{"gene":"RSPH3","run_date":"2026-06-10T07:46:28","timeline":{"discoveries":[{"year":2001,"finding":"RSP3 (RSPH3 ortholog in Chlamydomonas) is an A-kinase anchoring protein (AKAP) located in the radial spoke stalk; RII-binding domain mapped to amino acids 144-180, predicted to form an amphipathic helix; amino acid substitutions L to P or VL to AA in the central residues abolish RII binding. RSP3 is positioned near inner arm dyneins where anchored PKA can regulate dynein activity.","method":"RII blot overlay on motility mutants, truncated RSP3 expression, amphipathic helix prediction, site-directed mutagenesis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro binding assays with mutagenesis, domain mapping with multiple truncations, replicated in subsequent independent studies","pmids":["11309423"],"is_preprint":false},{"year":1993,"finding":"RSP3 (RSPH3 ortholog in Chlamydomonas) binds directly to spokeless axonemes from pf14 cells but not to wild-type axonemes or purified microtubules; the axoneme-binding domain is located within amino acids 1-85 (minimal domain: aa 42-85); deletion of aa 1-85 abolishes axoneme binding; aa 18-87 are required for radial spoke assembly and flagellar motility.","method":"In vitro binding assay with synthesized RSP3 and pf14 axonemes, deletion mutants, fusion protein binding assays, Chlamydomonas transformation with mutagenized RSP3 genes","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro binding with domain mapping via deletion mutants and fusion proteins, validated in vivo by transformation","pmids":["8408197"],"is_preprint":false},{"year":2006,"finding":"Disruption of the PKA-binding (RII-binding) domain in Chlamydomonas RSP3 results in unregulated axonemal cAMP-dependent PKA activity; transformed pf14 cells with RII-domain mutant RSP3 display paralyzed or twitching flagella; PKA inhibitors rescue motility, confirming that misregulated PKA activity causes the motility defect. Radial spoke assembly is not grossly disrupted by the RII-domain mutation.","method":"Chlamydomonas transformation with PKA-binding domain mutant RSP3, reactivation assays of demembranated cells with and without PKA inhibitors, motility analysis","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic complementation with point-mutant rescue, PKA inhibitor pharmacology, multiple independent transformants, confirmed in live cells and cell models","pmids":["16571668"],"is_preprint":false},{"year":2008,"finding":"Chlamydomonas RSP3 forms a homodimer; the dimerization domain coincides with the N-terminal axoneme-binding domain. Each radial spoke is proposed to be built on an RSP3 dimer, allowing localization of multiple PKAs or AKAP-binding proteins per spoke.","method":"Chemical crosslinking, native gel electrophoresis, epitope-tagged RSP3 proteins, truncation analysis","journal":"Cell motility and the cytoskeleton","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple biochemical methods (crosslinking, native gel, epitope tagging) in single lab; no independent replication reported","pmids":["18157907"],"is_preprint":false},{"year":2009,"finding":"Human RSPH3 interacts with ERK2 (identified in a yeast two-hybrid screen for proteins with higher affinity for phosphorylated/active ERK2); RSPH3 is a substrate for ERK1/2; RSPH3 is also an AKAP that scaffolds the PKA holoenzyme by binding regulatory subunits RIIα and RIIβ; ERK1/2 activity and phosphorylation regulate RSPH3 binding to RII subunits.","method":"Yeast two-hybrid screen, co-immunoprecipitation, in vitro kinase assay (ERK1/2 phosphorylation of RSPH3), RII overlay binding assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus co-IP plus in vitro kinase assay in single lab, multiple orthogonal methods","pmids":["19684019"],"is_preprint":false},{"year":2012,"finding":"Chlamydomonas RSP3 forms a dimeric structural scaffold in the radial spoke complex, anchoring through two distinct amphipathic helices (AHs) the RIIa and Dpy-30 domains of four non-PKA spoke proteins involved in spoke assembly and modulation; one AH can bind both RIIa and Dpy-30 domains in vitro.","method":"In vitro pull-down assays, AH-RIIa/Dpy-30 domain interaction assays, structural/biochemical characterization of RSP3 truncations","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple in vitro binding assays with domain mutants in single lab, defining two distinct AH-mediated interactions","pmids":["23148234"],"is_preprint":false},{"year":2012,"finding":"LC8 dimers bind in tandem to the N-terminal region of RSP3 in Chlamydomonas, enhance RSP3 binding to axonemes, and are required for efficient radial spoke docking; perturbation of RSP3's LC8-binding sites results in asynchronous flagella with hypophosphorylated RSP3 and defective RSP3-axoneme association; LC8 is absent from the 12S RS precursor but present in axoneme-bound spokes.","method":"Co-sedimentation, pull-down assays, phosphorylation analysis, flagellar motility phenotype in RSP3 LC8-binding site mutants, IFT imaging","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — binding assays and mutagenesis plus in vivo phenotype in Chlamydomonas, single lab with multiple orthogonal methods","pmids":["22753897"],"is_preprint":false},{"year":2015,"finding":"RSPH3 mutations in humans (PCD patients) cause near-complete absence of detectable radial spokes in respiratory cilia combined with central complex defects; RSPH3 protein localizes within respiratory epithelial cilia and is undetectable in airway cells from RSPH3-mutant individuals; despite absence of RSPH3, RS-neck protein RSPH23 and RS-head proteins RSPH1 and RSPH4A remain present in cilia, indicating RSPH3 is specifically required for RS stalk/base assembly but not RS head attachment.","method":"Immunofluorescence localization of RSPH3 and other RS proteins in patient airway cells, electron microscopy of cilia ultrastructure, high-speed videomicroscopy, genetic mutation identification","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple patient families, immunofluorescence showing protein absence, EM ultrastructure, protein hierarchy established with multiple RS protein markers","pmids":["26073779"],"is_preprint":false},{"year":2004,"finding":"In Ciona intestinalis, RSP3 co-purifies with Hsp40 and a spoke-head protein homolog (RSP4/6 homolog) as a complex