Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

CaMKII phosphorylation of neuroligin-1 regulates excitatory synapses


Neuroligins are postsynaptic cell adhesion molecules that are important for synaptic function through their trans-synaptic interaction with neurexins (NRXNs). The localization and synaptic effects of neuroligin-1 (NL-1, also called NLGN1) are specific to excitatory synapses with the capacity to enhance excitatory synapses dependent on synaptic activity or Ca2+/calmodulin kinase II (CaMKII). Here we report that CaMKII robustly phosphorylates the intracellular domain of NL-1. We show that T739 is the dominant CaMKII site on NL-1 and is phosphorylated in response to synaptic activity in cultured rodent neurons and sensory experience in vivo. Furthermore, a phosphodeficient mutant (NL-1 T739A) reduces the basal and activity-driven surface expression of NL-1, leading to a reduction in neuroligin-mediated excitatory synaptic potentiation. To the best of our knowledge, our results are the first to demonstrate a direct functional interaction between CaMKII and NL-1, two primary components of excitatory synapses.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: NL-1 T739 is phosphorylated by CaMKII in vitro.
Figure 2: NL-1 T739 is phosphorylated by CaMKII in vitro and in hererologous cells as detected by a phosphorylation state–specific antibody.
Figure 3: Phosphorylation of NL-1 T739 in neurons.
Figure 4: T739A reduces the surface expression and synaptic enhancement of NL-1.
Figure 5: Activity-dependent increase in NL-1 surface expression is diminished in NL-1 T739A.
Figure 6: Synaptic enhancement by NL-1 is reduced by either glutamate receptor blockade or the T739A mutation.
Figure 7: Synaptic activity dynamically regulates T739 phosphorylation in vivo.


  1. 1

    Yamagata, M., Sanes, J.R. & Weiner, J.A. Synaptic adhesion molecules. Curr. Opin. Cell Biol. 15, 621–632 (2003).

    CAS  PubMed  Google Scholar 

  2. 2

    Dean, C. & Dresbach, T. Neuroligins and neurexins: linking cell adhesion, synapse formation and cognitive function. Trends Neurosci. 29, 21–29 (2006).

    CAS  PubMed  Google Scholar 

  3. 3

    Dalva, M.B., McClelland, A.C. & Kayser, M.S. Cell adhesion molecules: signalling functions at the synapse. Nat. Rev. Neurosci. 8, 206–220 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Craig, A.M. & Kang, Y. Neurexin-neuroligin signaling in synapse development. Curr. Opin. Neurobiol. 17, 43–52 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Südhof, T.C. Neuroligins and neurexins link synaptic function to cognitive disease. Nature 455, 903–911 (2008).

    PubMed  PubMed Central  Google Scholar 

  6. 6

    Chih, B., Engelman, H. & Scheiffele, P. Control of excitatory and inhibitory synapse formation by neuroligins. Science 307, 1324–1328 (2005).

    CAS  PubMed  Google Scholar 

  7. 7

    Dean, C. et al. Neurexin mediates the assembly of presynaptic terminals. Nat. Neurosci. 6, 708–716 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Graf, E.R., Zhang, X., Jin, S.X., Linhoff, M.W. & Craig, A.M. Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins. Cell 119, 1013–1026 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Levinson, J.N. et al. Neuroligins mediate excitatory and inhibitory synapse formation: involvement of PSD-95 and neurexin-1β in neuroligin-induced synaptic specificity. J. Biol. Chem. 280, 17312–17319 (2005).

    CAS  PubMed  Google Scholar 

  10. 10

    Nam, C.I. & Chen, L. Postsynaptic assembly induced by neurexin-neuroligin interaction and neurotransmitter. Proc. Natl. Acad. Sci. USA 102, 6137–6142 (2005).

    CAS  PubMed  Google Scholar 

  11. 11

    Wittenmayer, N. et al. Postsynaptic Neuroligin1 regulates presynaptic maturation. Proc. Natl. Acad. Sci. USA 106, 13564–13569 (2009).

    CAS  PubMed  Google Scholar 

  12. 12

    Ichtchenko, K. et al. Neuroligin 1: a splice site-specific ligand for β-neurexins. Cell 81, 435–443 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Irie, M. et al. Binding of neuroligins to PSD-95. Science 277, 1511–1515 (1997).

    CAS  PubMed  Google Scholar 

  14. 14

    Iida, J., Hirabayashi, S., Sato, Y. & Hata, Y. Synaptic scaffolding molecule is involved in the synaptic clustering of neuroligin. Mol. Cell. Neurosci. 27, 497–508 (2004).

    CAS  PubMed  Google Scholar 

  15. 15

    Poulopoulos, A. et al. Neuroligin 2 drives postsynaptic assembly at perisomatic inhibitory synapses through gephyrin and collybistin. Neuron 63, 628–642 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Ichtchenko, K., Nguyen, T. & Südhof, T.C. Structures, alternative splicing, and neurexin binding of multiple neuroligins. J. Biol. Chem. 271, 2676–2682 (1996).

