Apelin-13 induces lordosis behavior in estradiol-primed ovariectomized rats via the activation of multiple protein kinases in the ventromedial hypothalamus.

Glycosylated Shikonin derivative Z-1-2 inhibits triple-negative breast Cancer growth and metastasis through glycerophospholipid metabolism modulation

Medulloblastoma (MB) is the most common childhood brain cancer, with Group 3 (G3) as the most aggressive subgroup, being prone to relapse and treatment resistance. A small subset of stem-like cells contributes to this recurrence, but the mechanisms behind their transformation are not fully understood. In this study, we employed therapeutically relevant in vitro and in vivo chemoradiotherapy (CRT) models of G3 MB and discovered a significant activation of SRC kinase following CRT treatment, while other kinases such as AKT and ERK were unaffected. Remarkably, SRC activation was exclusive to G3 MB cells and was absent in the less aggressive Sonic Hedgehog and Group 4 MB, as well as in normal brain cells. SRC activation in CRT-treated G3 MB cell and tumors corresponded with increased stemness, as evidenced by elevated levels of stemness factors SOX2, NOTCH1, OCT4, Nanog and phosphorylated STAT3, alongside a reduction in the differentiation marker βIII-tubulin/TUBB3. Conversely, SRC knockout or pharmacological inhibition promoted differentiation and reduced aggressiveness in CRT-resistant G3 MB cells, which could be rescued by re-expression of SRC in SRC knockout cells. Additionally, SRC inhibition significantly reduced the viability of CRT-treated G3 MB cells by inducing both apoptosis and necroptosis, while sparing the proliferation and stem-like properties of normal neural stem cells, indicating a promising toxicity profile. Importantly, in a therapeutically relevant orthotopic G3 MB model, administration of the re-purposed blood-brain-barrier permeable SRC inhibitor, Saracatinib, in conjunction with CRT, significantly reduced tumor burden and improved animal survival compared to CRT treatment alone without any neurotoxic side effects. Overall, our results underscore the pivotal role of SRC in enhancing stemness and aggressive behavior in CRT-resistant recurrent G3 MB. Targeting SRC not only promotes cell death through apoptosis and necroptosis but also encourages differentiation, positioning it as a promising therapeutic target for rapid clinical interventions.

New World arenaviruses (NWAs) that cause viral hemorrhagic fever, such as Junín virus, have few therapeutic options. Entry of these viruses into cells is mediated by binding to cell surface receptors, followed by endocytosis and trafficking to a low pH compartment. We showed previously that Signal Regulatory Protein Alpha (SIRPA), a critical cell surface receptor that inhibits macrophage phagocytic activity, decreases internalization by NWAs as well as other pathogenic RNA viruses that traffic to low pH compartments. Here we demonstrate that proteins involved in the SIRPA/integrin signaling axis, including Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2), src family kinases (SFKs), particularly FYN, focal adhesion kinase (FAK), and alpha-integrin play a role in viral endocytosis and that SIRPA inhibits virus entry through blocking this pathway. In addition to defining a role for integrins in viral entry, these studies also provide additional insight into SIRPA's interference in processes dependent on integrin signaling, including phagocytosis. Moreover, using drugs that block the integrin signaling pathway in vitro and in vivo , we show that there are additional steps that may be targeted therapeutically for inhibiting infection by RNA viruses that traffic to acidic compartments.