extracted from axonemes; immunoelectron microscopy localizes Hsp40 to the distal portion of the spoke stalk at the junction between spoke head and stalk; a spoke-head protein LRR37 is absent from this complex, indicating the complex constitutes the spoke stalk.","method":"KCl/KI extraction of axonemes, gel filtration, ion exchange chromatography, peptide mass fingerprinting (MALDI-TOF MS), immunoelectron microscopy","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical co-purification with MS identification and immunoEM localization, single lab study in ascidian model","pmids":["15563603"],"is_preprint":false},{"year":2018,"finding":"In vivo IFT imaging in Chlamydomonas shows RSP3 (stalk protein) and RSP4 (head protein) mostly co-migrate on IFT trains; IFT of RSP4 depends on RSP3; during repair of spoke-head-deficient axonemes, RSP4 is added onto pre-existing RSP3-containing stalks with little exchange of RSP3, indicating RSP3 and RSP4 are transported together but separate during spoke repair.","method":"In vivo fluorescence imaging of FP-tagged RSP3 and RSP4, IFT co-migration analysis, axoneme repair assays by mating flagella mutants","journal":"Cytoskeleton (Hoboken, N.J.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live-cell IFT imaging with FP-tagged proteins, genetic rescue experiments, single lab","pmids":["30070024"],"is_preprint":false},{"year":2019,"finding":"In Drosophila, dRSPH3 interacts with RSBP15 through RSBP15's DD_R_PKA superfamily domain; RSBP15 co-localizes with dRSPH3 in sperm flagella; loss of RSBP15 causes loss of dRSPH3 (and dRSPH1, dRSPH4a, dRSPH9) from sperm, disrupts the 9+2 axoneme structure, and causes male sterility, indicating RSBP15 stabilizes dRSPH3 to maintain radial spoke complex integrity.","method":"Co-immunoprecipitation, co-localization by immunofluorescence, RSBP15 knockout in Drosophila, electron microscopy of axoneme ultrastructure","journal":"Journal of genetics and genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus KO phenotype with EM ultrastructure, single lab, Drosophila model","pmids":["31281031"],"is_preprint":false},{"year":2019,"finding":"Drosophila Combover (Cmb) binds to the axonemal component Radial spoke protein 3 (dRsp3); knockdown of dRsp3 causes similar sperm individualization defects as cmb mutants (failure of actin cone synchronous movement along flagella), suggesting Cmb coordinates the individualization machinery with the axoneme via RSP3.","method":"Co-immunoprecipitation (Cmb binds RSP3), RNAi knockdown of dRsp3 with individualization phenotype assay","journal":"Development (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP interaction plus parallel loss-of-function phenotype, single lab, Drosophila model","pmids":["31391193"],"is_preprint":false},{"year":2022,"finding":"ARMC2/PF27 is a cargo adapter required for IFT of radial spokes in Chlamydomonas; tagged ARMC2 and RSP3 co-migrate on anterograde IFT trains; in armc2/pf27 mutants, IFT of RSP3 (and radial spokes) is abolished and spokes are limited to the proximal flagellar region; after tip unloading, RSP3 attaches to the axoneme while ARMC2 diffuses back.","method":"Live fluorescence IFT imaging of tagged ARMC2 and RSP3, armc2/pf27 mutant analysis, axoneme immunofluorescence","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-migration by live imaging plus mutant phenotype with localization data, single lab","pmids":["34982025"],"is_preprint":false},{"year":2014,"finding":"Mouse RSP3 (RSPH3) accumulates at the perinuclear region of CHO and 293T cells; it is not co-localized with ER or Golgi markers; in vivo it localizes to the proximal cytoplasmic dilation of the leading process of migrating cortical neurons; it concentrates in ependymal cilia as a ciliary component; overexpression by in utero electroporation increases the percentage of neurons reaching the upper cortical plate.","method":"Immunofluorescence/organelle marker co-localization, in utero electroporation overexpression, in vivo neuron migration analysis","journal":"Journal of molecular histology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, localization by immunofluorescence with overexpression phenotype but no mechanistic pathway placement","pmids":["25079589"],"is_preprint":false},{"year":2015,"finding":"Mouse RSP3 (RSPH3) is a nucleocytoplasmic shuttling protein; two nuclear localization signals and one nuclear export signal were identified in RSP3 by deletion mutant analysis; full-length RSP3-EGFP is mainly cytoplasmic in CHO cells; RSP3 localizes to primary cilia of radial glial cells; overexpression in the developing cerebral cortex by in utero electroporation promotes neurogenesis and thickens layer II/III of neocortex.","method":"Deletion mutant analysis of NLS/NES, fluorescent fusion protein localization, in utero electroporation overexpression, cortical layer analysis","journal":"Histochemistry and cell biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, domain mapping and localization with overexpression phenotype, no direct mechanistic pathway","pmids":["26082196"],"is_preprint":false},{"year":2023,"finding":"IQUB interacts with RSPH3 (and CEP295NL, GSTM1, ODF1) in the yeast two-hybrid system; in Iqub KO and knockin mice, sperm display radial spoke defects; functional assays suggest IQUB recruits calmodulin to inhibit the activity of an RSPH3/p-ERK1/2 complex (described as a non-typical AKAP), thereby facilitating normal radial spoke assembly. Co-immunoprecipitation confirmed IQUB-RSPH3 interaction in vivo.","method":"Yeast two-hybrid, co-immunoprecipitation, KO and KI mouse models, scanning/transmission electron microscopy, western blot","journal":"Human reproduction (Oxford, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus co-IP plus KO mouse phenotype with EM, single lab with multiple orthogonal methods","pmids":["36355624"],"is_preprint":false},{"year":2023,"finding":"LRRC23 interacts with RSPH3 in vitro (demonstrated by co-immunoprecipitation), indicating LRRC23 is associated with the radial spoke complex in humans; LRRC23 loss-of-function disrupts radial spoke integrity.","method":"Co-immunoprecipitation (in vitro interaction), patient mutation analysis, immunofluorescence","journal":"Clinical genetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP experiment in single lab, limited