{"gene":"AKAP10","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":1997,"finding":"D-AKAP2 (AKAP10) was identified as a dual-specific AKAP that binds both type I (RIα) and type II (RIIα) regulatory subunits of PKA; the R-binding domain resides at the C-terminus (residues 333–372) and interacts with the N-terminal dimerization domain of RIα and RIIα. A putative RGS domain was also identified near the N-terminus, suggesting a potential link to Gα protein signaling.","method":"Yeast two-hybrid screen; coprecipitation assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — reciprocal coprecipitation with defined domain mapping; foundational paper with 197 citations, replicated subsequently","pmids":["9326583"],"is_preprint":false},{"year":2001,"finding":"Full-length human D-AKAP2 (AKAP10, 662 residues) localizes predominantly to mitochondria; in vivo association with PKA in mouse brain was confirmed by cAMP-agarose pull-down. The protein contains two putative RGS domains and shows tissue-specific alternative splicing or post-translational modifications.","method":"Immunocytochemistry, immunohistochemistry, subcellular fractionation, cAMP-agarose pull-down assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal localization methods (fractionation + imaging) plus in vivo pull-down; 91 citations","pmids":["11248059"],"is_preprint":false},{"year":2002,"finding":"DXMS and limited proteolysis revealed that D-AKAP2 (AKAP10) has two distinctly folded domains: one containing the putative RGS domain and one containing the PKA binding site (highly protected from deuterium exchange) plus a PDZ-binding motif that is more solvent-accessible, indicating a multi-domain scaffold architecture.","method":"Deuterium exchange-mass spectrometry (DXMS); limited proteolysis","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — structural/biophysical method with mutagenesis-level resolution; 64 citations","pmids":["12206784"],"is_preprint":false},{"year":2003,"finding":"D-AKAP2 (AKAP10) interacts via its C-terminal PDZ-binding motif with the PDZ domain protein PDZK1 (PDZ domain 4) and also with NHERF-1 (with ~4-fold lower affinity), localizing it to the subapical pole of renal proximal tubular cells and anchoring PKA near the NaPi-IIa transporter for PTH-mediated regulation.","method":"Yeast two-hybrid, pull-down assays, co-immunoprecipitation from transfected OK cells, immunohistochemistry","journal":"Kidney international","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP plus pull-down with defined domain, confirmed in cells and tissue","pmids":["14531807"],"is_preprint":false},{"year":2007,"finding":"Heterozygous disruption of Akap10 (deleting the final 51 aa) in mice increases contractile response of cardiac cells to cholinergic signals, causes cardiac arrhythmias, and premature death, establishing AKAP10 as a regulator of cardiac rhythm and autonomic (cholinergic) signaling in a dominant interfering manner.","method":"Gene-trap mESC differentiation into cardiac myocytes; mouse knockout phenotyping (contractility assays, ECG/arrhythmia monitoring)","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — clean KO with defined cellular and in vivo phenotype; 60 citations","pmids":["17485678"],"is_preprint":false},{"year":2009,"finding":"The two tandem RGS domains of D-AKAP2 (AKAP10) bind the small GTPases Rab4 (preferentially GTP-bound form) and Rab11 — the first demonstration of RGS domains interacting with small GTPases. D-AKAP2 co-localizes with Rab4/Rab11 on endosomes, regulates Rab11-compartment morphology, and knockdown by RNAi redistributes Rab11 and transferrin receptor to the cell periphery and accelerates transferrin recycling.","method":"Co-immunoprecipitation, GTP-pulldown, fluorescence microscopy, RNAi knockdown with transferrin recycling assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP plus functional RNAi rescue with defined recycling phenotype; multiple orthogonal methods","pmids":["19797056"],"is_preprint":false},{"year":2010,"finding":"Crystal structures of RIα D/D domain alone and in complex with the D-AKAP2 (AKAP10) AKB helix revealed that: (1) RIα presents an extensive surface through a well-formed N-terminal helix; (2) the helical register of D-AKAP2 shifts compared to the RIIα:D-AKAP2 complex, making RIα binding mechanistically distinct; (3) a