Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 May 17;10(20):eadi7024.
doi: 10.1126/sciadv.adi7024. Epub 2024 May 17.

Phosphorylation of Doc2 by EphB2 modulates Munc13-mediated SNARE complex assembly and neurotransmitter release

Hong Zhang  1 , Mengshi Lei  1 , Yu Zhang  1 , Hao Li  2   3 , Zhen He  4 , Sheng Xie  1 , Le Zhu  1 , Shen Wang  1 , Jianfeng Liu  1 , Yan Li  4 , Youming Lu  2   3 , Cong Ma  1   2
Affiliations

Phosphorylation of Doc2 by EphB2 modulates Munc13-mediated SNARE complex assembly and neurotransmitter release

Hong Zhang et al. Sci Adv. .

Abstract

At the synapse, presynaptic neurotransmitter release is tightly controlled by release machinery, involving the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and Munc13. The Ca2+ sensor Doc2 cooperates with Munc13 to regulate neurotransmitter release, but the underlying mechanisms remain unclear. In our study, we have characterized the binding mode between Doc2 and Munc13 and found that Doc2 originally occludes Munc13 to inhibit SNARE complex assembly. Moreover, our investigation unveiled that EphB2, a presynaptic adhesion molecule (SAM) with inherent tyrosine kinase functionality, exhibits the capacity to phosphorylate Doc2. This phosphorylation attenuates Doc2 block on Munc13 to promote SNARE complex assembly, which functionally induces spontaneous release and synaptic augmentation. Consistently, application of a Doc2 peptide that interrupts Doc2-Munc13 interplay impairs excitatory synaptic transmission and leads to dysfunction in spatial learning and memory. These data provide evidence that SAMs modulate neurotransmitter release by controlling SNARE complex assembly.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.. Doc2B interacts with Munc13-1.
(A) Schematic diagram showing domain organization and variant fragments of Doc2B and Munc13-1. (B) Binding of MUN to GST-Doc2B FL or its variant fragments measured by GST pull-down experiments and quantification of the binding. (C) ITC-based measurements of MUN binding to GST-Doc2B 13 to 37 (Mid, left) and to GST-Doc2B 13 to 80 (Mid-L, right). (D) ITC-based measurements of the binding affinities between GST-Doc2B FL or its variant fragments and MUN. (E) 2D 1H-15N HSQC spectra of 13C/15N-labeled Doc2B 1 to 80 before (black) and after (red) addition of MUN. Cross-peaks of residues that are chosen for mutation to detect MUN binding are labeled along with their corresponding residue number (cyan). (F) Peak intensity alteration of 13C/15N-labeled Doc2B 1 to 80 protein expressed as ratio between integrated peak volumes after (V) and before (V0) addition of unlabeled MUN. (G) Binding of Doc2B FL or its variant mutations to GST-MUN measured by GST pull-down experiments and quantification of the binding. (H) Binding of MUN or its variant fragments to GST-Doc2B FL measured by GST pull-down experiments and quantification of the binding. Red asterisk shows the band of bound MUN-BC. (I) Binding of MUN or its variant mutations to GST-Doc2B FL measured by GST pull-down experiments and quantification of the binding. Data of GST pull-down experiments are processed by ImageJ (National Institutes of Health) and presented as means ± SEM (n = 3). Statistical significance and P values were determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. *P < 0.05; ***P < 0.001; ****P < 0.0001; ns, not significant.
Fig. 2.
Fig. 2.. Doc2B inhibits MUN-catalyzed SNARE complex assembly and membrane fusion.
(A) Illustration of the FRET assay for detecting MUN-catalyzed SNARE complex assembly starting from the Munc18-1/Syx1 (1 to 261) complex in the presence of Syb2 (29 to 96), SN25, and MUN. FRET signal between BDPY-labeled Syb2 S61C (donor) and TMR-labeled SN25 S187C (acceptor) was monitored. (B to D) MUN-catalyzed SNARE complex assembly by addition of Doc2B Mid, Doc2B Mid-L, or Doc2B FL, respectively (B), addition of different concentrations of Doc2B Mid (C), and addition of MUN NFAA or Doc2B I20A, which disrupts Doc2B-MUN interaction (D). Decrease of donor fluorescence at 1500 s is shown in the column at the right of the chart. (E) Illustration of Munc13-catalyzed lipid mixing between liposomes bearing Syb2 (1 to 116) and liposomes bearing the Munc18-1/Syx1 (1 to 288) complex in the presence of SN25, Syt1 C2AB, Ca2+, and Munc13-1 (C1C2BMUN). Donor (NBD) fluorescence was monitored at 538 nm. (F to H) Munc13-catalyzed lipid mixing by addition of Doc2B Mid, Doc2B Mid-L, or Doc2B FL, respectively (F); addition of C1C2BMUN NFAA or Doc2B I20A (G). Munc13-catalyzed lipid mixing at 1000 s is shown in (H). F1, fluorescence intensity observed as a function time; F0, initial fluorescence intensity. Data are presented as means ± SEM (n = 3). Statistical significance and P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05; ***P < 0.001; ****P < 0.0001.