mechanistic follow-up on RSPH3 specifically","pmids":["37804054"],"is_preprint":false},{"year":2025,"finding":"In Chlamydomonas, the ida1 mutation (defective I1/f dynein heavy chain, abolishing I1 dynein assembly) is epistatic to the RSP3 RII-binding domain mutation (388); the 388;ida1 double mutant displays an ida1-like motility phenotype rather than a 388-like phenotype, placing I1 dynein downstream of RSP3's PKA-anchoring function in the signaling pathway regulating ciliary motility.","method":"Genetic epistasis analysis, double mutant construction and motility phenotype characterization in Chlamydomonas","journal":"microPublication biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — classical genetic epistasis with defined double mutants and motility phenotype readout, single study","pmids":["41487906"],"is_preprint":false},{"year":2025,"finding":"RSPH3 is identified as a component of radial spoke 1 (RS1) stalk in human and mouse sperm; in IQUB-deficient sperm, RS1 (but not RS2 or RS3) is absent, and RSPH3 along with RSPH6A, RSPH9, DYDC1, NME5, DNAJB13, PPIL6, AK8, ROPN1L, RSPH14, DYNLL1, and IQUB constitute RS1 components.","method":"Protein mass spectrometry, western blotting, IQUB knockout mice, electron microscopy","journal":"Cell communication and signaling : CCS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomics plus KO mouse model with EM to define RS1 composition, single lab","pmids":["39849482"],"is_preprint":false}],"current_model":"RSPH3 (RSP3) is a highly conserved radial spoke stalk protein that serves as a structural scaffold and A-kinase anchoring protein (AKAP): its N-terminal domain (aa 1-85) mediates homodimerization and direct docking to axonemal doublet microtubules, while two amphipathic helices anchor the RIIa and Dpy-30 domains of PKA regulatory subunits and non-PKA spoke proteins; anchored PKA regulates axonemal dynein activity (with I1 dynein acting downstream of RSP3's PKA-anchoring function), and ERK1/2 phosphorylates RSPH3 to modulate its PKA-binding; in humans, RSPH3 is essential for radial spoke assembly (its loss abolishes detectable spokes while leaving RS head proteins intact), and it forms part of the RS1 stalk complex in sperm flagella where it interacts with IQUB, LRRC23, and other partners to maintain sperm motility."},"narrative":{"mechanistic_narrative":"RSPH3 (RSP3) is a highly conserved radial spoke stalk protein that functions as a structural scaffold and A-kinase anchoring protein (AKAP) coordinating PKA-dependent regulation of axonemal dynein activity in motile cilia and flagella [PMID:11309423, PMID:8408197]. Its N-terminal domain (minimal region aa 42–85) docks the protein directly onto spokeless axonemal doublet microtubules and is required for radial spoke assembly and motility, and this same region mediates RSPH3 homodimerization so that each spoke is built on an RSP3 dimer [PMID:8408197, PMID:18157907]. The central RII-binding amphipathic helix (aa 144–180) anchors the PKA holoenzyme; disrupting this helix leaves spoke assembly grossly intact but produces unregulated axonemal PKA activity and paralyzed/twitching flagella that are rescued by PKA inhibitors, establishing that RSPH3-anchored PKA tunes motility [PMID:11309423, PMID:16571668], with I1/f dynein acting genetically downstream of this PKA-anchoring function [PMID:41487906]. Beyond PKA, RSP3 uses two distinct amphipathic helices to anchor the RIIa and Dpy-30 domains of non-PKA spoke proteins [PMID:23148234], and recruits LC8 dimers to its N-terminus to enhance axoneme docking [PMID:22753897]. Human RSPH3 is the molecular adaptor for the spoke stalk: in primary ciliary dyskinesia patients its loss abolishes detectable radial spokes and produces central-complex defects, yet RS-neck (RSPH23) and RS-head (RSPH1, RSPH4A) proteins persist, showing RSPH3 is specifically required for stalk/base assembly rather than head attachment [PMID:26073779]. In sperm flagella RSPH3 is a component of the RS1 stalk, interacting with IQUB, LRRC23, and additional spoke proteins to maintain spoke integrity and motility [PMID:36355624, PMID:39849482]. Trafficking of RSPH3 into the axoneme proceeds via intraflagellar transport using the cargo adapter ARMC2/PF27, with RSP3 and the head protein RSP4 co-migrating on IFT trains before separating during spoke repair [PMID:30070024, PMID:34982025].","teleology":[{"year":1993,"claim":"Establishing how the radial spoke attaches to the axoneme, RSP3 was shown to bind directly to spokeless axonemes through a discrete N-terminal domain, defining it as the structural anchor of the spoke.","evidence":"in vitro binding of synthesized RSP3 and deletion mutants to pf14 axonemes, with rescue by Chlamydomonas transformation","pmids":["8408197"],"confidence":"High","gaps":["Did not resolve the microtubule docking interface at residue resolution","Did not establish the dimeric state of the docking domain"]},{"year":2001,"claim":"To explain how PKA signaling reaches axonemal dyneins, RSP3 was identified as an AKAP with a mapped RII-binding amphipathic helix positioned near inner arm dyneins.","evidence":"RII blot overlay on motility mutants, RSP3 truncations, and site-directed mutagenesis of the amphipathic helix","pmids":["11309423"],"confidence":"High","gaps":["Did not show the functional consequence of anchored PKA on motility","Did not identify the dynein substrates regulated by anchored PKA"]},{"year":2006,"claim":"Disrupting the RII-binding domain revealed that RSP3-anchored PKA must be spatially restrained, since unregulated axonemal PKA activity paralyzes flagella independently of spoke assembly.","evidence":"transformation of pf14 with RII-domain mutant RSP3, demembranated cell reactivation with PKA inhibitors, motility analysis","pmids":["16571668"],"confidence":"High","gaps":["Did not identify the dynein targets of misregulated PKA","Did not map the cAMP source controlling axonemal PKA"]},{"year":2008,"claim":"Defining the stoichiometry of the spoke, RSP3 was shown to homodimerize via its N-terminal axoneme-binding domain, implying each spoke can anchor multiple PKA/AKAP-binding partners.","evidence":"chemical crosslinking, native gel electrophoresis, and truncation analysis of epitope-tagged RSP3","pmids":["18157907"],"confidence":"Medium","gaps":["No independent replication of the