Science Signaling

NUDT2 is an emerging candidate for therapeutic intervention in cancer, and its inhibition or loss is known to elevate adenosine-containing dinucleoside polyphosphates (ApnNs), including diadenosine tetraphosphate (Ap4A). Ap4A is a stress- and immune-associated nucleotide metabolite proposed to act as a second messenger, raising the possibility that NUDT2 targeting may unintentionally affect important nucleotide-sensitive signaling pathways. One such pathway is cGAS-STING signaling, a central innate immune axis that detects cytosolic DNA, produces the nucleotide second messenger 2'3'-cGAMP, and drives type I interferon responses. Because cGAS-STING also contributes to antitumor immunity and is being pharmacologically targeted in cancer, we asked whether sustained Ap4A accumulation perturbs this pathway. We systematically evaluated Ap4A and related dinucleoside polyphosphates across the cGAS-STING-TBK1 axis using biophysical, enzymatic, structural, and cellular approaches. Contrary to a previous model, STING did not bind Ap4A, Ap3A, or Ap4G, despite robust binding of canonical cyclic dinucleotides. Although cGAS bound these nucleotides with micromolar affinities, DNA-activated cGAMP synthesis was inhibited only at high, supra-substrate ratios. Similarly, TBK1 inhibition required extreme Ap4A ratios beyond physiologically relevant levels. In a THP-1 cell model, NUDT2 knockout caused strong Ap4A accumulation, but the resulting intracellular dinucleoside polyphosphate levels remained below the ratios required to inhibit cGAS or TBK1 in vitro. This study thus distinguishes biochemical possibility from physiological relevance and argues that NUDT2-linked Ap4A accumulation is unlikely to directly compromise cGAS-STING pathway activity.

The inhibitor of {kappa}B kinase (IKK) complex integrates diverse cellular inflammatory responses, and induces transcription factor NF-{kappa}B. The molecular mechanism by which IKK becomes catalytically active in response to signaling remains unclear despite structural knowledge of the individual IKK1/, IKK2/{beta}, and NEMO/IKK{gamma} protein components within its hetero-oligomeric assembly. Cryo-EM of the IKK2/{beta} homodimer bound to an associating NEMO/IKK{gamma} protein fragment, reveals multiple conformers. Mutual exclusivity of dimeric conformers, canonical versus alternate, is reflected in and dependent upon order-to-disorder transition of the canonical 6-helical bundle dimerization interface. Correlation of this unusual structural plasticity of IKK2/{beta} with its biochemical and cellular activities suggests mechanistic possibilities for how association with its partner scaffold protein NEMO/IKK{gamma} and polyubiquitin chains might dictate catalytic activation of IKK through distinct IKK2/{beta} conformers.

Src family kinases (SFKs) share highly conserved catalytic domains yet display distinct biological functions, raising the question of how substrate specificity is achieved. Here, we investigate the molecular basis of differential ITAM recognition by Lck and Src, combining cellular assays with structural analysis and docking simulations. In-cell assays demonstrated that, contrary to Lck, Src was completely incapable of phosphorylating the TCR ITAMs when ectopically expressed in a T cell environment. Domain-swapping experiments further revealed that substitution of the Src kinase domain with that of Lck was sufficient to confer ITAM phosphorylation and trigger downstream TCR signaling responses, whereas exchange of adaptor domains had minimal effect. Comparative structural analysis revealed that, despite their overall conserved fold, Lck exhibits a more open and solvent accessible pocket located between the N- and C-lobes of the kinase domain, adjacent to the activation loop, compared to Src. Consistent with this, docking simulations showed that Lck accommodates ITAM peptides in multiple favourable conformations, whereas Src displays a markedly reduced number of non-productive binding poses. Residue-level contact analysis identified a defined interaction surface in Lck, spanning the inter-lobal regions and activation loop. Our results highlight the importance of kinase domain conformational landscape in shaping substrate selectivity and have implications for the rational design of selective SFK inhibitors.

