Positive surface charge of GluN1 N-terminus mediates the direct interaction with EphB2 and NMDAR mobility

Localization of the N-methyl-D-aspartate type glutamate receptor (NMDAR) to dendritic spines is essential for excitatory synaptic transmission and plasticity. Rather than remaining trapped at synaptic sites, NMDA receptors undergo constant cycling into and out of the postsynaptic density. Receptor movement is constrained by protein-protein interactions with both the intracellular and extracellular domains of the NMDAR. The role of extracellular interactions on the mobility of the NMDAR is poorly understood. Here we demonstrate that the positive surface charge of the hinge region of the N-terminal domain in the GluN1 subunit of the NMDAR is required to maintain NMDARs at dendritic spine synapses and mediates the direct extracellular interaction with a negatively charged phospho-tyrosine on the receptor tyrosine kinase EphB2. Loss of the EphB-NMDAR interaction by either mutating GluN1 or knocking down endogenous EphB2 increases NMDAR mobility. These findings begin to define a mechanism for extracellular interactions mediated by charged domains.


Reporting Summary
Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, seeAuthors & Referees and theEditorial Policy Checklist .

Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection

Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. Leica LAS X Imaging Software was used to collect confocal and FRAP images.
ImageJ was used to analyze data, GraphPad Prism was used for statistical analysis, Adaptive Poisson-Boltzmann Solver plugin in PyMol was used to determine surface potential of published crystal structures.
Provide your data availability statement here.

October 2018
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Sample sizes were determined so that experiments be replicated to ensure confidence in the results and were based on previous studies (Dalva et al., 2000, Kayser et al., 2008, Nolt et al., 2011, and Hruska et al., 2015. For all experiments there was enough statistical power to detect the corresponding effect size.
All data acquired were included in analyses.
Each experiment was replicated a minimum of three times and data was reliably reproduced with each replication attempt.
Data were acquired and analyzed based on the standards in the field, however, no method of randomization was used to determine how sample were allocated to experimental groups and processed.
Data were collected blind to condition and analyzed blind to condition.