dimerization model","No structural model of the dimer interface"]},{"year":2009,"claim":"Extending the regulatory logic to mammals, human RSPH3 was shown to be both an ERK1/2 substrate and an RIIalpha/RIIbeta-binding AKAP, linking MAPK signaling to PKA scaffolding.","evidence":"yeast two-hybrid screen for phospho-ERK2 binders, co-IP, in vitro ERK1/2 kinase assay, and RII overlay binding","pmids":["19684019"],"confidence":"Medium","gaps":["Phosphosites on RSPH3 not mapped","Functional consequence of ERK phosphorylation in cilia not tested in vivo"]},{"year":2012,"claim":"The scaffolding repertoire of RSP3 was broadened by showing it uses two distinct amphipathic helices to anchor RIIa and Dpy-30 domains of non-PKA spoke proteins, and recruits LC8 to strengthen axoneme docking.","evidence":"in vitro pull-downs with RSP3 truncations defining AH–RIIa/Dpy-30 interactions; co-sedimentation, mutagenesis, and motility phenotyping of LC8-binding mutants","pmids":["23148234","22753897"],"confidence":"Medium","gaps":["Spatial arrangement of the multiple anchored partners within the spoke unresolved","Whether one AH binds both domains simultaneously in vivo not shown"]},{"year":2015,"claim":"Connecting RSPH3 to human disease, its loss in PCD patients was shown to abolish radial spokes while sparing neck and head proteins, defining RSPH3 as specifically required for stalk/base assembly.","evidence":"immunofluorescence of RS proteins in patient airway cells, ciliary EM, high-speed videomicroscopy, and mutation identification across families","pmids":["26073779"],"confidence":"High","gaps":["Mechanism of central-complex defect secondary to RS loss not defined","Order of stalk-protein assembly relative to RSPH3 not resolved"]},{"year":2022,"claim":"Resolving how RSP3 is delivered to the spoke, ARMC2/PF27 was identified as the IFT cargo adapter required for RSP3 transport, with RSP3 attaching to the axoneme upon tip unloading.","evidence":"live IFT imaging of tagged ARMC2 and RSP3, armc2/pf27 mutant analysis, and axoneme immunofluorescence (with co-migration imaging of RSP3 and RSP4 in companion work)","pmids":["34982025","30070024"],"confidence":"Medium","gaps":["Molecular contacts between ARMC2 and RSP3 not mapped","How RSP3 transfers from adapter to axoneme not defined"]},{"year":2025,"claim":"In sperm flagella, RSPH3 was defined as a core RS1 stalk component whose integrity depends on partners IQUB and LRRC23, linking RS1 assembly to male fertility.","evidence":"yeast two-hybrid, co-IP, proteomics, and IQUB KO/KI mouse models with EM; genetic epistasis placing I1 dynein downstream of RSP3 PKA-anchoring in Chlamydomonas","pmids":["36355624","39849482","37804054","41487906"],"confidence":"Medium","gaps":["Whether RS1-specific assembly logic generalizes to respiratory cilia not established","Structural basis of IQUB/calmodulin inhibition of the RSPH3/p-ERK complex not resolved"]},{"year":null,"claim":"How RSPH3's reported nucleocytoplasmic shuttling and neuronal-migration functions relate to its axonemal scaffolding role remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["Non-ciliary roles rest on overexpression/localization in single labs without mechanistic pathway placement [#13, #14]","No structural model of the full-length human RSPH3 scaffold","Substrate dyneins directly tuned by RSPH3-anchored PKA not identified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,4,5]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[1,3,7]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[1,6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,2]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[7,13]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1,6]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[7,12]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,2,4]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[15,18]}],"complexes":["radial spoke","radial spoke 1 (RS1) stalk","PKA holoenzyme (AKAP scaffold)"],"partners":["PRKAR2A","PRKAR2B","MAPK1","DYNLL1","IQUB","LRRC23","ARMC2","RSP4"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q86UC2","full_name":"Radial spoke head protein 3 homolog","aliases":["A-kinase anchor protein RSPH3","Radial spoke head-like protein 2"],"length_aa":560,"mass_kda":63.7,"function":"Functions as part of axonemal radial spoke complexes that play an important part in the motility of sperm and cilia (By similarity). Functions as a protein kinase A-anchoring protein that scaffolds the cAMP-dependent protein kinase holoenzyme. May serve as a point of convergence for MAPK and PKA signaling in cilia (PubMed:19684019)","subcellular_location":"Cytoplasm, cytoskeleton, cilium axoneme; Cytoplasm, cytoskeleton, flagellum axoneme","url":"https://www.uniprot.org/uniprotkb/Q86UC2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RSPH3","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1089,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RSPH3","total_profiled":1310},"omim":[{"mim_id":"620708","title":"LEUCINE-RICH REPEAT-CONTAINING PROTEIN 23; LRRC23","url":"https://www.omim.org/entry/620708"},{"mim_id":"620557","title":"IQ MOTIF- AND UBIQUITIN DOMAIN-CONTAINING PROTEIN; IQUB","url":"https://www.omim.org/entry/620557"},{"mim_id":"616481","title":"CILIARY DYSKINESIA, PRIMARY, 32; CILD32","url":"https://www.omim.org/entry/616481"},{"mim_id":"615876","title":"RADIAL SPOKE HEAD 3; RSPH3","url":"https://www.omim.org/entry/615876"},{"mim_id":"612010","title":"CELIAC DISEASE, SUSCEPTIBILITY TO, 12; CELIAC12","url":"https://www.omim.org/entry/612010"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Flagellar centriole","reliability":"Approved"},{"location":"Mid piece","reliability":"Approved"},{"location":"Annulus","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RSPH3"},"hgnc":{"alias_symbol":["dJ111C20.1","RSP3"],"prev_symbol":["RSHL2"]},"alphafold":{"accession":"Q86UC2","domains":[{"cath_id":"1.20.5","chopping":"311-423","consensus_level":"medium","plddt":96.1086,"start":311,"end":423},{"cath_id":"1.20.5","chopping":"426-472","consensus_level":"high","plddt":88.6147,"start":426,"end":472}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86UC2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q86UC2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q86UC2-F1-predicted_aligned_error_v6.png","plddt_mean":64.