redox-sensitive disulfide in RIα affects AKAP binding affinity.","method":"X-ray crystallography; structural comparison with mutagenesis-informed analysis","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with functional validation; 104 citations","pmids":["20159461"],"is_preprint":false},{"year":2014,"finding":"Crystal structure of the D-AKAP2:PKA RII:PDZK1 ternary complex showed that the disordered C-terminal segment of D-AKAP2 nucleates a polyvalent scaffold by presenting an α-helix to PKA RII (AKB motif) and a β-strand to PDZK1 simultaneously; PKA binary complex formation is a prerequisite for high-affinity PDZK1 interaction, linking PKA signaling to transporter regulation without direct membrane-protein contact.","method":"X-ray crystallography of ternary complex; structural analysis","journal":"Protein science : a publication of the Protein Society","confidence":"High","confidence_rationale":"Tier 1 — ternary complex crystal structure with mechanistic interpretation","pmids":["25348485"],"is_preprint":false}],"current_model":"AKAP10 (D-AKAP2) is a dual-specific scaffold protein that anchors PKA (via a C-terminal AKB helix that binds RIα/RIIα through distinct helical registers) to mitochondria and endosomal/apical membranes, couples PKA to transporter regulation through a PDZ-motif interaction with PDZK1, modulates endocytic recycling via its tandem RGS domains that bind GTP-Rab4 and Rab11, and regulates cardiac cholinergic/autonomic signaling such that its disruption causes arrhythmias and premature death."},"narrative":{"teleology":[{"year":1997,"claim":"The discovery that D-AKAP2 binds both RIα and RIIα via a C-terminal domain established it as the first characterized dual-specificity AKAP and raised the question of how a single helix accommodates two structurally distinct R-subunit classes.","evidence":"Yeast two-hybrid screen with RI/RII baits followed by reciprocal coprecipitation with domain truncations","pmids":["9326583"],"confidence":"High","gaps":["Structural basis for dual specificity was unknown","Function of the N-terminal putative RGS domain was uncharacterized","Subcellular localization of full-length protein was not determined"]},{"year":2001,"claim":"Localization of full-length D-AKAP2 to mitochondria and confirmation of in vivo PKA association in brain placed the scaffold at a defined organelle, but left its non-PKA functions unresolved.","evidence":"Immunocytochemistry, subcellular fractionation, and cAMP-agarose pull-down from mouse brain","pmids":["11248059"],"confidence":"High","gaps":["Mitochondrial targeting mechanism was not identified","RGS domain ligands remained unknown","Functional consequence of mitochondrial PKA anchoring was not tested"]},{"year":2002,"claim":"Biophysical mapping by deuterium exchange revealed that D-AKAP2 consists of two independently folded domains — an RGS-containing region and a PKA-binding/PDZ-motif region — establishing a modular scaffold architecture that could integrate distinct signaling inputs.","evidence":"DXMS and limited proteolysis of purified D-AKAP2","pmids":["12206784"],"confidence":"High","gaps":["Identity of PDZ-domain binding partners was unknown","Whether the two domains function independently or cooperatively was unclear"]},{"year":2003,"claim":"Identification of PDZK1 as a binding partner for D-AKAP2's PDZ motif explained how the scaffold positions PKA near apical membrane transporters such as NaPi-IIa, linking it to PTH-regulated phosphate transport in the kidney.","evidence":"Yeast two-hybrid, pull-down, and co-immunoprecipitation in OK cells plus immunohistochemistry of renal tissue","pmids":["14531807"],"confidence":"High","gaps":["Whether PKA phosphorylation of NaPi-IIa is directly mediated through this complex was not shown","Relative contribution of NHERF-1 versus PDZK1 interaction in vivo was unresolved"]},{"year":2007,"claim":"Gene-trap disruption of Akap10 in mice demonstrated that loss of the PKA-anchoring domain causes enhanced cholinergic responsiveness, cardiac arrhythmias, and premature death, providing the first in vivo evidence that AKAP10 scaffolding is essential for normal cardiac autonomic signaling.","evidence":"Gene-trap knockout mice phenotyped by ECG monitoring, contractility assays, and mESC-derived cardiomyocyte analysis","pmids":["17485678"],"confidence":"High","gaps":["Molecular