Pyk2 and Src are non-receptor tyrosine kinases that assemble into a complex to promote mutual activation. The Pyk2 FERM domain mediates autoinhibition by direct interaction with the kinase. Src autoinhibition is maintained by intramolecular interactions of the SH3 and SH2 domains with internal ligands, including an inhibitory C-terminal phosphotyrosine. FERM disengagement permits Pyk2 autophosphorylation of the FERM-kinase linker to generate a scaffolding site for Src recruitment. Although Src-mediated phosphorylation of the Pyk2 activation loop is well-established, the mechanism by which complex formation activates Src remains unclear. We reconstituted defined phosphorylation and regulatory states of both kinases, combining site-directed mutagenesis with phosphosite-resolved activity profiling to dissect the reciprocal regulation. Src phosphorylates the Pyk2 activation loop via an ordered, self-primed dual phosphorylation mechanism. Although activated Pyk2 productively phosphorylates the Src activation loop, Pyk2-Src complex formation does not significantly increase activation loop phosphorylation above the rate of Src autophosphorylation alone. Conversely, autoinhibited Src strictly requires Pyk2 scaffolding engagement to productively phosphorylate Pyk2. Pyk2 therefore relieves Src autoinhibition by presenting competing scaffolding sites to disengage intramolecular SH3 and SH2 conformational constraints. The results distinguish activation loop phosphorylation from the conformational competence required for substrate phosphorylation. We propose that autophosphorylated Pyk2 functions principally as a conformational activator and scaffolding platform, opening Src for phosphatase-mediated removal of the inhibitory C-terminal phosphosite and committing both Pyk2 and Src to downstream substrate phosphorylation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/739604v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@16915f9org.highwire.dtl.DTLVardef@21b937org.highwire.dtl.DTLVardef@6d641org.highwire.dtl.DTLVardef@dc6d6c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Because ErbB receptors play distinct roles in regulating diverse cellular functions, the mechanisms governing ErbB receptor activation are likely to be more diverse than previously recognized. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] positively regulates ErbB1 kinase activity, but the role of PI(4,5)P2 in regulating other ErbB family members in living cells remains poorly understood. We show that disruption of PI(4,5)P2 binding enhances ErbB4 oligomerization and kinase activity while reducing both processes in ErbB1. Analysis of chimeric receptors identified the juxtamembrane (JM) regions of ErbB1 and ErbB4 as key determinants of their distinct responses to PI(4,5)P2 during receptor oligomerization and kinase activation. Furthermore, the JM-kinase module of ErbB1 is more active in the presence of PI(4,5)P2, whereas that of ErbB4 is activated by the disruption of PI(4,5)P2 binding. In contrast, the JM-kinase module of ErbB2 exhibits weak dependence on PI(4,5)P2. ErbB2 preferentially promotes ErbB4 oligomerization over ErbB1 oligomerization, thereby enhancing ErbB4 activation. Collectively, these findings identify plasma membrane PI(4,5)P2 availability and ErbB2 abundance as two factors that jointly govern ErbB receptor oligomerization and activation.

Toll-like receptor (TLR) signaling must be activated rapidly and then terminated to support host defense without sustained inflammation. We developed a rule-based model of mouse macrophage TLR4 signaling at the molecular-interaction level using measured protein copy numbers, RNA-seq-based abundance estimates, literature- and structure-informed reaction rates, and 979 dynamic experimental constraints. The trained model reproduced much of the TLR4-induced NF-{kappa}B and MAP kinase response but consistently failed to capture deactivation of MyD88, TRAF6-associated species, and IKK/{beta}. The recurrent model failure conveyed important biological information, localizing missing regulation to the proximal MyD88-IRAK-TRAF6 module and guiding experimental evaluation of IKK{varepsilon} and its scaffold TANK. Loss of IKK{varepsilon} enhanced transcriptional, cytokine, MAP kinase, and NF-{kappa}B responses to MyD88-specific TLR ligands. TANK deficiency produced a similar cellular phenotype and abolished stimulus-induced IKK{varepsilon} phosphorylation. Deficiency of either protein increased IRAK1 and TRAF6 ubiquitination without increasing MyD88 ubiquitination, placing the inhibitory checkpoint at or immediately downstream of the IRAK1-TRAF6 ubiquitin-signaling node. Overlapping but non-identical in vivo phenotypes further supported a shared regulatory axis with additional protein-specific functions. Our study presents a model-experiment discovery cycle where quantitative pathway discordance identifies missing biology and reveals a TANK-dependent IKK{varepsilon} checkpoint that restrains MyD88-driven inflammation.

Protein kinases are critical regulators of cellular signaling, but precise modulation of their activity remains challenging due to their high structural conservation. Here, we present de novo designed genetically encoded miniproteins capable of activating or inhibiting focal adhesion kinase (FAK) by directly targeting the kinase domain itself. Among 96 binders designed to stabilize distinct conformational states of FAK, 33 modulated kinase activity. Biochemical characterization of the four most potent modulators revealed that two designs inhibit FAK with low-nanomolar IC50 values while the remaining two potentiated FAK activity by more than two-fold. When expressed in cells, the modulators preserved the same inhibitory and activating effects observed in vitro, establishing that designed conformational binders can directly tune FAK signaling in living cells. Taking advantage of the high similarity between kinases, we redesigned the FAK inhibitors to inhibit Src kinase. Our approach establishes a versatile platform for selective and genetically encoded kinase control as a way to rewire cell signaling and as a starting point for the discovery of novel modulatory sites of kinases.

Osimertinib is the current standard-of-care for treatment-naive patients with EGFR mutation-positive advanced/metastatic non-small cell lung cancer (NSCLC), however resistance inevitably emerges. Osimertinib does not eradicate all cancer cells even in culture, leaving a long-lasting sub-population of Drug Tolerant Persister (DTP) cells that is common to many chemotherapeutics. The DTP population is non-proliferative and seemingly dormant, but resistant clones eventually emerge from the DTP state. Here we show that extensive DNA replication occurs in the DTP state and cells frequently progress through the cell cycle, though cell death is also frequent, such that cell division and cell loss are balanced and the population remains approximately constant. Cell cycling occurs with aberrant gene expression and abnormal DNA replication pattern, leading to DNA damage, extrachromosomal circular DNA formation and mitotic defects, such that replicating DTP cells are hypersensitive to low doses of ATM and ATR inhibitors. Our findings suggest that the DTP state is highly mutagenic and that targeting DNA repair in DTP cells has the potential to prevent the emergence of resistance through de novo mutations.

How cells deform, sense, and respond to mechanical cues drives physiological and disease processes ranging from development to cancer metastasis; however, unbiased approaches to identify mechanical mediators are lacking. We screened 1280 compounds to identify modulators of cancer cell deformability using a cellular filtration assay and identified 92 compounds that significantly reduced deformability of ovarian cancer cells; top hits also reduced migration and invasion. Connectivity mapping of the top 21 compounds identified NUDT5 (Nudix hydrolase 5) as a predicted mechanical mediator; transcriptomic analyses implicated NUDT5 in mechanobiology and metabolic processes. We confirmed that NUDT5 mediates intracellular ATP and cellular mechanical behaviors, including morphology and deformability. In ovarian cancer, increased NUDT5 levels were associated with higher tumor stage and worse patient survival; NUDT5 inhibition reduced migration and colony formation in vitro and peritoneal tumor burden in mice. These findings establish deformability-based screening as a platform for discovering mechanical mediators and identify NUDT5 as a therapeutic target in ovarian cancer. TeaserScreening cells based on deformability provides an unbiased approach to identify NUDT5 as a mediator of cell mechanics