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RSPH3","jax_strain_url":"https://www.jax.org/strain/search?query=RSPH3"},"sequence":{"accession":"Q86UC2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q86UC2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q86UC2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86UC2"}},"corpus_meta":[{"pmid":"16738661","id":"PMC_16738661","title":"Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit.","date":"2006","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/16738661","citation_count":185,"is_preprint":false},{"pmid":"16607017","id":"PMC_16607017","title":"Flagellar motility contributes to cytokinesis in Trypanosoma brucei and is modulated by an evolutionarily conserved dynein regulatory system.","date":"2006","source":"Eukaryotic cell","url":"https://pubmed.ncbi.nlm.nih.gov/16607017","citation_count":138,"is_preprint":false},{"pmid":"29703884","id":"PMC_29703884","title":"The Ustilago maydis repetitive effector Rsp3 blocks the antifungal activity of mannose-binding maize proteins.","date":"2018","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/29703884","citation_count":98,"is_preprint":false},{"pmid":"11309423","id":"PMC_11309423","title":"Flagellar radial spoke protein 3 is an A-kinase anchoring protein (AKAP).","date":"2001","source":"The Journal of cell 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RSP3 is positioned near inner arm dyneins where anchored PKA can regulate dynein activity.\",\n      \"method\": \"RII blot overlay on motility mutants, truncated RSP3 expression, amphipathic helix prediction, site-directed mutagenesis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro binding assays with mutagenesis, domain mapping with multiple truncations, replicated in subsequent independent studies\",\n      \"pmids\": [\"11309423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"RSP3 (RSPH3 ortholog in Chlamydomonas) binds directly to spokeless axonemes from pf14 cells but not to wild-type axonemes or purified microtubules; the axoneme-binding domain is located within amino acids 1-85 (minimal domain: aa 42-85); deletion of aa 1-85 abolishes axoneme binding; aa 18-87 are required for radial spoke assembly and flagellar motility.\",\n      \"method\": \"In vitro binding assay with synthesized RSP3 and pf14 axonemes, deletion mutants, fusion protein binding assays, Chlamydomonas transformation with mutagenized RSP3 genes\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro binding with domain mapping via deletion mutants and fusion proteins, validated in vivo by transformation\",\n      \"pmids\": [\"8408197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Disruption of the PKA-binding (RII-binding) domain in Chlamydomonas RSP3 results in unregulated axonemal cAMP-dependent PKA activity; transformed pf14 cells with RII-domain mutant RSP3 display paralyzed or twitching flagella; PKA inhibitors rescue motility, confirming that misregulated PKA activity causes the motility defect. Radial spoke assembly is not grossly disrupted by the RII-domain mutation.\",\n      \"method\": \"Chlamydomonas transformation with PKA-binding domain mutant RSP3, reactivation assays of demembranated cells with and without PKA inhibitors, motility analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic complementation with point-mutant rescue, PKA inhibitor pharmacology, multiple independent transformants, confirmed in live cells and cell models\",\n      \"pmids\": [\"16571668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Chlamydomonas RSP3 forms a homodimer; the dimerization domain coincides with the N-terminal axoneme-binding domain. Each radial spoke is proposed to be built on an RSP3 dimer, allowing localization of multiple PKAs or AKAP-binding proteins per spoke.\",\n      \"method\": \"Chemical crosslinking, native gel electrophoresis, epitope-tagged RSP3 proteins, truncation analysis\",\n      \"journal\": \"Cell motility and the cytoskeleton\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biochemical methods (crosslinking, native gel, epitope tagging) in single lab; no independent replication reported\",\n      \"pmids\": [\"18157907\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Human RSPH3 interacts with ERK2 (identified in a yeast two-hybrid screen for proteins with higher affinity for phosphorylated/active ERK2); RSPH3 is a substrate for ERK1/2; RSPH3 is also an AKAP that scaffolds the PKA holoenzyme by binding regulatory subunits RIIα and RIIβ; ERK1/2 activity and phosphorylation regulate RSPH3 binding to RII subunits.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, in vitro kinase assay (ERK1/2 phosphorylation of RSPH3), RII overlay binding assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus co-IP plus in vitro kinase assay in single lab, multiple orthogonal methods\",\n      \"pmids\": [\"19684019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Chlamydomonas RSP3 forms a dimeric structural scaffold in the radial spoke complex, anchoring through two distinct amphipathic helices (AHs) the RIIa and Dpy-30 domains of four non-PKA spoke proteins involved in spoke assembly and modulation; one AH can bind both RIIa and Dpy-30 domains in vitro.\",\n      \"method\": \"In vitro pull-down assays, AH-RIIa/Dpy-30 domain interaction assays, structural/biochemical characterization of RSP3 truncations\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple in vitro binding assays with domain mutants in single lab, defining two distinct AH-mediated interactions\",\n      \"pmids\": [\"23148234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"LC8 dimers bind in tandem to the N-terminal region of RSP3 in Chlamydomonas, enhance RSP3 binding to axonemes, and are required for efficient radial spoke docking; perturbation of RSP3's LC8-binding sites results in asynchronous flagella with hypophosphorylated RSP3 and defective RSP3-axoneme association; LC8 is absent from the 12S RS precursor but present in axoneme-bound spokes.