pathway downstream of PKA in cardiomyocytes was not delineated","Whether cardiac phenotype involves RGS-domain or PDZ-motif functions was not tested","Cell-type specificity of the cardiac requirement was not resolved"]},{"year":2009,"claim":"The demonstration that D-AKAP2's tandem RGS domains bind GTP-Rab4 and Rab11 — an unprecedented RGS–small-GTPase interaction — and regulate endosomal recycling revealed that AKAP10 integrates PKA signaling with membrane trafficking.","evidence":"Co-immunoprecipitation, GTP-pulldown, colocalization microscopy, and RNAi knockdown with transferrin recycling assays","pmids":["19797056"],"confidence":"High","gaps":["Whether the RGS domains possess GAP activity toward Rab4/Rab11 was not established","Coupling between Rab-binding and PKA-anchoring functions on the same scaffold was not tested","Structural basis of RGS–Rab recognition was not determined"]},{"year":2010,"claim":"Crystal structures of the RIα D/D domain bound to the D-AKAP2 AKB helix resolved how dual specificity arises: the helix shifts its register compared to the RIIα complex, and a redox-sensitive disulfide in RIα modulates binding affinity, introducing a regulatory dimension to AKAP–PKA interaction.","evidence":"X-ray crystallography of RIα D/D alone and in complex with the D-AKAP2 AKB peptide","pmids":["20159461"],"confidence":"High","gaps":["Physiological relevance of redox-regulated AKAP binding was not tested in cells","Whether the helical register shift alters downstream signaling output was unknown"]},{"year":2014,"claim":"The ternary complex crystal structure of D-AKAP2–PKA RII–PDZK1 showed that PKA binding nucleates a polyvalent scaffold where an intrinsically disordered segment adopts both α-helical (AKB) and β-strand (PDZ ligand) conformations, and that binary PKA engagement is prerequisite for high-affinity PDZK1 recruitment, establishing an ordered assembly mechanism.","evidence":"X-ray crystallography of the reconstituted ternary complex","pmids":["25348485"],"confidence":"High","gaps":["Whether ordered assembly occurs in vivo and is regulated by cAMP levels was not shown","How PKA catalytic subunit release from this ternary complex affects transporter phosphorylation was not addressed"]},{"year":null,"claim":"The relationship between AKAP10's Rab-binding and PKA-anchoring activities on the same molecule, the structural basis of RGS–Rab recognition, and the downstream PKA substrates mediating the cardiac phenotype remain unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No structure of the RGS–Rab complex exists","PKA substrates relevant to cardiac rhythm regulation via AKAP10 are unidentified","Whether Rab-dependent trafficking and PKA anchoring are coordinated on the same scaffold in vivo is untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,3,5,7]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[1]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[5]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,4,6]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[5]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[3]}],"complexes":["D-AKAP2–PKA RII–PDZK1 ternary complex"],"partners":["PRKAR1A","PRKAR2A","PDZK1","RAB4A","RAB11A","SLC9A3R1"],"other_free_text":[]},"mechanistic_narrative":"AKAP10 (D-AKAP2) is a multi-domain scaffold protein that anchors cAMP-dependent protein kinase (PKA) to specific subcellular compartments and coordinates PKA signaling with endocytic recycling and transporter regulation. Its C-terminal AKB helix binds the dimerization/docking domains of both RIα and RIIα regulatory subunits of PKA through distinct helical registers, and a redox-sensitive disulfide in RIα modulates this interaction [PMID:9326583, PMID:20159461]. The tandem N-terminal RGS domains bind GTP-loaded Rab4 and Rab11 on recycling endosomes, controlling transferrin receptor trafficking and endosomal compartment morphology [PMID:19797056], while a C-terminal PDZ-binding motif recruits PDZK1 to form a ternary complex with PKA RII that couples phosphorylation to apical membrane transporter regulation in renal proximal tubule cells [PMID:14531807, PMID:25348485]. Heterozygous disruption of Akap10 in mice increases cardiac myocyte sensitivity to cholinergic stimulation, causes arrhythmias, and leads to premature death, establishing AKAP10 as a critical regulator of cardiac autonomic signaling [PMID:17485678]."},"prefetch_data":{"uniprot":{"accession":"O43572","full_name":"A-kinase anchor protein 10, mitochondrial","aliases":["Dual specificity A kinase-anchoring protein 2","D-AKAP-2","Protein kinase A-anchoring protein 10","PRKA10"],"length_aa":662,"mass_kda":73.8,"function":"Differentially targeted protein that binds to type I and II regulatory subunits of protein kinase A and anchors them to the mitochondria or the plasma membrane. Although the physiological relevance between PKA and AKAPS with mitochondria is not fully understood, one idea is that BAD, a proapoptotic member, is phosphorylated and inactivated by mitochondria-anchored PKA. It cannot be excluded too that it may facilitate PKA as well as G protein signal transduction, by acting as an adapter for assembling multiprotein complexes. With its RGS domain, it could lead to the interaction to G-alpha proteins, providing a link between the signaling machinery and the downstream kinase (By similarity)","subcellular_location":"Mitochondrion; Membrane; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/O43572/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/AKAP10","classification":"Not Classified","n_dependent_lines":9,"n_total_lines":1208,"dependency_fraction":0.0074503311258278145},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ARFGEF1","stoichiometry":10.0},{"gene":"ARFGEF2","stoichiometry":10.0}],"url":"https://opencell.sf.czbiohub.org/search/AKAP10","total_profiled":1310},"omim":[{"mim_id":"604694","title":"A-KINASE ANCHOR PROTEIN 10; AKAP10","url":"https://www.omim.org/entry/604694"},{"mim_id":"152430","title":"LONGEVITY 1","url":"https://www.omim.org/entry/152430"},{"mim_id":"115080","title":"CARDIAC CONDUCTION DEFECT","url":"https://www.omim.org/entry/115080"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/AKAP10"},"hgnc":{"alias_symbol":["D-AKAP2","PRKA10","MGC9414"],"prev_symbol":[]},"alphafold":{"accession":"O43572","domains":[{"cath_id":"1.10.167.10","chopping":"124-177_285-378","consensus_level":"medium","plddt":91.7281,"start":124,"end":378},{"cath_id":"1.10.167.10","chopping":"379-516","consensus_level":"medium","plddt":90.8638,"start":379,"end":516}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O43572","model_url":"https://alphafold.ebi.ac.uk/files/AF-O43572-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O43572-F1-predicted_aligned_error_v6.png","plddt_mean":64.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=AKAP10","jax_strain_url":"https://www.jax.org/strain/search?query=AKAP10"},"sequence":{"accession":"O43572","fasta_url":"https://rest.uniprot.org/uniprotkb/O43572.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O43572/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O43572"}},"corpus_meta":[{"pmid":"9326583","id":"PMC_9326583","title":"D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain.","date":"1997","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/9326583","citation_count":197,"is_preprint":false},{"pmid":"20159461","id":"PMC_20159461","title":"Structure of D-AKAP2:PKA RI complex: insights into AKAP specificity and selectivity.","date":"2010","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/20159461","citation_count":104,"is_preprint":false},{"pmid":"11248059","id":"PMC_11248059","title":"Cloning and mitochondrial localization of full-length D-AKAP2, a protein kinase A anchoring protein.","date":"2001","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/11248059","citation_count":91,"is_preprint":false},{"pmid":"12206784","id":"PMC_12206784","title":"Domain organization of D-AKAP2 revealed by enhanced deuterium exchange-mass spectrometry (DXMS).","date":"2002","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/12206784","citation_count":64,"is_preprint":false},{"pmid":"17485678","id":"PMC_17485678","title":"Gene-trapped mouse embryonic stem cell-derived cardiac myocytes and human genetics implicate AKAP10 in heart rhythm regulation.","date":"2007","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/17485678","citation_count":60,"is_preprint":false},{"pmid":"19797056","id":"PMC_19797056","title":"D-AKAP2 interacts with Rab4 and Rab11 through its RGS domains and regulates transferrin receptor recycling.