    CAS  PubMed  Google Scholar 

  17. 17

    Song, J.Y., Ichtchenko, K., Südhof, T.C. & Brose, N. Neuroligin 1 is a postsynaptic cell-adhesion molecule of excitatory synapses. Proc. Natl. Acad. Sci. USA 96, 1100–1105 (1999).

    CAS  PubMed  Google Scholar 

  18. 18

    Varoqueaux, F., Jamain, S. & Brose, N. Neuroligin 2 is exclusively localized to inhibitory synapses. Eur. J. Cell Biol. 83, 449–456 (2004).

    CAS  PubMed  Google Scholar 

  19. 19

    Budreck, E.C. & Scheiffele, P. Neuroligin-3 is a neuronal adhesion protein at GABAergic and glutamatergic synapses. Eur. J. Neurosci. 26, 1738–1748 (2007).

    PubMed  Google Scholar 

  20. 20

    Hoon, M. et al. Neuroligin-4 is localized to glycinergic postsynapses and regulates inhibition in the retina. Proc. Natl. Acad. Sci. USA 108, 3053–3058 (2011).

    CAS  PubMed  Google Scholar 

  21. 21

    Chubykin, A.A. et al. Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2. Neuron 54, 919–931 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Ko, J. et al. Neuroligin-1 performs neurexin-dependent and neurexin-independent functions in synapse validation. EMBO J. 28, 3244–3255 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Shipman, S.L. et al. Functional dependence of neuroligin on a new non-PDZ intracellular domain. Nat. Neurosci. 14, 718–726 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Kwon, H.B. et al. Neuroligin-1–dependent competition regulates cortical synaptogenesis and synapse number. Nat. Neurosci. 15, 1667–1674 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Kim, J. et al. Neuroligin-1 is required for normal expression of LTP and associative fear memory in the amygdala of adult animals. Proc. Natl. Acad. Sci. USA 105, 9087–9092 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Blundell, J. et al. Neuroligin-1 deletion results in impaired spatial memory and increased repetitive behavior. J. Neurosci. 30, 2115–2129 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Dahlhaus, R. & El-Husseini, A. Altered neuroligin expression is involved in social deficits in a mouse model of the fragile X syndrome. Behav. Brain Res. 208, 96–105 (2010).

    CAS  PubMed  Google Scholar 

  28. 28

    Jung, S.Y. et al. Input-specific synaptic plasticity in the amygdala is regulated by neuroligin-1 via postsynaptic NMDA receptors. Proc. Natl. Acad. Sci. USA 107, 4710–4715 (2010).

    CAS  Google Scholar 

  29. 29

    Choi, Y.B. et al. Neurexin-neuroligin transsynaptic interaction mediates learning-related synaptic remodeling and long-term facilitation in aplysia. Neuron 70, 468–481 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Shipman, S.L. & Nicoll, R.A. A subtype-specific function for the extracellular domain of neuroligin 1 in hippocampal LTP. Neuron 76, 309–316 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Dresbach, T., Neeb, A., Meyer, G., Gundelfinger, E.D. & Brose, N. Synaptic targeting of neuroligin is independent of neurexin and SAP90/PSD95 binding. Mol. Cell. Neurosci. 27, 227–235 (2004).

    CAS  PubMed  Google Scholar 

  32. 32

    Schapitz, I.U. et al. Neuroligin 1 is dynamically exchanged at postsynaptic sites. J. Neurosci. 30, 12733–12744 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Gutiérrez, R.C. et al. Activity-driven mobilization of post-synaptic proteins. Eur. J. Neurosci. 30, 2042–2052 (2009).

    PubMed  Google Scholar 

  34. 34

    Kim, J.H. & Huganir, R.L. Organization and regulation of proteins at synapses. Curr. Opin. Cell Biol. 11, 248–254 (1999).

    CAS  PubMed  Google Scholar 

  35. 35

    Lisman, J., Schulman, H. & Cline, H. The molecular basis of CaMKII function in synaptic and behavioural memory. Nat. Rev. Neurosci. 3, 175–190 (2002).

    CAS  PubMed  Google Scholar 

  36. 36

    Bolliger, M.F. et al. Unusually rapid evolution of Neuroligin-4 in mice. Proc. Natl. Acad. Sci. USA 105, 6421–6426 (2008).

    CAS  PubMed  Google Scholar 

  37. 37

    Peixoto, R.T. et al. Transsynaptic signaling by activity-dependent cleavage of neuroligin-1. Neuron 76, 396–409 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Suzuki, K. et al. Activity-dependent proteolytic cleavage of neuroligin-1. Neuron 76, 410–422 (2012).

    CAS  PubMed  Google Scholar 

  39. 39

    Prange, O., Wong, T.P., Gerrow, K., Wang, Y.T. & El-Husseini, A. A balance between excitatory and inhibitory synapses is controlled by PSD-95 and neuroligin. Proc. Natl. Acad. Sci. USA 101, 13915–13920 (2004).

    CAS  PubMed  Google Scholar 

  40. 40

    Futai, K. et al. Retrograde modulation of presynaptic release probability through signaling mediated by PSD-95–neuroligin. Nat. Neurosci. 10, 186–195 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Shipman, S.L. & Nicoll, R.A. Dimerization of postsynaptic neuroligin drives synaptic assembly via transsynaptic clustering of neurexin. Proc. Natl. Acad. Sci. USA 109, 19432–19437 (2012).