\",\n      \"method\": \"Co-sedimentation, pull-down assays, phosphorylation analysis, flagellar motility phenotype in RSP3 LC8-binding site mutants, IFT imaging\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — binding assays and mutagenesis plus in vivo phenotype in Chlamydomonas, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"22753897\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RSPH3 mutations in humans (PCD patients) cause near-complete absence of detectable radial spokes in respiratory cilia combined with central complex defects; RSPH3 protein localizes within respiratory epithelial cilia and is undetectable in airway cells from RSPH3-mutant individuals; despite absence of RSPH3, RS-neck protein RSPH23 and RS-head proteins RSPH1 and RSPH4A remain present in cilia, indicating RSPH3 is specifically required for RS stalk/base assembly but not RS head attachment.\",\n      \"method\": \"Immunofluorescence localization of RSPH3 and other RS proteins in patient airway cells, electron microscopy of cilia ultrastructure, high-speed videomicroscopy, genetic mutation identification\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple patient families, immunofluorescence showing protein absence, EM ultrastructure, protein hierarchy established with multiple RS protein markers\",\n      \"pmids\": [\"26073779\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"In Ciona intestinalis, RSP3 co-purifies with Hsp40 and a spoke-head protein homolog (RSP4/6 homolog) as a complex extracted from axonemes; immunoelectron microscopy localizes Hsp40 to the distal portion of the spoke stalk at the junction between spoke head and stalk; a spoke-head protein LRR37 is absent from this complex, indicating the complex constitutes the spoke stalk.\",\n      \"method\": \"KCl/KI extraction of axonemes, gel filtration, ion exchange chromatography, peptide mass fingerprinting (MALDI-TOF MS), immunoelectron microscopy\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical co-purification with MS identification and immunoEM localization, single lab study in ascidian model\",\n      \"pmids\": [\"15563603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In vivo IFT imaging in Chlamydomonas shows RSP3 (stalk protein) and RSP4 (head protein) mostly co-migrate on IFT trains; IFT of RSP4 depends on RSP3; during repair of spoke-head-deficient axonemes, RSP4 is added onto pre-existing RSP3-containing stalks with little exchange of RSP3, indicating RSP3 and RSP4 are transported together but separate during spoke repair.\",\n      \"method\": \"In vivo fluorescence imaging of FP-tagged RSP3 and RSP4, IFT co-migration analysis, axoneme repair assays by mating flagella mutants\",\n      \"journal\": \"Cytoskeleton (Hoboken, N.J.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell IFT imaging with FP-tagged proteins, genetic rescue experiments, single lab\",\n      \"pmids\": [\"30070024\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In Drosophila, dRSPH3 interacts with RSBP15 through RSBP15's DD_R_PKA superfamily domain; RSBP15 co-localizes with dRSPH3 in sperm flagella; loss of RSBP15 causes loss of dRSPH3 (and dRSPH1, dRSPH4a, dRSPH9) from sperm, disrupts the 9+2 axoneme structure, and causes male sterility, indicating RSBP15 stabilizes dRSPH3 to maintain radial spoke complex integrity.\",\n      \"method\": \"Co-immunoprecipitation, co-localization by immunofluorescence, RSBP15 knockout in Drosophila, electron microscopy of axoneme ultrastructure\",\n      \"journal\": \"Journal of genetics and genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus KO phenotype with EM ultrastructure, single lab, Drosophila model\",\n      \"pmids\": [\"31281031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Drosophila Combover (Cmb) binds to the axonemal component Radial spoke protein 3 (dRsp3); knockdown of dRsp3 causes similar sperm individualization defects as cmb mutants (failure of actin cone synchronous movement along flagella), suggesting Cmb coordinates the individualization machinery with the axoneme via RSP3.\",\n      \"method\": \"Co-immunoprecipitation (Cmb binds RSP3), RNAi knockdown of dRsp3 with individualization phenotype assay\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP interaction plus parallel loss-of-function phenotype, single lab, Drosophila model\",\n      \"pmids\": [\"31391193\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ARMC2/PF27 is a cargo adapter required for IFT of radial spokes in Chlamydomonas; tagged ARMC2 and RSP3 co-migrate on anterograde IFT trains; in armc2/pf27 mutants, IFT of RSP3 (and radial spokes) is abolished and spokes are limited to the proximal flagellar region; after tip unloading, RSP3 attaches to the axoneme while ARMC2 diffuses back.\",\n      \"method\": \"Live fluorescence IFT imaging of tagged ARMC2 and RSP3, armc2/pf27 mutant analysis, axoneme immunofluorescence\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-migration by live imaging plus mutant phenotype with localization data, single lab\",\n      \"pmids\": [\"34982025\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Mouse RSP3 (RSPH3) accumulates at the perinuclear region of CHO and 293T cells; it is not co-localized with ER or Golgi markers; in vivo it localizes to the proximal cytoplasmic dilation of the leading process of migrating cortical neurons; it concentrates in ependymal cilia as a ciliary component; overexpression by in utero electroporation increases the percentage of neurons reaching the upper cortical plate.\",\n      \"method\": \"Immunofluorescence/organelle marker co-localization, in utero electroporation overexpression, in vivo neuron migration analysis\",\n      \"journal\": \"Journal of molecular histology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, localization by immunofluorescence with overexpression phenotype but no mechanistic pathway placement\",\n      \"pmids\": [\"25079589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Mouse RSP3 (RSPH3) is a nucleocytoplasmic shuttling protein; two nuclear localization signals and one nuclear export signal were identified in RSP3 by deletion mutant analysis; full-length RSP3-EGFP is mainly cytoplasmic in CHO cells; RSP3 localizes to primary cilia of radial glial cells; overexpression in the developing cerebral cortex by in utero electroporation promotes neurogenesis and thickens layer II/III of neocortex.