T cell-based immunotherapies have remained ineffective against high-grade serous ovarian carcinoma (HGSOC). The metabolic environment of HGSOC suppresses the activity of cellular therapies, however, the metabolites that enhance or suppress T cell antitumor activity are not fully understood. Here, a pooled CRISPR-Cas9 knockout screen in primary human T cells cultured with patient-derived ascites was used to identify metabolic enzymes that inhibit effector cytokine production and cytolytic function. The screen identified PRDX1 as a negative regulator of T cell effector function. Targeted deletion of PRDX1 increased the frequency of IFN-{gamma}-producing T cells, enhanced glucose uptake, increased mitochondrial mass, and improved T cell viability under suppressive ascites conditions. Mechanistically, PRDX1 deficiency increased intracellular reactive oxygen species (ROS) and impaired autophagic flux. The effects of PRDX1 deletion enhanced aspects of T cell function, while its effects on chimeric antigen receptor (CAR)-T cell cytotoxicity were donor dependent. Collectively, this study identifies PRDX1 as a regulator of T cell activation, metabolism, and effector function in the inhibitory physiological suppressive environment of HGSOC ascites.

BackgroundJanus kinase (JAK)-Signal Transducer and Activator of Transcription (STAT) pathway is a key regulator of inflammatory signaling in ulcerative colitis (UC). While most studies have focused on JAK/tyrosine kinase 2 (TYK2) inhibitors effects on immune cell-mediated responses, their direct epithelial impact remains less known. We investigated epithelial-specific transcriptional responses to JAK/TYK2 inhibitors using patient-derived intestinal epithelial organoids (IEOs) under UC-relevant conditions. MethodsColonic IEOs from UC patients were pretreated with various concentrations of tofacitinib, upadacitinib, filgotinib, brepocitinib, and deucravacitinib for 1 hour prior to stimulation with IFN{beta}, IFN{gamma}, or IFN{lambda}1 for Western blot analysis of STAT1/3 and TYK2 phosphorylation. For transcriptomic profiling, IEOs were pretreated with upadacitinib or deucravacitinib for 16 hours, followed by 8-hour stimulation with IFN{gamma}, IFN{lambda}1, TNF, or IFN{gamma} + TNF. Bulk RNA sequencing assessed differential gene expression, and multiplex assays quantified chemokine secretion. Ki67 immunohistochemistry on colonic biopsies from healthy controls, and UC patients with and without JAK inhibitors-treatment were assessed for epithelial proliferation. ResultsIFNs induced distinct STAT1/3 and TYK2 activation, with IFN{beta}/{gamma} eliciting stronger phosphorylation than IFN{lambda}1. All JAK/TYK2 inhibitors regulated pSTAT1/3 and pTYK2, with upadacitinib most strongly inhibiting pSTAT1/3 and deucravacitinib selectively targeting pTYK2. Transcriptomic analysis revealed extensive cytokine-driven gene regulation, with IFN{gamma} + TNF eliciting the strongest response. Enrichment analysis highlighted upregulation of IFN signaling, antigen presentation, and innate immune pathways, alongside downregulation of cell-cycle processes. Drug-response profiling showed minimal transcriptional changes with upadacitinib and deucravacitinib alone. Upadacitinib broadly modulated IFNs and IFN{gamma} + TNF-regulated genes, attenuating JAK-STAT, NF{kappa}B, antiviral, and cell death pathways, while restoring genes linked to mucosal healing. Upadacitinib also reduced IFNs and IFN{gamma} + TNF-driven chemokine genes and protein secretion. In contrast, deucravacitinib showed selective, potent inhibition of inflammatory genes under IFN{lambda}1-stimulation. Both inhibitors minimally impacted TNF-driven pathways. Ki67 immunohistochemistry confirmed enhanced epithelial proliferation in JAK inhibitor-treated UC patients regardless of mucosal inflammation status. ConclusionsOur findings provide novel evidence that JAK/TYK2 inhibitors influence epithelial transcriptional programs associated with inflammation and mucosal healing. Upadacitinib demonstrated broader modulation of cytokine-driven gene networks compared to TYK2-selective inhibition. These findings provide insight into epithelial-specific drug actions and support precision approaches for UC therapy.