Dasatinib exhibits broad therapeutic potentials for several diseases; however, its clinical application is limited by safety concerns and unfavorable pharmacokinetics. Structural optimization of dasatinib yielded PDD-87. Preclinical studies evaluated kinase inhibition, antiproliferative activity in leukemia and lymphoma cell lines, antitumor effects in a xenograft model, pharmacokinetics and metabolism in vitro and in rodents, and exploratory safety in mice. Here we show that PDD-87 strongly inhibits ABL and BTK along with their key mutants, and SRC family kinases (IC₅₀ <1 nM), displaying potent antiproliferative effects against several leukemia and lymphoma cell lines. In a K-562G mouse xenograft model, PDD-87 significantly reduces tumor growth in a dose-dependent manner. Compared to dasatinib, PDD-87 exhibits lower clearance (405 vs 989 mL/hr/kg), reduced volume of distribution (0.9 vs 2.4 mL/hr/kg), higher plasma exposure (2521 vs 190 hr*pmol/mL for oral dose), and improved oral bioavailability (18.9 vs 3.9%) in rats. PDD-87 demonstrates lower metabolic clearance than dasatinib in human, mouse, rat, and dog liver microsomes. Metabolically, PDD-87 undergoes hydroxylation on the 2-chloro-6-methylphenyl ring, followed by sulfation, and glucuronidation. Significantly, PDD-87 shows markedly lower lung accumulation than dasatinib, suggesting a reduced risk of pulmonary toxicity. The lung/plasma ratios of Cmax and AUClast for PDD-87 are 2 and 4, respectively, compared to 12 and 22 for dasatinib. Additionally, PDD-87 is well-tolerated in mice at doses up to 200 mg/day for two weeks, with no overt toxicity. PDD-87 is a highly potent, selective kinase inhibitor with improved pharmacokinetics and safety potentials, holding promise for broader clinical applications.

Alcohol-associated liver disease (ALD) remains a major public health challenge with limited treatment options. NF-κB-driven inflammation in Kupffer cells (KCs) plays a central role in ALD, but the upstream regulators remain poorly understood. Here, we identify the tyrosine kinase SRC as a key mediator of ALD. Chronic ethanol exposure activates SRC in KCs, which directly phosphorylates ubiquitin-conjugating enzyme 9 (UBC9), the only E2 SUMO enzyme, at tyrosine-68 (Y68). This modification enhances NF-κB signaling and increases proinflammatory cytokines (TNF-α, IL-6, and IL-1β). These cytokines then promote hepatic lipogenesis through SREBP1c- and CEBPβ-dependent induction of FASN and ACC. Inhibition of UBC9 phosphorylation by gene editing or SRC inhibitor reduces NF-κB-dependent inflammation and lessens ethanol-induced liver injury in mouse models. These findings uncover a previously unrecognized SRC-UBC9-NF-κB axis that drives inflammation in ALD and highlight it as a potential therapeutic target in liver disease.

Kidney fibrosis is a hallmark of chronic kidney disease (CKD), yet its underlying mechanisms remain incompletely understood. Retinoic acid receptor responder protein 1 (RARRES1) is largely restricted to podocytes in healthy kidneys but was upregulated within the tubulointerstitium in CKD. However, its functional contribution to kidney fibrosis remains unclear. To assess the link between tubulointerstitial RARRES1 expression, estimated glomerular filtration rate (eGFR), and fibrosis severity in CKD patients, we analyzed multiple clinical datasets. RARRES1 upregulation in proximal tubular epithelial cells was confirmed in human CKD samples by immunofluorescence and RNAscope assays. Kidney fibrosis was evaluated in proximal tubule-specific Rarres1 knockout mice and RARRES1-overexpressing mice following CKD induction, including unilateral ureteral obstruction (UUO) and folic acid-induced nephropathy. Mechanistically, mass spectrometry and co-immunoprecipitation, combined with truncation mutants, uncovered interactions among soluble RARRES1, KHDRBS1, Src kinase, and phosphorylated STAT3. Pharmacological blockade of STAT3 or Src kinase were used to evaluate the reversal of RARRES1-induced fibrotic phenotype. Multiple datasets revealed that tubulointerstitial RARRES1 expression correlated with decreased eGFR and increased fibrosis severity in CKD patients. Immunofluorescence and RNAscope confirmed RARRES1 upregulation specifically in proximal tubular epithelial cells in CKD. Proximal tubule-specific knockout of Rarres1 significantly attenuated kidney fibrosis in two independent CKD models. Conversely, RARRES1-overexpressing mice showed aggravated kidney fibrosis compared to controls in the UUO model. Soluble RARRES1 was identified as the key pathogenic form, with its plasma levels correlating with declining kidney function in CKD patients. Mechanistically, soluble RARRES1 bound KHDRBS1, recruited Src kinase, and induced STAT3 phosphorylation at Tyr705, leading to upregulation of pro-fibrotic factors. Inhibition of STAT3 or Src kinase partially reversed the fibrotic phenotype induced by RARRES1 overexpression. Our findings demonstrated that RARRES1 played an important role in regulating kidney fibrosis through the KHDRBS1-Src-p-STAT3 signaling axis.

