unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extended GPR174 function in Tregs to control of CTLA-4/IL-10 expression and macrophage polarization, linking the receptor to inflammatory tissue outcomes.\",\n      \"evidence\": \"Gpr174 KO mice, LPS sepsis and CLP models, Treg\\u2013macrophage co-culture, flow cytometry and ELISA\",\n      \"pmids\": [\"30850582\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct signaling pathway linking GPR174 loss to CTLA-4/IL-10 not dissected here\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Mapped a downstream transcriptional axis (G\\u03b1s/cAMP/PKA/EGR1/AREG) through which GPR174 in Tregs regulates angiogenesis and macrophage polarization.\",\n      \"evidence\": \"Treg-specific Gpr174 KO mice, hindlimb ischemia, AREG manipulation, EGR1 nuclear translocation, cAMP/PKA inhibition\",\n      \"pmids\": [\"36473866\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct molecular link between PKA and EGR1 nuclear exclusion not fully resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Provided the atomic basis for LysoPS recognition and Gs coupling, including lateral membrane ligand entry and confirmation of LysoPS as the constitutively bound endogenous ligand driving high basal signaling.\",\n      \"evidence\": \"Cryo-EM of LysoPS-bound and ligand-free GPR174-Gs complexes, mutagenesis, cAMP assays versus D1R, lipid specificity tests\",\n      \"pmids\": [\"36823105\", \"37737235\", \"38048360\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Gi-bound conformation not resolved in these structures\", \"Structural basis of constitutive activity versus regulated signaling not fully separated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked the LysoPS/GPR174 cAMP-PKA-CREB axis to a disease setting by showing it drives ESCC invasion and metastasis.\",\n      \"evidence\": \"LC-MS LysoPS measurement, migration/invasion assays, nuclear-cytoplasmic fractionation, nude mouse metastasis model, GPR174 overexpression\",\n      \"pmids\": [\"40229851\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Causal requirement of endogenous GPR174 (vs overexpression) in patient tumors not established\", \"Single lab\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Resolved the mechanistic basis of Gs versus Gi selectivity by defining a continuous hydration network bridging the sodium pocket, conserved activation motifs, and the G protein interface.\",\n      \"evidence\": \"High-resolution cryo-EM of GPR174-Gs and GPR174-Gi complexes, molecular dynamics, and functional coupling assays\",\n      \"pmids\": [\"42096402\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"How physiological cues bias cells toward Gs versus Gi engagement not addressed\", \"Link between hydration-network mutations and immune phenotypes untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How GPR174 integrates two ligands (LysoPS and CCL21) and switches between G\\u03b1s and G\\u03b1i outputs to produce context- and sex-specific immune outcomes remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Mechanism of ligand-dependent G protein switching in native cells unknown\", \"Molecular basis of testosterone-driven G\\u03b1i bias undefined\", \"Relative physiological contribution of constitutive versus ligand-driven signaling unclear\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 8, 9]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [8, 9]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [8, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 4, 12]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 3, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"GNAS\",\n      \"GNAI\",\n      \"CCL21\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}