Repetitive cytosolic Ca2+ spikes in pancreatic acinar cells, elicited by low (physiological) concentrations of acetylcholine (ACh), cholecystokinin (CCK) and gastrin releasing peptide (GRP), control secretion of digestive enzymes, whereas high-intensity stimulation induces sustained Ca2+ elevation initiating acute pancreatitis. Since inositol trisphosphate (IP3) was discovered as an intracellular Ca2+ releasing messenger, it has been assumed that a major class of G-protein coupled receptors relies on this pathway. We have now compared the mechanisms of action of the three physiological stimulants, all acting on different receptors, but each coupled to the IP3 pathway. Low concentrations of CCK and GRP cannot elicit Ca2+ signals without co-operation of an additional intracellular mechanism. CCK-elicited Ca2+ signalling requires activation of intracellular receptors for nicotinic acid adenine dinucleotide phosphate (NAADP), whereas this is not the case for the action of GRP that nevertheless relies on the operation of CD38, the enzyme involved in the synthesis of both cyclic ADP ribose and NAADP. Even Ca2+ signals elicited by ACh are partially dependent on CD38. It is engagement of these additional non-IP3 pathways that allows low concentrations of secretagogues to elicit safe Ca2+ spiking and therefore secretion, obviating the need for potentially toxic high levels of secretagogues.

CD8+ T cells in solid cancers progressively lose anti-tumor activity, yet the cell-intrinsic mechanisms driving this loss of function remain incompletely defined. Here, we performed matched proteomic and transcriptomic profiling of dysfunctional and bystander CD8+ tumor-infiltrating T cells isolated from primary tumors of treatment-naive non-small cell lung cancer patients. Proteomic analysis revealed widespread discordance with mRNA expression, with 8% of all quantified proteins displaying differential expression exclusively at the protein level. Genetic perturbation of such differentially expressed proteins identified the chromatin remodeler CHD4 and fatty acid synthase (FASN) as cell-intrinsic regulators of T cell function. CHD4 deletion resulted in altered gene-regulatory networks that promoted effector differentiation and enhanced cytokine production. In contrast, FASN deletion preserved mitochondrial fitness and sustained T cell functionality under chronic T cell receptor stimulation. Together, these findings demonstrate that proteomic profiling uncovers regulators of T cell functionality that are not apparent from transcriptomic analyses alone, highlighting an additional layer of regulatory control.

BRAF mutant colorectal cancer (CRC) remains difficult to treat despite the clinical use of combined BRAF and EGFR inhibition, highlighting a need to define tumour-intrinsic mechanisms that limit therapeutic response. Here, using genome-wide CRISPR-Cas9 screening in BRAF-mutant CRC cells, we identify MEN1, encoding the chromatin-associated protein Menin, as a selective determinant of sensitivity to combined encorafenib and cetuximab (EC). MEN1 loss markedly enhanced EC-mediated inhibition of cell proliferation and ERK activity while having comparatively little effect in untreated cells, and re-expression of Menin restored resistance. Transcriptomic and chromatin profiling revealed that Menin supports the transcriptional response associated with MAPK signalling. Menin occupied promoters of MAPK/BRAF-responsive genes and EC treatment caused widespread displacement of Menin from chromatin. Phosphoproteomic analysis demonstrated extensive remodelling of MAPK signalling following EC treatment, whereas proximity proteomics showed that the Menin-associated protein complexes remained largely intact despite loss of Menin chromatin occupancy. Importantly, MLL1 loss did not reproduce the sensitising effect of MEN1 deletion, and pharmacological Menin inhibition with revumenib failed to phenocopy either genetic MEN1 loss or acute Menin degradation, indicating that this phenotype is independent of Menin-MLL activity. Together, these findings identify a previously unrecognised, MLL-independent role for Menin in buffering the response of BRAF-mutant CRC cells to MAPK pathway inhibition and suggest targeting Menin, rather than disruption of its interaction with MLL, may provide a strategy for enhancing the response to BRAF-targeted therapy for CRC.