Recent studies have reported a correlation between SRC and EGFR as key factors contributing to tumor aggressiveness in cancers, such as glioblastoma, colon, breast, and lung cancers. Resistance to therapy remains a major obstacle in cancer treatment. Therefore, the discovery of novel compounds with inhibitory potential is crucial. In this study, urea- and pyrimidine-containing compounds structurally similar to osimertinib were designed as potential inhibitors of both SRC and EGFR kinases, with the aim of identifying compounds that may also overcome resistance conferred by mutations. The compounds were synthesized through the development of new synthetic routes. Their structure-activity relationships (SAR) were evaluated using in vitro enzyme inhibition assays, cell culture experiments, molecular docking, and molecular dynamics studies. Compounds 19, 20, and 21, which bear substitutions at the third position of the indole ring, inhibited SRC kinase with 77.75-89.22% activity. These compounds also demonstrated notable cytotoxicity against the PC3 cell line, with IC50 values of 7.89, 6.92, and 9.85 μM, respectively, comparable to reference compounds cisplatin (IC50 = 5.16 μM) and dasatinib (IC50 = 0.9 μM). Notably, compound 20 was active against both EGFR and SRC kinases, with IC50 values of 3.91 μM and 0.00058 μM, respectively. Compound 20 also exhibited the strongest cytotoxic effect on prostate cancer cells (IC50 = 6.92 μM). Further analyses indicated that compound 20 induced apoptosis in cancer cells by increasing the levels of caspase-3, caspase-8, and Bax, while reducing Bcl-2 expression. Molecular docking and dynamics studies revealed strong interactions of compound 20 with the target receptors. Docking and biological activity studies indicated that compound 20 (1-(2- Fluoro-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidine-2-yl)amino)phenyl)-3- phenylurea) is a promising dual inhibitor of both EGFR and SRC kinases. In silico analyses further support the potential therapeutic efficacy of compound 20. Overall, compound 20 emerged as the most promising candidate from this study, warranting further investigation for its therapeutic potential.

Endoplasmic reticulum stress-mediated epithelial-mesenchymal transition contributes to cardiac biomarker-induced kidney injury.

Specialized high-capacity mitochondria fuel cell invasion.

Arenaviruses encompass a diverse group of infectious agents, some of which, like Lassa virus (LASV), pose significant threats to global public health due to their acute virulence, while others, such as lymphocytic choriomeningitis virus (LCMV), are less pathogenic but still clinically relevant. Both Old World arenaviruses LASV and LCMV exploit host tyrosine kinase signaling to establish infection, though the molecular mechanisms remain incompletely understood. In this study, phosphorylated receptor tyrosine kinase antibody array screening revealed that recombinant LASV (rLCMV-LASV GP) infection specifically activates Src family kinases (SFKs), Fyn and Lyn, with their phosphorylation levels markedly elevated during early infection. Subsequent co-immunoprecipitation assays confirmed rLCMV-LASV GP-induced tyrosine phosphorylation of Fyn and Lyn activation. RNAi experiments demonstrated that knockdown of Fyn/Lyn significantly suppressed rLCMV-LASV GP and LCMV nucleocapsid protein expression, viral RNA synthesis, and viral production, highlighting the critical role of SFKs in viral infection. Further investigations showed that saracatinib, an Src inhibitor, exhibited broad-spectrum antiviral activity against LCMV strains, as well as the authentic LASV strain Lassa_HX strain. In vivo studies revealed that saracatinib substantially reduced viral loads in splenic and hepatic tissues and alleviated infection-associated histopathological damage. These findings identify Src family kinase Fyn/Lyn as novel host targets for arenavirus and provide new targets of prevention and treatment for arenaviral infection.IMPORTANCEArenaviruses, including the highly pathogenic Lassa virus (LASV) and clinically relevant lymphocytic choriomeningitis virus (LCMV), pose severe global health threats due to limited therapeutic options. This study unveils a critical host-pathogen interaction mechanism by which Src family kinases (SFKs) Fyn and Lyn facilitate LASV and LCMV infection. Through phosphorylation profiling and functional validation, we establish that viral activation of these kinases drives arenavirus infection. Moreover, the FDA-approved Src inhibitor saracatinib exhibits broad-spectrum efficacy, suppressing LCMV strains as well as the authentic LASV in vitro, while reducing LCMV viral loads and histopathology in vivo. These findings redefine tyrosine kinases as host targets, offering a novel host-directed antiviral strategy by repurposing existing clinical compounds.