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19797056","citation_count":53,"is_preprint":false},{"pmid":"14531807","id":"PMC_14531807","title":"PDZK1: II. an anchoring site for the PKA-binding protein D-AKAP2 in renal proximal tubular cells.","date":"2003","source":"Kidney international","url":"https://pubmed.ncbi.nlm.nih.gov/14531807","citation_count":48,"is_preprint":false},{"pmid":"19496216","id":"PMC_19496216","title":"AKAP10 (I646V) functional polymorphism predicts heart rate and heart rate variability in apparently healthy, middle-aged European-Americans.","date":"2009","source":"Psychophysiology","url":"https://pubmed.ncbi.nlm.nih.gov/19496216","citation_count":22,"is_preprint":false},{"pmid":"25348485","id":"PMC_25348485","title":"D-AKAP2:PKA RII:PDZK1 ternary complex structure: insights from the nucleation of a polyvalent scaffold.","date":"2014","source":"Protein science : a publication of the Protein Society","url":"https://pubmed.ncbi.nlm.nih.gov/25348485","citation_count":8,"is_preprint":false},{"pmid":"21701445","id":"PMC_21701445","title":"Possible counter effect in newborns of 1936A>G (I646V) polymorphism in the AKAP10 gene encoding A-kinase-anchoring protein 10.","date":"2011","source":"Journal of perinatology : official journal of the California Perinatal Association","url":"https://pubmed.ncbi.nlm.nih.gov/21701445","citation_count":7,"is_preprint":false},{"pmid":"26110499","id":"PMC_26110499","title":"Genetic association of AKAP10 gene polymorphism with reduced risk of preterm birth.","date":"2015","source":"Journal of perinatology : official journal of the California Perinatal Association","url":"https://pubmed.ncbi.nlm.nih.gov/26110499","citation_count":5,"is_preprint":false},{"pmid":"23092224","id":"PMC_23092224","title":"1936A→G (I646 V) polymorphism in the AKAP10 gene encoding A-kinase-anchoring protein 10 in very long-lived poles is similar to that in newborns.","date":"2012","source":"Experimental aging research","url":"https://pubmed.ncbi.nlm.nih.gov/23092224","citation_count":2,"is_preprint":false},{"pmid":"19462906","id":"PMC_19462906","title":"[Genotyping of AKAP10 gene 2073A/G single nucleotide polymorphism by TaqMan probe real-time PCR].","date":"2009","source":"Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition","url":"https://pubmed.ncbi.nlm.nih.gov/19462906","citation_count":1,"is_preprint":false},{"pmid":"22817328","id":"PMC_22817328","title":"Association of 1936A > G in AKAP10 (A-kinase anchoring protein 10) and blood pressure in Polish full-term newborns.","date":"2012","source":"Blood pressure","url":"https://pubmed.ncbi.nlm.nih.gov/22817328","citation_count":1,"is_preprint":false},{"pmid":"23095189","id":"PMC_23095189","title":"Polymorphism 1936A > G in the AKAP10 gene (encoding A-kinase-anchoring protein 10) is associated with higher cholesterol cord blood concentration in Polish full-term newsborns.","date":"2013","source":"Journal of perinatal medicine","url":"https://pubmed.ncbi.nlm.nih.gov/23095189","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8594,"output_tokens":2081,"usd":0.028498},"stage2":{"model":"claude-opus-4-6","input_tokens":5417,"output_tokens":2509,"usd":0.134715},"total_usd":0.163213,"stage1_batch_id":"msgbatch_01TqNM6p6FsoMBT2jzQTwKBr","stage2_batch_id":"msgbatch_01Ya473eco8sTHbvRymQaU8o","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1997,\n      \"finding\": \"D-AKAP2 (AKAP10) was identified as a dual-specific AKAP that binds both type I (RIα) and type II (RIIα) regulatory subunits of PKA; the R-binding domain resides at the C-terminus (residues 333–372) and interacts with the N-terminal dimerization domain of RIα and RIIα. A putative RGS domain was also identified near the N-terminus, suggesting a potential link to Gα protein signaling.\",\n      \"method\": \"Yeast two-hybrid screen; coprecipitation assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal coprecipitation with defined domain mapping; foundational paper with 197 citations, replicated subsequently\",\n      \"pmids\": [\"9326583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Full-length human D-AKAP2 (AKAP10, 662 residues) localizes predominantly to mitochondria; in vivo association with PKA in mouse brain was confirmed by cAMP-agarose pull-down. The protein contains two putative RGS domains and shows tissue-specific alternative splicing or post-translational modifications.