\",\n      \"method\": \"Deletion mutant analysis of NLS/NES, fluorescent fusion protein localization, in utero electroporation overexpression, cortical layer analysis\",\n      \"journal\": \"Histochemistry and cell biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, domain mapping and localization with overexpression phenotype, no direct mechanistic pathway\",\n      \"pmids\": [\"26082196\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"IQUB interacts with RSPH3 (and CEP295NL, GSTM1, ODF1) in the yeast two-hybrid system; in Iqub KO and knockin mice, sperm display radial spoke defects; functional assays suggest IQUB recruits calmodulin to inhibit the activity of an RSPH3/p-ERK1/2 complex (described as a non-typical AKAP), thereby facilitating normal radial spoke assembly. Co-immunoprecipitation confirmed IQUB-RSPH3 interaction in vivo.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, KO and KI mouse models, scanning/transmission electron microscopy, western blot\",\n      \"journal\": \"Human reproduction (Oxford, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus co-IP plus KO mouse phenotype with EM, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"36355624\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"LRRC23 interacts with RSPH3 in vitro (demonstrated by co-immunoprecipitation), indicating LRRC23 is associated with the radial spoke complex in humans; LRRC23 loss-of-function disrupts radial spoke integrity.\",\n      \"method\": \"Co-immunoprecipitation (in vitro interaction), patient mutation analysis, immunofluorescence\",\n      \"journal\": \"Clinical genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP experiment in single lab, limited mechanistic follow-up on RSPH3 specifically\",\n      \"pmids\": [\"37804054\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In Chlamydomonas, the ida1 mutation (defective I1/f dynein heavy chain, abolishing I1 dynein assembly) is epistatic to the RSP3 RII-binding domain mutation (388); the 388;ida1 double mutant displays an ida1-like motility phenotype rather than a 388-like phenotype, placing I1 dynein downstream of RSP3's PKA-anchoring function in the signaling pathway regulating ciliary motility.\",\n      \"method\": \"Genetic epistasis analysis, double mutant construction and motility phenotype characterization in Chlamydomonas\",\n      \"journal\": \"microPublication biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — classical genetic epistasis with defined double mutants and motility phenotype readout, single study\",\n      \"pmids\": [\"41487906\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RSPH3 is identified as a component of radial spoke 1 (RS1) stalk in human and mouse sperm; in IQUB-deficient sperm, RS1 (but not RS2 or RS3) is absent, and RSPH3 along with RSPH6A, RSPH9, DYDC1, NME5, DNAJB13, PPIL6, AK8, ROPN1L, RSPH14, DYNLL1, and IQUB constitute RS1 components.\",\n      \"method\": \"Protein mass spectrometry, western blotting, IQUB knockout mice, electron microscopy\",\n      \"journal\": \"Cell communication and signaling : CCS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomics plus KO mouse model with EM to define RS1 composition, single lab\",\n      \"pmids\": [\"39849482\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RSPH3 (RSP3) is a highly conserved radial spoke stalk protein that serves as a structural scaffold and A-kinase anchoring protein (AKAP): its N-terminal domain (aa 1-85) mediates homodimerization and direct docking to axonemal doublet microtubules, while two amphipathic helices anchor the RIIa and Dpy-30 domains of PKA regulatory subunits and non-PKA spoke proteins; anchored PKA regulates axonemal dynein activity (with I1 dynein acting downstream of RSP3's PKA-anchoring function), and ERK1/2 phosphorylates RSPH3 to modulate its PKA-binding; in humans, RSPH3 is essential for radial spoke assembly (its loss abolishes detectable spokes while leaving RS head proteins intact), and it forms part of the RS1 stalk complex in sperm flagella where it interacts with IQUB, LRRC23, and other partners to maintain sperm motility.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RSPH3 (RSP3) is a highly conserved radial spoke stalk protein that functions as a structural scaffold and A-kinase anchoring protein (AKAP) coordinating PKA-dependent regulation of axonemal dynein activity in motile cilia and flagella [#0, #1]. Its N-terminal domain (minimal region aa 42–85) docks the protein directly onto spokeless axonemal doublet microtubules and is required for radial spoke assembly and motility, and this same region mediates RSPH3 homodimerization so that each spoke is built on an RSP3 dimer [#1, #3]. The central RII-binding amphipathic helix (aa 144–180) anchors the PKA holoenzyme; disrupting this helix leaves spoke assembly grossly intact but produces unregulated axonemal PKA activity and paralyzed/twitching flagella that are rescued by PKA inhibitors, establishing that RSPH3-anchored PKA tunes motility [#0, #2], with I1/f dynein acting genetically downstream of this PKA-anchoring function [#17]. Beyond PKA, RSP3 uses two distinct amphipathic helices to anchor the RIIa and Dpy-30 domains of non-PKA spoke proteins [#5], and recruits LC8 dimers to its N-terminus to enhance axoneme docking [#6]. Human RSPH3 is the molecular adaptor for the spoke stalk: in primary ciliary dyskinesia patients its loss abolishes detectable radial spokes and produces central-complex defects, yet RS-neck (RSPH23) and RS-head (RSPH1, RSPH4A) proteins persist, showing RSPH3 is specifically required for stalk/base assembly rather than head attachment [#7]. In sperm flagella RSPH3 is a component of the RS1 stalk, interacting with IQUB, LRRC23, and additional spoke proteins to maintain spoke integrity and motility [#15, #18]. Trafficking of RSPH3 into the axoneme proceeds via intraflagellar transport using the cargo adapter ARMC2/PF27, with RSP3 and the head protein RSP4 co-migrating on IFT trains before separating during spoke repair [#9, #12].\",\n  \"teleology\": [\n    {\n      \"year\": 1993,\n      \"claim\": \"Establishing how the radial spoke attaches to the axoneme, RSP3 was shown to bind directly to spokeless axonemes through a discrete N-terminal domain, defining it as the structural anchor of the spoke.