Microscale engagement of the hemi-immunoreceptor tyrosine-based activation motif-containing receptor Dectin-1 by fungal particles activates Src-family kinases (SFKs) and Syk, drives second-messenger generation, and induces downstream Erk and Akt signaling and proinflammatory responses in macrophages. To avoid inappropriate activation in the absence of a pathogenic threat, macrophages restrict signaling in response to low-valency ligands. To examine how SFKs regulate this sensitivity threshold, we compared signaling induced by pharmacological SFK activation with signaling triggered by depleted zymosan, a high-valency β-glucan particle that engages Dectin-1 to form a phagocytic synapse. We found that particulate engagement of Dectin-1 protected the inhibitory ITIM-associated phosphatase SHIP1 from phosphorylation by SFKs, allowing robust activation of Erk and Akt and proinflammatory induction. In contrast, receptor-independent SFK activation induced phosphorylation of SHIP1 and failed to amplify signaling downstream of PLCγ2 and PI3K. Although multiple SFKs could phosphorylate SHIP1, Lyn uniquely maintained the basal set-point of SHIP1 phosphorylation, thereby keeping PIP3 levels low and suppressing basal Erk and Akt signaling. This Lyn-dependent regulation was essential for suppressing Akt activation and balancing signaling through the Erk and Akt pathways in the absence of a phagocytic synapse. In contrast, antimicrobial responses to particulate stimuli, including second-messenger signaling, Erk/Akt, and proinflammatory outputs, did not strictly require Lyn expression. These findings highlight the unique role of Lyn in limiting spurious proinflammatory signaling and shed light on a mechanism by which macrophages selectively respond to high-valency particulate ligands that override this basal inhibitory program.