Protein secretion represents a key functional output of cellular signalling, capturing dynamic responses to stimulation and pharmacological perturbation that shape immune behaviour. In macrophages, activation of Toll-like receptors (TLRs) drives tightly regulated secretion programmes that mediate inflammatory responses and provide a biologically meaningful readout of pathway activity. Whilst mass spectrometry (MS)-based secretomics enables unbiased profiling of these processes, broader application in drug discovery remains constrained by sample preparation workflows that limit scalability. Here, we describe a plate-based in-solution digestion and solid-phase extraction (ISD-SPE) workflow that enables 96-well processing of conditioned media for integrated proteome and secretome analysis from the same sample well. Benchmarking against a precipitation-based approach demonstrated comparable proteomic depth with improved quantitative reproducibility and robust performance across multiple plates. Coupled with dia-PASEF acquisition, this workflow enabled in-depth profiling of macrophage responses to TLR activation, resolving receptor-specific secretory programmes following TLR3, TLR4 and TLR7/8 activation. Extension of the approach to concentration-response studies enabled quantitative characterisation of pharmacological perturbation across intracellular and extracellular protein landscapes, revealing both shared and compartment-specific responses to TLR inhibition, as well as differences in apparent potency linked to secretion dynamics. Together, this workflow provides a scalable strategy for integrated analysis of intracellular signalling and downstream protein secretion, enabling systems-level characterisation of inflammatory responses and compound mechanisms of action.

Clinical-grade RAS inhibitors raise an unresolved question as to whether KRAS-alleles impose constraints on adaptive resistance that can be exploited therapeutically. Using daraxonrasib (RMC-6236), a multi-selective RAS(ON) inhibitor, we compared resistance mechanisms between KRASG12D and KRASG12R, alleles with fundamentally different RAS network dynamics. Daraxonrasib inhibited KRASMUT primarily through steric occlusion of effector binding, while engaging RASWT only modestly ([~]20%). KRASG12R is marked by its inability to transactivate RASWT, and it was observed that daraxonrasib resistant KRASG12R PDAC cells utilize EGFR/RASWT-GTP signaling as the dominant adaptive route. In contrast, KRASG12D resistance arose through retained KRASG12D-GTP signaling, with a decrease of cyclophilin A (CypA) protein, the binding partner required for daraxonrasib activity. The shift from KRASG12R dependence to the EGFR/RASWT conferred sensitivity to trametinib. We confirmed this clinically: a KRASG12R PDAC patient who progressed after 10 months on daraxonrasib showed intratumoral EGFR/RASWT activation, and rapid 3D-bioprinted patient-derived toroid modeling predicted sensitivity to trametinib-based combination therapy. Given the aggressive disease trajectory and lack of response to the two immediately preceding lines of therapy, sixth-line trametinib-based combination therapy achieved approximately 5 months of disease control. This patient ultimately achieved 40 months of overall survival, far exceeding the 8-12 month median for metastatic PDAC. Collectively, these data establish a framework in which allele-specific RAS network topology dictates the adaptive resistance landscape, enabling rational selection of targeted therapies with meaningful clinical benefit in metastatic PDAC. STATEMENT OF SIGNIFICANCEDaraxonrasib resistance mechanisms have allele-specific routes: CypA becomes downregulated in KRASG12D and reliance on EGFR/RASWT in KRASG12R. Rapid patient-derived toroids identified sixth-line targeted therapy strategies with an overall survival of 40 months.

Anti-angiogenic cancer therapies attempt to withhold necessary nutrients and oxygen from growing tumors by targeting the major promoters of endothelial cell (EC) angiogenesis: vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR-2). Unfortunately, these treatments are often insufficient, even when coupled with chemotherapies, and fail to significantly increase survival rates. The tumor microenvironment (TME) is mechanically distinct compared to normal tissue, including increased matrix deformations or strains caused by cancer-associated fibroblasts (CAFs). In this report, we detail the specific and independent roles of two tyrosine residues, Y1054 and Y1214, on mechanical activation of VEGFR-2. Furthermore, we characterize CAF biochemical and mechanical signaling and demonstrate how ECs exhibit decreased vessel growth when co-cultured with CAFs and treated with a contractility inhibitor. Using non-phosphorylatable VEGFR-2 mutants, we reveal Y1054 and Y1214 are each necessary for EC angiogenesis, particularly in response to strain. Overall, this research highlights the need to study how mechanics in the TME promote vessel growth and thus tumor progression, which is important to consider when developing future anti-angiogenic therapies.