\",\n      \"evidence\": \"in vitro binding of synthesized RSP3 and deletion mutants to pf14 axonemes, with rescue by Chlamydomonas transformation\",\n      \"pmids\": [\"8408197\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the microtubule docking interface at residue resolution\", \"Did not establish the dimeric state of the docking domain\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"To explain how PKA signaling reaches axonemal dyneins, RSP3 was identified as an AKAP with a mapped RII-binding amphipathic helix positioned near inner arm dyneins.\",\n      \"evidence\": \"RII blot overlay on motility mutants, RSP3 truncations, and site-directed mutagenesis of the amphipathic helix\",\n      \"pmids\": [\"11309423\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not show the functional consequence of anchored PKA on motility\", \"Did not identify the dynein substrates regulated by anchored PKA\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Disrupting the RII-binding domain revealed that RSP3-anchored PKA must be spatially restrained, since unregulated axonemal PKA activity paralyzes flagella independently of spoke assembly.\",\n      \"evidence\": \"transformation of pf14 with RII-domain mutant RSP3, demembranated cell reactivation with PKA inhibitors, motility analysis\",\n      \"pmids\": [\"16571668\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the dynein targets of misregulated PKA\", \"Did not map the cAMP source controlling axonemal PKA\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defining the stoichiometry of the spoke, RSP3 was shown to homodimerize via its N-terminal axoneme-binding domain, implying each spoke can anchor multiple PKA/AKAP-binding partners.\",\n      \"evidence\": \"chemical crosslinking, native gel electrophoresis, and truncation analysis of epitope-tagged RSP3\",\n      \"pmids\": [\"18157907\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No independent replication of the dimerization model\", \"No structural model of the dimer interface\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extending the regulatory logic to mammals, human RSPH3 was shown to be both an ERK1/2 substrate and an RIIalpha/RIIbeta-binding AKAP, linking MAPK signaling to PKA scaffolding.\",\n      \"evidence\": \"yeast two-hybrid screen for phospho-ERK2 binders, co-IP, in vitro ERK1/2 kinase assay, and RII overlay binding\",\n      \"pmids\": [\"19684019\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphosites on RSPH3 not mapped\", \"Functional consequence of ERK phosphorylation in cilia not tested in vivo\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"The scaffolding repertoire of RSP3 was broadened by showing it uses two distinct amphipathic helices to anchor RIIa and Dpy-30 domains of non-PKA spoke proteins, and recruits LC8 to strengthen axoneme docking.\",\n      \"evidence\": \"in vitro pull-downs with RSP3 truncations defining AH–RIIa/Dpy-30 interactions; co-sedimentation, mutagenesis, and motility phenotyping of LC8-binding mutants\",\n      \"pmids\": [\"23148234\", \"22753897\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Spatial arrangement of the multiple anchored partners within the spoke unresolved\", \"Whether one AH binds both domains simultaneously in vivo not shown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Connecting RSPH3 to human disease, its loss in PCD patients was shown to abolish radial spokes while sparing neck and head proteins, defining RSPH3 as specifically required for stalk/base assembly.\",\n      \"evidence\": \"immunofluorescence of RS proteins in patient airway cells, ciliary EM, high-speed videomicroscopy, and mutation identification across families\",\n      \"pmids\": [\"26073779\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of central-complex defect secondary to RS loss not defined\", \"Order of stalk-protein assembly relative to RSPH3 not resolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolving how RSP3 is delivered to the spoke, ARMC2/PF27 was identified as the IFT cargo adapter required for RSP3 transport, with RSP3 attaching to the axoneme upon tip unloading.\",\n      \"evidence\": \"live IFT imaging of tagged ARMC2 and RSP3, armc2/pf27 mutant analysis, and axoneme immunofluorescence (with co-migration imaging of RSP3 and RSP4 in companion work)\",\n      \"pmids\": [\"34982025\", \"30070024\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular contacts between ARMC2 and RSP3 not mapped\", \"How RSP3 transfers from adapter to axoneme not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"In sperm flagella, RSPH3 was defined as a core RS1 stalk component whose integrity depends on partners IQUB and LRRC23, linking RS1 assembly to male fertility.\",\n      \"evidence\": \"yeast two-hybrid, co-IP, proteomics, and IQUB KO/KI mouse models with EM; genetic epistasis placing I1 dynein downstream of RSP3 PKA-anchoring in Chlamydomonas\",\n      \"pmids\": [\"36355624\", \"39849482\", \"37804054\", \"41487906\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether RS1-specific assembly logic generalizes to respiratory cilia not established\", \"Structural basis of IQUB/calmodulin inhibition of the RSPH3/p-ERK complex not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RSPH3's reported nucleocytoplasmic shuttling and neuronal-migration functions relate to its axonemal scaffolding role remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Non-ciliary roles rest on overexpression/localization in single labs without mechanistic pathway placement [#13, #14]\", \"No structural model of the full-length human RSPH3 scaffold\", \"Substrate dyneins directly tuned by RSPH3-anchored PKA not identified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 4, 5]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [1, 3, 7]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [1, 6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [7, 13]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [7, 12]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 2, 4]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [15, 18]}\n    ],\n    \"complexes\": [\n      \"radial spoke\",\n      \"radial spoke 1 (RS1) stalk\",\n      \"PKA holoenzyme (AKAP scaffold)\"\n    ],\n    \"partners\": [\n      \"PRKAR2A\",\n      \"PRKAR2B\",\n      \"MAPK1\",\n      \"DYNLL1\",\n      \"IQUB\",\n      \"LRRC23\",\n      \"ARMC2\",\n      \"RSP4\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}