Among the diverse mechanisms driving cancer progression, dysregulated kinase signaling and immune evasion mechanisms are particularly critical, necessitating the development of therapies that simultaneously target kinase-dependent tumor survival pathways and reinvigorate antitumor immunity. Oleuropein (OP), a natural polyphenol with established chemopreventive activity, exhibits limited potency and bioavailability. To overcome these limitations, a series of structurally optimized semi-synthetic OP analogs were developed and screened for their anticancer activity. The most prominent, Ole-4, was further evaluated for its antitumor mechanism of action. Ten OP analogs were screened against a panel of human and mouse cancer cell lines. The most active, Ole-4 was further assessed for its cytostatic and pro-apoptotic effects in breast cancer cells using proliferation assays, cell-cycle analysis, and apoptosis quantification. Kinome profiling was performed to identify alterations in protein tyrosine (PTK) and serine/threonine (STK) kinase activity induced by Ole-4. C57BL/6J mice bearing subcutaneous B16.F1 melanoma tumors and BALB/c mice bearing CT26 colon and D2F2/E2 breast carcinomas, were treated intraperitoneally with Ole-4 to evaluate its in vivo efficacy. Immune activation and tumor infiltration by effector cells were analyzed by flow cytometry and immunohistochemistry. In vitro, the OP analog Ole-4 exhibited potent cytotoxicity against breast, ovarian, melanoma, cervical and colon carcinoma cell lines. Specifically in HER2+ breast cancer cells, Ole-4 significantly inhibited proliferation by inducing S-phase arrest and triggering apoptosis. These effects correlated with kinome profiling data, indicating sustained functional suppression of the STKs Akt and P70S6KB, key regulators of cell survival and growth. Likewise, Ole-4 reduced the activity of Src, ROCK1, and EphA2 kinases which are critically implicated in tumor metastasis, and enhanced the RAF/ERK and Jak1b signaling axes, closely linked to stress-adaptive responses. In vivo, Ole-4 suppressed melanoma, colon and breast carcinoma growth in mice subcutaneously inoculated with syngeneic B16.F1, CT26 and D2F2/E2 cancer cells. Additionally, Ole-4 generated immune cell-mediated antitumor cytotoxicity in treated mice, and importantly, increased tumor infiltration by cytotoxic T and natural killer cells. Our findings suggest that Ole-4 exerts direct anticancer effects, likely mediated via STK-dependent mechanisms, while indirectly it enhances immune effector responses, sensitizing tumor cells to immune-mediated apoptosis. Based on these tumor inhibitory and immunomodulatory properties, Ole-4 emerges as a promising candidate for designing next-generation anticancer drugs.

Polymorphonuclear neutrophils (PMNs) serve as frontline defenders against injury and infection, eliminating pathogens and initiating mucosal tissue repair. However, excessive PMN transepithelial migration (TEpM) contributes to chronic mucosal inflammatory disorders, including inflammatory bowel disease. PMN pro-inflammatory and pro-repair functions are regulated by incompletely defined signaling cascades involving kinases and phosphatases. Here, we determined how the protein tyrosine phosphatase CD45/PTPRC regulates PMN trafficking and effector functions in the gut. Pharmacologic inhibition of CD45 significantly reduced PMN colonic TEpM in vitr o and in vivo and decreased intestinal PMN trafficking was observed in transgenic mice with PMN-specific deletion of CD45 ( MRP8-Cre;Cd45 fl /fl ). Beyond limiting TEpM, CD45 depletion impaired key antimicrobial functions, including degranulation and phagocytosis, indicating broader effects on PMN effector activity. Importantly, recovery from dextran sodium sulfate (DSS)-induced colitis and biopsy-induced colonic wounding was delayed in MRP8-Cre;Cd45 fl /fl mice, linking altered PMN function to defective mucosal healing. Mechanistically, CD45 depletion reduced surface expression of the β2 integrin CD11b/CD18 and inactivated the Src family kinase member Lyn. Together, data highlight a novel CD45-CD11b-Lyn signaling axis that regulates PMN trafficking and effector functions in the intestine and identify CD45 as a promising target for modulating PMN function to promote mucosal tissue repair.

Background/Objectives: Epstein-Barr virus (EBV) is associated with a subset of gastric carcinomas characterized by latency programs that promote survival of infected cells. EBV-encoded BamH I A rightward transcript (BART) microRNAs contribute to apoptosis resistance in infected epithelial cells. This study investigated whether dasatinib, a Src family kinase (SFK) inhibitor, selectively targets EBV-positive gastric epithelial cells and examined the molecular mechanisms underlying this effect. Methods: EBV-positive and EBV-negative gastric epithelial cell models were analyzed to evaluate cell viability, apoptosis induction, signaling pathways, and viral gene regulation. BART miRNA expression was quantified by RT-qPCR, and promoter activity was examined using luciferase reporter assays. Downstream target gene expression was analyzed at both the transcript and protein levels. Recombinant EBV lacking BZLF1 or LMP2A was used to assess the contributions of lytic activation and LMP2A-associated signaling. Results: Dasatinib preferentially reduced viability and induced apoptosis in EBV-positive gastric epithelial cells compared with EBV-negative counterparts. Treatment suppressed phosphorylation of Src and ERK and reduced expression of the anti-apoptotic proteins BCL-xL and MCL1. Apoptosis was also observed in cells infected with LMP2A-deficient EBV, suggesting that the effect cannot be fully explained by inhibition of LMP2A-associated signaling. Dasatinib inhibited BART miRNA promoter activity and reduced pri-, pre-, and mature miR-BART levels, accompanied by increased expression of pro-apoptotic target genes including CASZ1a, OCT1, ARID2, TP53INP1, and DAB2. In parallel, dasatinib suppressed BZLF1 promoter activity without evidence of lytic reactivation. Conclusions: Dasatinib promotes apoptosis in EBV-positive gastric epithelial cells in association with coordinated suppression of SFK signaling and EBV-encoded BART miRNA expression, accompanied by derepression of pro-apoptotic cellular genes. These findings reveal a previously underappreciated vulnerability of EBV-positive epithelial cells and suggest that targeting host kinase signaling pathways that regulate viral microRNAs may represent a potential therapeutic strategy for EBV-associated malignancies.