Activation of Natural Killer (NK) cells depends on the stimulation of a broad range of receptors. Here we use murine NIH3T3 target cells expressing defined human NK cell ligands to stimulate different aspects of NK cell activity and identify how distinct ligand-receptor interactions regulate different aspects of NK cell activation. B7H6, MICA, and CD20-bound obinutuzumab engaging NKp30, NKG2D, or CD16, respectively, were found to be potent inducers of activity in pre-stimulated NK cells. Combination of these ligands with CD48 to also stimulate 2B4 significantly increased degranulation and cytokine secretion, while PVR engaging DNAM-1 showed favorable effects only in combination with B7H6. Activating NK cell receptors were downregulated in a ligand-specific fashion, whereas CD16 and NKp46 were found to be downregulated depending on NK cell activation induced by other receptors. Co-stimulation via 2B4 in combination with either NKp30, NKG2D, or CD16 significantly enhanced NK cells to serially degranulate and kill multiple targets. Our systematic analysis unravels the complexity of different activating NK cell receptors and provides strategies to specifically tune NK cell reactivities for therapeutic applications.

Melanocortin 2 receptor (MC2R) is a G protein-coupled receptor (GPCR) for adrenocorticotropic hormone (ACTH), and its trafficking and signalling are associated with the melanocortin receptor accessory protein (MRAP). Mutations in either MC2R or MRAP disrupt this signalling and cause familial glucocorticoid deficiency. Here, we combine native mass spectrometry (MS) and hydrogen deuterium exchange mass spectrometry (HDX MS) to uncover how MRAP association and post-translational modification status shape the conformations of MC2R. Using native MS, we demonstrate that MC2R associates with MRAP or when MRAP is depleted the protein is extensively palmitoylated at the C-terminus. ACTH binding is restricted to the MC2R MRAP complex. By contrast antagonists shift the equilibrium toward MRAP-independent receptor populations. Our HDX MS analysis shows that ACTH binding induces global stabilisation of MC2R and the MRAP N terminus, consistent with reinforcement of the receptor accessory protein interface. Antagonist binding by contrast destabilises this interface and increases dynamics in transmembrane helix 2 (TM2). Notably, TM2 destabilisation is retained when the MRAP complex is depleted. Together, MRAP association and palmitoylation define distinct MC2R assemblies with ligand-dependent dynamics, suggesting new ways to influence MC2R pharmacology.

RAS GTPases regulate cellular activity through the selective activation of effectors, yet identifying proteins directly recruited by small GTPases in cells remains challenging. Current approaches rely on extracellular stimulation or prolonged expression of constitutively active mutants, which trigger secondary signalling and negative feedback pathways. Most of the RAS superfamily also lack known upstream activators, limiting investigation of their biological functions. Here we develop SPaRTa (Sequestered Protein activation through RAS-TEV actuation), an inducible system in which activated GTPases are maintained in a sequestered state by tethered effector-binding domains that can be released by rapamycin-induced reconstitution of split-TEV protease. We first applied SPaRTa to KRAS, as despite being one of the most intensely studied proteins in biology fundamental questions regarding its effector engagement remain unresolved. KRAS became activated within minutes of proteolytic release and stimulated rapid MAPK activity. Direct visualization of effector recruitment revealed distinct responses: BRAF was rapidly recruited to the plasma membrane (PM), whereas AFDN and RASSF5 accumulated gradually. In contrast, PI3K and SHOC2 were not recruited despite robust KRAS activation, though EGF stimulation efficiently re-localized PI3K to the membrane. This suggests that activation of KRAS alone is insufficient to stimulate PI3K. Sequestration shapes signalling as both AFDN and RASSF5 are subcellularly partitioned in the nucleus, and only prolonged KRAS activation re-located these effectors to the PM. Inducible activation of a designed RHOG SPaRTa resulted in ELMO1 recruitment and robust lamellipodia formation. Our system thus provides a broadly applicable platform for defining direct GTPase-effector interactions and signalling dynamics in cells.