    CAS  PubMed  Google Scholar 

  42. 42

    Philpot, B.D., Sekhar, A.K., Shouval, H.Z. & Bear, M.F. Visual experience and deprivation bidirectionally modify the composition and function of NMDA receptors in visual cortex. Neuron 29, 157–169 (2001).

    CAS  PubMed  Google Scholar 

  43. 43

    Tropea, D., Majewska, A.K., Garcia, R. & Sur, M. Structural dynamics of synapses in vivo correlate with functional changes during experience-dependent plasticity in visual cortex. J. Neurosci. 30, 11086–11095 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Giannone, G. et al. Neurexin-1β binding to Neuroligin-1 triggers the preferential recruitment of PSD-95 versus gephyrin through tyrosine phosphorylation of Neuroligin-1. Cell Rep. 3, 1996–2007 (2013).

    CAS  PubMed  Google Scholar 

  45. 45

    Esteban, J.A. et al. PKA phosphorylation of AMPA receptor subunits controls synaptic trafficking underlying plasticity. Nat. Neurosci. 6, 136–143 (2003).

    CAS  PubMed  Google Scholar 

  46. 46

    Yan, J. et al. Analysis of the neuroligin 3 and 4 genes in autism and other neuropsychiatric patients. Mol. Psychiatry 10, 329–332 (2005).

    CAS  PubMed  Google Scholar 

  47. 47

    Jamain, S. et al. Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism. Nat. Genet. 34, 27–29 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Etherton, M.R., Tabuchi, K., Sharma, M., Ko, J. & Südhof, T.C. An autism-associated point mutation in the neuroligin cytoplasmic tail selectively impairs AMPA receptor–mediated synaptic transmission in hippocampus. EMBO J. 30, 2908–2919 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Comoletti, D. et al. The Arg451Cys-neuroligin-3 mutation associated with autism reveals a defect in protein processing. J. Neurosci. 24, 4889–4893 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Linhoff, M.W. et al. An unbiased expression screen for synaptogenic proteins identifies the LRRTM protein family as synaptic organizers. Neuron 61, 734–749 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references


We are grateful to A. Sanz-Clemente and A. Scimemi for technical assistance and for discussions on the project and manuscript. We thank the NINDS sequencing facility and light imaging facility for their expertise. This research was supported by the National Institute of Neurological Disorders and Stroke Intramural Research Program (M.A.B., T.H., J.D.B., Y.L., J.S.D. and K.W.R.) and the National Institute of Mental Health grant number 5 R37 MH038256 (S.L.S., B.E.H. and R.A.N.).

Author information




M.A.B. designed experiments, performed all biochemical and imaging experiments, conducted electrophysiology experiments in disassociated hippocampal cultures and executed data analysis. M.A.B. and K.W.R. wrote the manuscript. S.L.S. and B.E.H. designed and conducted all electrophysiology experiments in slice cultures. T.H. designed constructs and aided in biochemistry and imaging experiments. Y.L. performed and analyzed all mass spectrometry data. J.D.B. aided in animal and biochemical experiments. K.W.R., J.S.D. and R.A.N. helped design experiments and supervised the project.

Corresponding author

Correspondence to Katherine W Roche.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 NL-1 and GluA1 c-tails have similar reaction kinetics.

(a) GST-NL-1 or GluA1 were incubated with purified CaMKII and [γ-P32]ATP and analyzed by autoradiography. Reactions were stopped at their marked time. (b) Protein concentrations are plotted as a ratio of phosphorylated NL-1 (P-32) to total NL-1 (CBB) normalized to the 1 min reaction condition. Saturation of phosphorylated NL-1 and GluA1 occurred at approximately 10 min. Total protein was visualized by CBB protein staining, in a,b. Full-length blots are presented in Supplementary Figure 4 when applicable.

Supplementary Figure 2 NL-1 T739A does not affect presynaptic release probability.

Paired-pulse ratio (PPR), second EPSC over first EPSC for consecutive stimuli separated by 40 ms. Example traces normalized at first EPSC for (a) NL-1. (b) NL-1 T739A. (c) Second EPSC over first ± SEM. NL-1 and NL-1 T739A had similar PPRs (P > 0.05, n = 5). Scale bars represent 25 ms.

Supplementary Figure 3 Titration of phosphorylated NL-1 with pT739-Ab

(a) Protein concentrations from adult WT or NL-1 KO brains were titrated with pT739-Ab. (b) Protein concentrations are plotted as ratio of phosphorylated (IP) to total NL-1 (input) normalized to the 0.2 mg protein condition for an individual brain. Saturation of the pT739-Ab occurs between 0.5 and 1 mg of protein.

Supplementary Figure 4 Full-length blots of cropped blots from the manuscript.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 (PDF 2937 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Bemben, M., Shipman, S., Hirai, T. et al. CaMKII phosphorylation of neuroligin-1 regulates excitatory synapses. Nat Neurosci 17, 56–64 (2014).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing