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Adhesion GPCR-induced ectocytosis mediates intercellular GPCR signal propagation

Nature Chemical Biology (2026)Cite this article

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Abstract

Cell–cell communications involve signal transmission from sending cells to receiving cells expressing specific receptors. Extracellular vesicles (EVs) mediate this process by transporting diverse biomolecules. G-protein-coupled receptors (GPCRs) are canonical membrane receptors that integrate various extracellular signals into intracellular responses. However, whether and how GPCRs engage in EV-mediated communications remain elusive. Here, we report that adhesion GPCRs (aGPCRs) induce the formation of migrasomes and retractosomes, two newly identified EV subtypes, through their extracellular adhesion-like domains and G12/13-protein signaling. Remarkably, activated receptors undergo ectocytosis into these EVs and are subsequently internalized by receiving cells, eliciting de novo G-protein activation. We further demonstrate that cancer-cell-derived migrasomes transfer aGPCRs such as GPR56 to endothelial cells in vitro and in vivo, thereby enhancing angiogenic potential. Together, our findings uncover that aGPCRs promote migrasome formation and provide a novel mechanism of cell–cell communications through EV-mediated intercellular spread of active GPCRs.

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Fig. 1: aGPCRs promote migrasome and retractosome formation.
Fig. 2: Adhesion-like domain is required for GPR56-induced migrasome formation.
Fig. 3: Activation of GPR56 induces migrasome formation through G12/13 protein.
Fig. 4: Migrasomes mediate the intercellular transport of GPR56.
Fig. 5: Vesicular-transported GPR56 receptors activate G-protein signaling in the recipient cells.
Fig. 6: Cancer-derived EVs transport GPR56 to promote angiogenesis.

Data availability

All data generated and analyzed in this study are included in the article and its Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank the Research Core Facilities of Life Science (Huazhong University of Science and Technology) for their assistance in functional measurements and animal experiments. We thank the group of L. Chen at Guangzhou CSR Biotech for assistance with live-cell imaging and image analysis using HIS-SIM. This work was supported by grants from the National Key R&D Program of China (2022YFE0116600 and 2021ZD0203302, to J. L.) and National Natural Science Foundation of China (32421003, 32330049 and 82320108021, to J.L.).

Author information

Author notes

  1. These authors contributed equally: Guobing Huang, Ni Li.

Authors and Affiliations

  1. Cellular Signaling Laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China

    Guobing Huang, Ni Li, Yiyang Chen, Xingrun Li, Chanjuan Xu & Jianfeng Liu

  2. Hubei Provincial Research Center for Basic Biological Sciences, Wuhan, China

    Guobing Huang, Ni Li, Yiyang Chen, Xingrun Li, Chanjuan Xu & Jianfeng Liu

  3. Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA

    X. Z. Shawn Xu

  4. Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China

    Chanjuan Xu & Jianfeng Liu

  5. Hubei Jiangxia Laboratory, Wuhan, China

    Jianfeng Liu

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  1. Guobing Huang
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  2. Ni Li
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  3. Yiyang Chen
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  4. Xingrun Li
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Contributions

J.L. and C.X. conceptualized and supervised the entire project. G.H. performed the live-cell imaging, EV purification, animal model construction and data analysis. N.L. conducted the TEM sample preparation, NTA and biochemical assays. Y.C. and N.L. were responsible for the stable cell line construction and signaling assays. X.L. assisted with the EV purification and plasmid construction. X.Z.S.X. contributed to the data interpretation and manuscript preparation. J.L., G.H. and C.X. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Chanjuan Xu or Jianfeng Liu.

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The authors declare no competing interests.

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Nature Chemical Biology thanks the anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 GPR56 induce the biogenesis of matrix-bound migrasomes and retractosomes.

a, Long-term live-cell imaging of HEK293 cells expressing GPR56-Venus, cultured on collagen-coated glass-bottom dishes, related to Supplementary Video 1. Scale bar, 10 μm. b, HEK293GPR56-mCherry cells were cultured on collagen-coated glass-bottom dishes and imaged by structured illumination microscopy (SIM). Scale bar, 10 μm. Insets show enlarged regions of interest (ROIs). White arrows in the top panels indicate the migrasomes, white arrows in the bottom panels indicate the retractosomes. c, Representative confocal images of HEK293GPR56-mCherry cells cultured on collagen-coated, Fibronectin-coated or non-coated glass-bottom dishes. Scale bar, 10 μm. Insets show enlarged ROIs. Scale bar, 10 μm.

Extended Data Fig. 2 Spatial organization of GPR56 in retraction fibers and migrasomes.

a-b, Representative confocal images of HEK293 cells co-expressing GPR56-Venus with ITGA5 (integrin α5)-mCherry (a) or TSPAN4-mCherry (b). Insets show enlarged regions. Scale bars, 10 μm. Images are representative of > 20 cells from three biological independent experiments. c, Representative confocal images of HEK293 cells co-expressing GPR56-Venus and TSPAN4-mCherry. Insets show enlarged regions of interest (ROIs). Scale bar, 10 μm. Images are representative of > 30 cells from three biologically independent experiments per condition. The white arrows indicate the migrasomes labeled by GPR56 but not TSPAN4. d, Representative TIRF-SIM images of HEK293 cells co-expressing GPR56-Venus and TSPAN4-mCherry. Scale bar, 10 μm. per condition. Insets show enlarged regions of interest (ROIs). Scale bar, 10 μm. Images are representative of > 30 cells from three biologically independent experiments per condition. The white arrows indicate the migrasomes labeled by GPR56 but not TSPAN4.

Extended Data Fig. 3 GPR56 promotes migrasomes and retractosomes formation in multiple cell lines.

Left panels: Representative confocal images of HeLa, A875 and U87 cells expressing PH-GFP or GPR56-Venus. Insets show enlarged regions. Scale bars, 10 μm. The right side demonstrates the associated statistical results. Data are presented as mean ± SEM; n = 41 (Hela-PH), n = 50 (Hela-GPR56), n = 42 (CHO-K1-PH), n = 42 (CHO-K1-GPR56), n = 54 (A875-PH), n = 52 (A875-GPR56), n = 44 (U87-PH), n = 41 (U87-GPR56) cells from three independent experiments. Statistical significance was determined by unpaired t test (two-tailed). ****P < 0.0001.

Source data

Extended Data Fig. 4 GPCRs are shed into migrasomes and retractosomes.

a, Left: Schematic of HaloGPR56 WT and HaloGPR56ΔC-tail truncate. Right: HEK293 cells expressing indicated constructs were labeled with Halo-Alexa Fluor 647 (100 nM, 30 min, 37 °C), washed, and imaged by confocal microscopy. HaloGPR56 and HaloGPR56ΔC-tail were cultured on dishes and labeled with Halo660 and imaged by confocal microscopy. Scale bar, 10 μm. Images are representative of >20 cells from three independent experiments. b, Representative confocal images of HEK293 cells co-expressing FYVE-Venus (an endosome marker) with Halo-GPR56. Insets show enlarged regions. Scale bars, 10 μm. Images are representative of >20 cells from three experiments. c, Schematic of the method used to isolate migrasomes and retractosomes adherent to the bottom of culture dishes. d, Representative TEM images of migrasomes and retractosomes collected from HEK293GPR56-mCherry cells according to (b). Scale bar, 200 nm. e, The mixture of migrasomes and retractosomes isolated from (b) were passed through a 0.45μm filter (HVLP08050, Millipore) to obtain retractosomes and quantified by Nanoparticle tracking analysis (NTA). f, Immunoblot analysis of migrasomes and retractosomes from HEK293GPR56-mCherry cells. # denotes three biologically independent experiments. Source numerical data and unprocessed blot are available in source data. g, U87 cells cultured on collagen were stained with anti-GPR56 antibody and WGA, then imaged by confocal microscopy. Insets show enlarged regions. Scale bar, 20 μm. h, Representative TEM images of EVs from U87 cells. Scale bar, 200 nm. i, Size distribution of retractosomes ( < 0.45 μm) from U87 cells measured by nanoparticle tracking analysis. j, Immunoblot analysis of migrasomes and retractosomes from U87 cells. # denotes three biologically independent experiments. Source numerical data and unprocessed blot are available in source data.

Source data

Extended Data Fig. 5 Ectocytosis of AT1R and GPR64 into migrasomes and retractosomes.

a-b, Confocal images of HEK293 cells co-expressing Halo-AT1R with GPR56-mCherry (a, upper panel), or co-expressing Halo-AT1R with TSPAN4-mCherry (a, lower panel), or co-expressing Halo-GPR64 with GPR56-mCherry (b, upper panel), or co-expressing Halo-GPR64 with TSPAN4-mCherry (b, lower panel). Scale bar, 10 μm. Images are representative of >20 cells from at least three biologically independent experiments.

Extended Data Fig. 6 GPR56-G12/13 downstream signaling regulates migrasomes formation.

a, HEK293T cells were transfected with GPR56 for 24 hours, cultured on 12-well plates, then treated with col3a1 (50 nM), AP19 (50 μM), TG2 plasmid (100 ng/well), or DHM (50 μM), before SRF-RE luciferase reporter assay. Data are presented as mean ± SEM from four biologically independent experiments and analyzed using ordinary one-way ANOVA with Tukey’s multiple comparisons test. P values from left to right: 0.0051**; < 0.0001****; < 0.0001****; 0.0026**. b, HEK293T cells were transfected with GPR56, GPR56F508A, or GPR56ΔC-tail for 24 hours before SRF-RE luciferase reporter assay. Data are presented as mean ± SEM from three biologically independent experiments and analyzed using ordinary one-way ANOVA with Tukey’s multiple comparisons test. < 0.0001****. c, Western blot analysis of Gα13 in cell body and migrasomes purified from HEK293GPR56-mCherry cells. # denotes three biologically independent experiments. Source numerical data and unprocessed blot are provided in source data. d, Representative confocal images of Gα12/13 knockout (KO) HEK293 cells expressing GPR56-mCherry alone, or together with Gα12 or Gα13. Scale bar, 10 μm. Quantification of migrasome area per cell is shown in the right panel as mean ± SEM (GPR56, n = 51; GPR56 + Gα12, n = 36; GPR56 + Gα13, n = 33) from three independent experiments. Statistical significance was determined by ordinary one-way ANOVA with Tukey’s test. ****P < 0.0001. e, Western blot analysis of GPR56 expression in U87 cells and U87 shGPR56#1, U87 shGPR56#2 stable cell lines. f, U87 cells, U87-shGPR56#1 cells, or U87-shGPR56#2 cells were cultured on the collagen-coated dishes with the treatment of DMSO, AP19, or DHM. Cells were stained with membrane dye (WGA) and observed by confocal microscopy. Scale bar, 20 μm. Quantification of migrasome area per cell is shown in the right panel. Data are presented as mean ± SEM from over 30 cells in three biologically independent experiments. The sample size (n) for each treatment is provided in the source data. Statistical analysis was performed by ordinary one-way ANOVA with Tukey’s multiple comparisons test. P values from left to right are: < 0.0001****; > 0.9999, n.s.; > 0.9999, n.s.; 0.9848, n.s.

Source data

Extended Data Fig. 7 Migrasomes transport GPR56 across cells.

a, Left panel: Schematic diagram illustrating the pH-dependent fluorescence of the pHluorin tag. The C-terminally tagged pHluorin on GPR56 fluoresces at the plasma membrane and in early endosomes, where the pH is neutral, but is quenched in acidic compartments such as lysosomes and multivesicular body. Right panel: Representative confocal images of HEK293-GPR56-pHluorin cells co-cultured with HEK293T cells expressing the membrane marker CAAX-mCherry for 12 hours. Scale bar, 10 μm. Insets show enlarged regions of interest (ROIs). b, HEK293T cells were incubated for 24 hours with purified migrasomes derived from HEK293GPR56-pHluorin cells, along with the treatment of DMSO (control), 50 μM dynasore, or 30 μM Pitstop2. Cells were stained with Hoechst and then imaged by confocal microscopy. Scale bar, 10 μm. Quantification of uptake of GPR56-positive puncta per cell is shown in the lower left panel. Data are presented as mean ± SEM from over 30 cells in three biologically independent experiments. Statistical significance was determined by ordinary one-way ANOVA with Tukey’s test. ****P < 0.0001

Source data

Extended Data Fig. 8 Vesicular transported GPR56 activate multiple G protein signaling in the recipient cells.

a, Representative confocal images of HEK293T cells expressing Gαq-GFP (green) alone, or expressing Gαq-GFP (green) with Halo-GPR56 (magenta), or expressing Gαq-GFP (green) treated with purified migrasomes (magenta) derived from HEK293GPR56-mCherry cells. Scale bar, 10 μm. Fluorescence intensity profiles along the indicated lines are shown on the right. Images are representative of >30 cells from three biologically independent experiments. b, BRET-based assessment of Gq activation in response to migrasome treatment, measured using the Gq-CASE biosensor. Data are presented as mean ± SEM from three biologically independent experiments, each performed in triplicate. Statistical significance was determined by ordinary one-way ANOVA with Tukey’s test. PBS versus Migs, **P = 0.0016; Migs versus (Migs + DHM), **P = 0.0030. c, HEK293T cells co-expressing SRF-RE with PM-p115 or Endo-p115, were co-transfected with GPR56 for 24 hours, followed by luciferase assay. Data are presented as mean ± SEM from three biologically independent experiments, each performed in triplicate. Statistical analysis was performed by ordinary one-way ANOVA with Tukey’s multiple comparisons test. ****P < 0.0001.

Source data

Extended Data Fig. 9 Characterization of Cancer-Associated GPR56 Mutants in migrasomes formation.

a, Lollipop plot of GPR56 mutations identified in glioblastoma (GBM), derived from the cBioPortal database. b, Cell expression levels of wild-type (WT) and mutants in transfected HEK293 cells, quantified by anti-Flag ELISA The transfection protocol (1 µg plasmid) was consistent with imaging experiments to ensure comparable expression conditions. Data are presented as mean ± SEM from three independent experiments. Statistical analysis was performed by ordinary one-way ANOVA with Tukey’s multiple comparisons test. P values from left to right are as follows: n.s., not significant, P = 0.9496; n.s., not significant, P = 0.1942; ****P < 0.0001; *P = 0.0207. c, SRF-RE luciferase reporter activity in HEK293T cells transfected with the indicated constructs for 24 hours. Data are presented as mean ± SEM from three biologically independent experiments, each performed in triplicate. Statistical significance was determined by ordinary one-way ANOVA with Tukey’s multiple comparisons test. P values from left to right are as follows: n.s., not significant, P = 0.7485; **P = 0.0032; ****P < 0.0001; **P = 0.0080. d-e, Representative confocal images of HEK293 cells co-expressing PH-GFP (yellow) and the indicated receptor constructs (cyan), cultured on collagen-coated dishes. Migrasome area per cell is quantified in (e). Data are presented as mean ± SEM from three biologically independent experiments. Statistical significance was determined by ordinary one-way ANOVA with Tukey’s multiple comparisons test. P values from left to right are as follows: *P = 0.0244; ***P = 0.0008; ****P < 0.0001; n.s., not significant, P = 0.5245.

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Extended Data Fig. 10 U87 cells deposit migrasomes containing GPR56 to promote angiogenesis.

a, Schematic of the large EVs and small EVs isolation workflow from U87 xenograft tumor tissues. b, Representative transmission electron microscopy (TEM) images of large and small EVs in (a). c, The size distribution of small EVs from U87 xenografts, as determined by nanoparticle tracking analysis (NTA). d, Western blot analysis of EVs isolated from U87 xenografts tumors. # denotes three biologically independent experiments. Source numerical data and unprocessed blot are available in source data. e, Representative confocal images of HUVEC cells expressing FYVE-Venus (the endosome marker) treated with purified migrasomes derived from U87GPR56-mCherry cells. Scale bar, 10 μm. f, Representative images and quantification of blood vessel tube formation by HUVECs cultured in matrix gel for 6 hours, treated with PBS, U87_migrasomes (10 μg/mL), U87-shGPR56_migrasomes (10 μg/mL), U87_migrasomes (10 μg/mL) pre-incubated with DHM (50 μM), or HUVECs expressing Endo-p115RGS treated with U87_migrasomes (10 μg/mL). Data are presented as mean ± SEM from three biologically independent experiments, each with four replicates. Statistical significance was determined using ordinary one-way ANOVA with Tukey’s multiple comparisons test. ****P < 0.0001. g, Immunofluorescence staining of tumor tissue sections from U87 xenografts with the treatment of PBS, U87_migrasomes (10 μg/mL), or U87-shGPR56_migrasomes (10 μg/mL). The left side displays representative histochemical staining images of CD31. Scale bar, 100 μm. The right side demonstrates the associated statistical results. Data are presented as mean ± SEM from n = 5 mice per group. Statistical significance was determined by ordinary one-way ANOVA with Tukey’s test. PBS vs WT_Migs, ****P < 0.0001; WT_Migs vs shGPR56_Migs, ***P = 0.006.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–6

Reporting Summary

Supplementary Video 1

Real-time monitoring of GPR56-induced migrasomes in HEK293 cells expressing GPR56–Venus by high-content analysis system equipped with a ×63 water-immersion objective, related to Extended Data Fig. 1.

Supplementary Video 2

Real-time monitoring of GPR56-induced migrasomes in HEK293 cells expressing GPR56–mCherry using TIRF-SIM, related to Supplementary Fig. 2.

Supplementary Data 1

Source data for supplementary figures.

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Huang, G., Li, N., Chen, Y. et al. Adhesion GPCR-induced ectocytosis mediates intercellular GPCR signal propagation. Nat Chem Biol (2026). https://doi.org/10.1038/s41589-026-02148-7

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