\",\n      \"method\": \"Immunocytochemistry, immunohistochemistry, subcellular fractionation, cAMP-agarose pull-down assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal localization methods (fractionation + imaging) plus in vivo pull-down; 91 citations\",\n      \"pmids\": [\"11248059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"DXMS and limited proteolysis revealed that D-AKAP2 (AKAP10) has two distinctly folded domains: one containing the putative RGS domain and one containing the PKA binding site (highly protected from deuterium exchange) plus a PDZ-binding motif that is more solvent-accessible, indicating a multi-domain scaffold architecture.\",\n      \"method\": \"Deuterium exchange-mass spectrometry (DXMS); limited proteolysis\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — structural/biophysical method with mutagenesis-level resolution; 64 citations\",\n      \"pmids\": [\"12206784\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"D-AKAP2 (AKAP10) interacts via its C-terminal PDZ-binding motif with the PDZ domain protein PDZK1 (PDZ domain 4) and also with NHERF-1 (with ~4-fold lower affinity), localizing it to the subapical pole of renal proximal tubular cells and anchoring PKA near the NaPi-IIa transporter for PTH-mediated regulation.\",\n      \"method\": \"Yeast two-hybrid, pull-down assays, co-immunoprecipitation from transfected OK cells, immunohistochemistry\",\n      \"journal\": \"Kidney international\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus pull-down with defined domain, confirmed in cells and tissue\",\n      \"pmids\": [\"14531807\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Heterozygous disruption of Akap10 (deleting the final 51 aa) in mice increases contractile response of cardiac cells to cholinergic signals, causes cardiac arrhythmias, and premature death, establishing AKAP10 as a regulator of cardiac rhythm and autonomic (cholinergic) signaling in a dominant interfering manner.\",\n      \"method\": \"Gene-trap mESC differentiation into cardiac myocytes; mouse knockout phenotyping (contractility assays, ECG/arrhythmia monitoring)\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined cellular and in vivo phenotype; 60 citations\",\n      \"pmids\": [\"17485678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The two tandem RGS domains of D-AKAP2 (AKAP10) bind the small GTPases Rab4 (preferentially GTP-bound form) and Rab11 — the first demonstration of RGS domains interacting with small GTPases. D-AKAP2 co-localizes with Rab4/Rab11 on endosomes, regulates Rab11-compartment morphology, and knockdown by RNAi redistributes Rab11 and transferrin receptor to the cell periphery and accelerates transferrin recycling.\",\n      \"method\": \"Co-immunoprecipitation, GTP-pulldown, fluorescence microscopy, RNAi knockdown with transferrin recycling assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus functional RNAi rescue with defined recycling phenotype; multiple orthogonal methods\",\n      \"pmids\": [\"19797056\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Crystal structures of RIα D/D domain alone and in complex with the D-AKAP2 (AKAP10) AKB helix revealed that: (1) RIα presents an extensive surface through a well-formed N-terminal helix; (2) the helical register of D-AKAP2 shifts compared to the RIIα:D-AKAP2 complex, making RIα binding mechanistically distinct; (3) a redox-sensitive disulfide in RIα affects AKAP binding affinity.\",\n      \"method\": \"X-ray crystallography; structural comparison with mutagenesis-informed analysis\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional validation; 104 citations\",\n      \"pmids\": [\"20159461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of the D-AKAP2:PKA RII:PDZK1 ternary complex showed that the disordered C-terminal segment of D-AKAP2 nucleates a polyvalent scaffold by presenting an α-helix to PKA RII (AKB motif) and a β-strand to PDZK1 simultaneously; PKA binary complex formation is a prerequisite for high-affinity PDZK1 interaction, linking PKA signaling to transporter regulation without direct membrane-protein contact.