Fig. 3.
Fig. 3.. Presynaptic EphB2 interacts with Doc2B and phosphorylates Doc2B at Y36.
(A) Immunostaining against Doc2B (green) and EphB2 (red) in the mouse hippocampus region. Nuclear DNA was labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 500 μm. (B) Separation of pre- and postsynaptic densities from purified synaptosomes. Syn, synaptosome; Pre, presynaptic elements; Post, postsynaptic elements; Extra, extrajunctional synaptic elements. (C and D) Western blot analysis of Doc2B-EphB2 interaction following cotransfection of mCherry-tagged Doc2B and Flag-tagged EphB2 in HEK293 cells. Cell lysates were immunoprecipitated by anti-mCherry antibody (C) or anti-Flag antibody (D), followed by immunoblotting with indicated antibody. (E) Schematic representation of EphB2 constructs. (F and G) Western blot analysis of Doc2B-EphB2 interaction following cotransfection of various combinations of plasmids containing EphB2 FL, EphB2-∆SP, EphB2-∆KSP, and/or mCherry-tagged Doc2B (F), EphB2 WT, KR, and/or mCherry-tagged Doc2B (G) in HEK293 cells. (H and I) In vitro phosphorylation assay using sumo-tagged EphB2 and GST-tagged Doc2B fragments (H) and GST-tagged Doc2B Mid or Y36F (I) in the presence or absence of ATP and APase. Asterisks show bands of GST-Doc2B Mid, Mid-L, and FL, respectively. CBB, Coomassie Brilliant Blue staining. (J and K) Western blot analysis of Doc2B phosphorylation following cotransfection of mCherry-tagged Doc2B WT or Y36F and/or EphB2 (J) and EphB2 WT, KR, or YYEE and/or mCherry-tagged Doc2B (K) in HEK293 cells. (L) Detection of Doc2B phosphorylation in cultured mouse cortex neurons by application of Fc (control) or preclustered Ephrin-B3-Fc. (M and N) Western blot analysis of Doc2B phosphorylation in EphB2+/+ or EphB2−/− mouse brains (M) in Doc2A-deficient neurons infected with Doc2B KD or control virus (N). Tubulin used as the reference protein. Data are presented as means ± SEM (n = 3). Statistical significance and P values were determined by Student’s t test (**P < 0.01; ****P < 0.0001).
Fig. 4.
Fig. 4.. Doc2B phosphorylation by EphB2 relieves inhibition of Doc2B on Munc13-catalyzed SNARE complex assembly and membrane fusion.
(A) Binding of Doc2B WT or its phosphomimetic/unphosphorylatable mutations to GST-MUN measured by GST pull-down experiments and quantification of the binding. (B) Binding of the MUN domain to GST-Doc2B fragments and EphB2 mixture measured by GST pull-down experiments in the presence or absence of ATP and quantification of the binding. (C and D) MUN-catalyzed SNARE complex assembly by addition of Doc2B WT, Y36D, or Y36F, respectively (C), and addition of Doc2B WT and EphB2 mixture in the presence or absence of ATP (D). Decrease of donor fluorescence at 1500 s is shown in the column at the right of the chart. (E and F) Munc13-catalyzed lipid mixing by addition of Doc2B WT, Y36D, or Y36F, respectively (E), and addition of Doc2B WT and EphB2 mixture in the presence or absence of ATP (F). Munc13-catalyzed lipid mixing at 1000 s is shown in the column at the right of the chart. F1, fluorescence intensity observed as a function time; F0, initial fluorescence intensity. Data are presented as means ± SEM (n = 3). Statistical significance and P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig. 5.
Fig. 5.. EphrinB3-EphB2 signaling regulates spontaneous release and synaptic augmentation.