Tyrosine kinases (TKs) are frequently mutated or overexpressed in cancer, and TK inhibitors (TKIs) are an important therapeutic modality against TK-driven cancers, but many patients show an underwhelming response to TKIs prescribed on the basis of tumor genotype. To find cell-intrinsic TK signaling patterns which might be predictive of poor response to TKI therapies, we used high-sensitivity multiplexed mass spectrometry to quantify endogenous levels of 1,222 phosphotyrosine (pY) sites across the proteomes of TK-driven human cancer cell lines with variable response to genotype-matched TKIs. In direct comparisons between TKI-tolerant and TKI-sensitive lines with a common driver TK, we found that TKI treatment was equally effective at blocking driver TK signaling, and higher basal activity of the driver TK did not always predict higher sensitivity to TKI. All tolerant lines showed a dampened proteome-wide pY response to TKI exposure compared to sensitive lines, suggesting that tumor cells with more robust TK signaling are less vulnerable to driver TK blockade. We found that each tolerant line depends on a unique set of compensatory TKs and signaling axes but are unified by hyperactivity of at least one of the SRC family kinases (SFKs) or the related ABL1/2 kinases, both at rest and under TKI treatment, despite the absence of SFK or ABL genetic mutations. In time- and dose-resolved drug combination experiments, SFK/ABL inhibitors were potently synergistic with all TKIs tested, demonstrating that elevated SFK/ABL signaling is a conserved bottleneck for maximal TKI efficacy which could be exploited therapeutically.

Keratinocyte carcinoma (KC) rates continue to increase, despite current prevention strategies. Effective treatment of actinic keratoses (AKs) can reduce the risk of subsequent KC; however, the prevalence of AKs precludes treating all individuals. Recently, we identified a subset of human papillomavirus 8 (HPV8)-associated AKs. Hence, further stratification is needed to better identify patients at high risk of KC. To determine the association of HPV8 with subsequent KC and identify specific treatment. Patients with a prior history of pathologist-proven AK were recruited. Histology samples were analysed for HPV8, and medical records were reviewed for KC. HPV8-associated human AK, cell lines and mouse models were interrogated for expression of Src family kinases. The in vitro and in vivo biologic effects of Src inhibition were determined using small interfering RNA and tirbanibulin. Sixty-one patients with AK without a history of antecedent KC were divided into those with (n = 31) and without HPV8 (n = 30) infection. Using multivariable Cox regression with data adjusted for sex, age and body site, patients with HPV8-associated AKs had a greater risk of subsequent KC (hazard ratio 5.5, 95% confidence interval 2.3-12.9; P < 0.001). Independently, HPV8-associated AKs were associated with invasive squamous cell [odds ratio (OR) 32.0; P < 0.001] and basal cell carcinoma (OR 4.5; P < 0.01), and included all nine patients with multiple KCs. HPV8-associated AKs showed elevated expression of phosphorylated Src kinase. Inhibition of Src reduced cell proliferation and blocked colony-forming efficiency. In vivo, Src inhibition restored epidermal thickness and hindered the development of skin tumours. The presence of koilocytes in AK histology is indicative of a subset of patients at risk of multiple KCs. Inhibiting Src kinase blocked this HPV8-associated keratinocyte proliferation and prevented skin tumours in a mouse model.