\",\n      \"method\": \"X-ray crystallography of ternary complex; structural analysis\",\n      \"journal\": \"Protein science : a publication of the Protein Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — ternary complex crystal structure with mechanistic interpretation\",\n      \"pmids\": [\"25348485\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"AKAP10 (D-AKAP2) is a dual-specific scaffold protein that anchors PKA (via a C-terminal AKB helix that binds RIα/RIIα through distinct helical registers) to mitochondria and endosomal/apical membranes, couples PKA to transporter regulation through a PDZ-motif interaction with PDZK1, modulates endocytic recycling via its tandem RGS domains that bind GTP-Rab4 and Rab11, and regulates cardiac cholinergic/autonomic signaling such that its disruption causes arrhythmias and premature death.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"AKAP10 (D-AKAP2) is a multi-domain scaffold protein that anchors cAMP-dependent protein kinase (PKA) to specific subcellular compartments and coordinates PKA signaling with endocytic recycling and transporter regulation. Its C-terminal AKB helix binds the dimerization/docking domains of both RIα and RIIα regulatory subunits of PKA through distinct helical registers, and a redox-sensitive disulfide in RIα modulates this interaction [PMID:9326583, PMID:20159461]. The tandem N-terminal RGS domains bind GTP-loaded Rab4 and Rab11 on recycling endosomes, controlling transferrin receptor trafficking and endosomal compartment morphology [PMID:19797056], while a C-terminal PDZ-binding motif recruits PDZK1 to form a ternary complex with PKA RII that couples phosphorylation to apical membrane transporter regulation in renal proximal tubule cells [PMID:14531807, PMID:25348485]. Heterozygous disruption of Akap10 in mice increases cardiac myocyte sensitivity to cholinergic stimulation, causes arrhythmias, and leads to premature death, establishing AKAP10 as a critical regulator of cardiac autonomic signaling [PMID:17485678].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"The discovery that D-AKAP2 binds both RIα and RIIα via a C-terminal domain established it as the first characterized dual-specificity AKAP and raised the question of how a single helix accommodates two structurally distinct R-subunit classes.\",\n      \"evidence\": \"Yeast two-hybrid screen with RI/RII baits followed by reciprocal coprecipitation with domain truncations\",\n      \"pmids\": [\"9326583\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis for dual specificity was unknown\",\n        \"Function of the N-terminal putative RGS domain was uncharacterized\",\n        \"Subcellular localization of full-length protein was not determined\"\n      ]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Localization of full-length D-AKAP2 to mitochondria and confirmation of in vivo PKA association in brain placed the scaffold at a defined organelle, but left its non-PKA functions unresolved.\",\n      \"evidence\": \"Immunocytochemistry, subcellular fractionation, and cAMP-agarose pull-down from mouse brain\",\n      \"pmids\": [\"11248059\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mitochondrial targeting mechanism was not identified\",\n        \"RGS domain ligands remained unknown\",\n        \"Functional consequence of mitochondrial PKA anchoring was not tested\"\n      ]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Biophysical mapping by deuterium exchange revealed that D-AKAP2 consists of two independently folded domains — an RGS-containing region and a PKA-binding/PDZ-motif region — establishing a modular scaffold architecture that could integrate distinct signaling inputs.\",\n      \"evidence\": \"DXMS and limited proteolysis of purified D-AKAP2\",\n      \"pmids\": [\"12206784\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Identity of PDZ-domain binding partners was unknown\",\n        \"Whether the two domains function independently or cooperatively was unclear\"\n      ]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identification of PDZK1 as a binding partner for D-AKAP2's PDZ motif explained how the scaffold positions PKA near apical membrane transporters such as NaPi-IIa, linking it to PTH-regulated phosphate transport in the kidney.