(A and B) Representative traces (A) and quantification of mEPSC frequency (B) in Doc2A−/− neurons (EGFP, n = 15; Doc2A WT, n = 15; Doc2B WT, n = 15). (C and D) Representative traces (C) and quantification of mEPSC frequency (D) in Doc2A−/− neurons expressing EGFP (n = 16), Doc2B WT (n = 16), Doc2B D303N (n = 16), Doc2B (81 to 412) (n = 16), or Doc2B I20A (n = 16). (E) Representative traces of mEPSC frequency in Doc2A+/+ (top) and Doc2A−/− (bottom) neurons after stimulation with EphrinB3. (F and G) Summary of the changes in mEPSC frequency after stimulation with EphrinB3 in Doc2A+/+ (n = 16) and Doc2A−/− (n = 18) neurons (F) and in Doc2A−/− neurons expressing EGFP (n = 16), Doc2B WT (n = 16), or Doc2B Y36F (n = 16) (G). Data were normalized to the average value during the control period before stimulation. (H) Representative traces of evoked EPSCs in synaptic augmentation at 20, 40, and 60 s in Doc2A+/+ and Doc2A−/− neurons. (I and J) Normalized peak amplitudes of evoked EPSCs in Doc2A+/+ (n = 20) and Doc2A−/− (n = 23) neurons (I) or in Doc2A−/− neurons expressing EGFP (n = 16), Doc2B WT (n = 16), Doc2B I20A (n = 17), Doc2B Y36D (n = 15), or Doc2B Y36F (n = 15) (J). Data are presented as means ± SEM. Recorded cells are from three independent experiments. Statistical significance and P values for (B), (D), (G), and (J) were determined by one-way ANOVA with Dunnett’s multiple comparison test, and those for (F) and (I) were determined by Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig. 6.
Fig. 6.. Doc2-Munc13 interaction is critical for synaptic transmission and learning and memory.
(A and B) Representative traces (A) and quantification of mEPSC frequency (B) in WT neurons expressing EGFP (n = 22), Mid-L (n = 20), Mid-L I20A (n = 21), Mid-L Y36D (n = 22), or Mid-L Y36F (n = 20). (C and D) Representative traces (C) and quantification of AP-evoked EPSC amplitude (D) in WT neurons expressing EGFP (n = 16), Mid-L (n = 16), Mid-L I20A (n = 16), Mid-L Y36D (n = 16), or Mid-L Y36F (n = 16). (E and F) Representative traces (E) and quantification of EPSCs evoked by 0.5 M sucrose (F) in WT neurons expressing EGFP (n = 16), Mid-L (n = 15), Mid-L I20A (n = 18), Mid-L Y36D (n = 15), or Mid-L Y36F (n = 17). (G and H) Schematic representation of the novel object recognition task (G) and quantification of the discrimination ratio of time (H). F, familiar object; N, novel object. (I) Representative hotspots of path tracings taken from training session at day 6 in Morris water maze test. (J and K) Average latency (J) and swim length (K) to reach a hidden platform plotted against the blocks of trials (days). (L) Representative hotspots of path tracings taken from the probe trial at day 8. (M and N) Quantification of the latency to reach the previously hidden platform area (M) and percentage of time spent in the target quadrant (N) on day 8. Data are presented as means ± SEM. For electrophysiological recordings, cells are from three independent experiments. For behavioral tests, 12 mice per group. Statistical significance and P values were determined by one-way ANOVA with Dunnett’s multiple comparison test (*P < 0.05; **P < 0.01).
Fig. 7.
Fig. 7.. Model of EphB2 retrograde signaling that modulates neurotransmitter release.
Doc2 initially associates with vesicles via its C2AB domain. In vesicle docking, Doc2 binds Munc13 via its Mid-L region, which mediates the association of Doc2-bound vesicles to Munc13-enriched fusion sites but renders vesicle priming by inhibiting SNARE complex assembly. In spontaneous release and/or synaptic augmentation, postsynaptic EphrinB3 clusters activate presynaptic EphB2, which causes Doc2 Mid-L (Y36) phosphorylation and the dissociation of Doc2 from Munc13 at the fusion sites. This dissociation promotes SNARE complex assembly and vesicle priming. Afterward, Doc2 C2AB is capable of binding the plasma membrane (PS and PIP2) and the assembled SNARE complex, and exerts its action to drive membrane fusion in response to Ca2+.
See this image and copyright information in PMC

References

    1. Südhof T. C., Neurotransmitter release: The last millisecond in the life of a synaptic vesicle. Neuron 80, 675–690 (2013). - PMC - PubMed
    1. Brunger A. T., Choi U. B., Lai Y., Leitz J., Zhou Q., Molecular mechanisms of fast neurotransmitter release. Annu. Rev. Biophys. 47, 469–497 (2018). - PMC - PubMed
    1. Rizo J., Molecular mechanisms underlying neurotransmitter release. Annu. Rev. Biophys. 51, 377–408 (2022). - PMC - PubMed
    1. Sutton R. B., Fasshauer D., Jahn R., Brunger A. T., Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution. Nature 395, 347–353 (1998). - PubMed
    1. Weber T., Zemelman B. V., McNew J. A., Westermann B., Gmachl M., Parlati F., Söllner T. H., Rothman J. E., SNAREpins: Minimal machinery for membrane fusion. Cell 92, 759–772 (1998). - PubMed

LinkOut - more resources

-