Chayote is a fruit that has been used for centuries to treat various diseases, including hypertension. However, how the chemical compounds derived from chayote work in treating hypertension remains unclear. Integrating molecular docking and network pharmacology to elucidate the active constituents and potential mechanisms of chayote in treating hypertension. Initially, 50 active compounds from chayote and 97 key targets related to hypertension were identified through network pharmacology analysis. Then, the results of molecular docking and simulations showed: gibberellin A4; gibberellin A7; gibberellin A29; gibberellin A38; gibberellin A44; stigmasta-3,5-dien-7-one; stigmasterol and routinely overcome hypertension through the regulation of ACE, AKT1, ALB, SRC, and TNF genes. These compounds and genes may be key factors of chayote fruit in treating hypertension. Pathway enrichment analysis showed that the antihypertensive effect of chayote is regulated by the gibberellin A7 and TNF signaling pathways. These pathways are primarily associated with anti-oxidative stress, anti-inflammatory responses, and β-cell protection. This study identified the active constituents and potential signaling pathways involved in the antihypertensive effect of chayote. Result: These findings provide a theoretical basis for understanding the mechanism of the antihypertensive effect of chayote. Furthermore, this study may help develop health supplements or natural antihypertensive drugs based on chayote.

Efferocytosis is mediated by MERTK in many tissues, but the signaling pathway and molecular mechanisms used by MERTK to engulf apoptotic cells is largely unknown. Here, using mass spectrometry and super-resolution microscopy, we identified 180 nm receptor complexes comprised of MERTK, β2 integrins and several associated signaling molecules. Efferocytosis was found to be dependent on both MERTK and β2 integrins, with MERTK inducing the conformational change of β2 integrins from low to high-affinity via a PI3K-dependent pathway, with the active integrins then mediating the expansion of an efferocytic synapse around the apoptotic cell. This synapse was highly structured, with MERTK retained by ligand-induced clustering in the synapse centre, while β2 integrins and actin form a Src family kinase and FAK-dependent expanding ring that defined the leading edge of the efferocytic synapse. These findings provide new insights into the function of this crucial homeostatic receptor and provide new insights into how MERTK mutations and signaling defects might contribute to inflammatory and autoimmune diseases.

G protein-coupled receptors (GPCRs) initiate G protein-mediated signaling pathways upon ligand binding and are desensitized via β-arrestin-dependent internalization. However, emerging evidence indicates that β-arrestin also mediates G protein-independent signaling pathways that lead to distinct cellular responses. This signaling complexity has led to the development of GPCR-biased ligands that selectively modulate either G protein- or β-arrestin-dependent pathways for drug discovery, offering precise control of cellular processes with minimal side effects. We have developed biased ligands for the 5-hydroxytryptamine receptor subtype 7 (5-HT7R), which is implicated in autism spectrum disorder (ASD). Notably, we observed that a G protein-biased ligand (2b), but not a β-arrestin-biased ligand (1 g), induced a characteristic ASD behavior in mice, suggesting differential signaling pathways between G protein- or β-arrestin-mediated pathways. We thus further explored the mechanisms of β-arrestin-biased signaling pathways of 5-HT7R. Our study revealed that 1 g induced a slow but sustained activation of proto-oncogene tyrosine-protein kinase Src (Src), demonstrating temporal specificity of β-arrestin-biased signaling. Src was recruited to 5-HT7R via β-arrestin-2, followed by the internalization and accumulation of this receptor complex at endosomes. The sustained colocalization of Src at the internalized receptor complex suggests a mechanism for the slow and sustained Src activation. These results highlight spatiotemporal regulation of Src activation inGPCR-biased signaling pathways, providing new insights into 5-HT7R-targeted therapeutics.