\",\n      \"evidence\": \"Yeast two-hybrid, pull-down, and co-immunoprecipitation in OK cells plus immunohistochemistry of renal tissue\",\n      \"pmids\": [\"14531807\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether PKA phosphorylation of NaPi-IIa is directly mediated through this complex was not shown\",\n        \"Relative contribution of NHERF-1 versus PDZK1 interaction in vivo was unresolved\"\n      ]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Gene-trap disruption of Akap10 in mice demonstrated that loss of the PKA-anchoring domain causes enhanced cholinergic responsiveness, cardiac arrhythmias, and premature death, providing the first in vivo evidence that AKAP10 scaffolding is essential for normal cardiac autonomic signaling.\",\n      \"evidence\": \"Gene-trap knockout mice phenotyped by ECG monitoring, contractility assays, and mESC-derived cardiomyocyte analysis\",\n      \"pmids\": [\"17485678\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Molecular pathway downstream of PKA in cardiomyocytes was not delineated\",\n        \"Whether cardiac phenotype involves RGS-domain or PDZ-motif functions was not tested\",\n        \"Cell-type specificity of the cardiac requirement was not resolved\"\n      ]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"The demonstration that D-AKAP2's tandem RGS domains bind GTP-Rab4 and Rab11 — an unprecedented RGS–small-GTPase interaction — and regulate endosomal recycling revealed that AKAP10 integrates PKA signaling with membrane trafficking.\",\n      \"evidence\": \"Co-immunoprecipitation, GTP-pulldown, colocalization microscopy, and RNAi knockdown with transferrin recycling assays\",\n      \"pmids\": [\"19797056\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether the RGS domains possess GAP activity toward Rab4/Rab11 was not established\",\n        \"Coupling between Rab-binding and PKA-anchoring functions on the same scaffold was not tested\",\n        \"Structural basis of RGS–Rab recognition was not determined\"\n      ]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Crystal structures of the RIα D/D domain bound to the D-AKAP2 AKB helix resolved how dual specificity arises: the helix shifts its register compared to the RIIα complex, and a redox-sensitive disulfide in RIα modulates binding affinity, introducing a regulatory dimension to AKAP–PKA interaction.\",\n      \"evidence\": \"X-ray crystallography of RIα D/D alone and in complex with the D-AKAP2 AKB peptide\",\n      \"pmids\": [\"20159461\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Physiological relevance of redox-regulated AKAP binding was not tested in cells\",\n        \"Whether the helical register shift alters downstream signaling output was unknown\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"The ternary complex crystal structure of D-AKAP2–PKA RII–PDZK1 showed that PKA binding nucleates a polyvalent scaffold where an intrinsically disordered segment adopts both α-helical (AKB) and β-strand (PDZ ligand) conformations, and that binary PKA engagement is prerequisite for high-affinity PDZK1 recruitment, establishing an ordered assembly mechanism.\",\n      \"evidence\": \"X-ray crystallography of the reconstituted ternary complex\",\n      \"pmids\": [\"25348485\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether ordered assembly occurs in vivo and is regulated by cAMP levels was not shown\",\n        \"How PKA catalytic subunit release from this ternary complex affects transporter phosphorylation was not addressed\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The relationship between AKAP10's Rab-binding and PKA-anchoring activities on the same molecule, the structural basis of RGS–Rab recognition, and the downstream PKA substrates mediating the cardiac phenotype remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No structure of the RGS–Rab complex exists\",\n        \"PKA substrates relevant to cardiac rhythm regulation via AKAP10 are unidentified\",\n        \"Whether Rab-dependent trafficking and PKA anchoring are coordinated on the same scaffold in vivo is untested\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 3, 5, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 4, 6]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"complexes\": [\n      \"D-AKAP2–PKA RII–PDZK1 ternary complex\"\n    ],\n    \"partners\": [\n      \"PRKAR1A\",\n      \"PRKAR2A\",\n      \"PDZK1\",\n      \"RAB4A\",\n      \"RAB11A\",\n      \"SLC9A3R1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}