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The GTPase RhoA is a major player in many different regulatory pathways. RhoA catalyzes GTP hydrolysis, and its catalysis is accelerated when RhoA forms heterodimers with proteins of the guanine nucleotide exchange factor (GEF) family. Neuroepithelial cell transforming gene 1 (Net1) is a RhoA-interacting GEF implicated in cancer, but the structural features supporting the RhoA/Net1 interaction are unknown. Taking advantage of a simple production and purification process, here we solved the structure of a RhoA/Net1 heterodimer with X-ray crystallography at 2-Å resolution. Using a panel of several techniques, including molecular dynamics simulations, we characterized the RhoA/Net1 interface. Moreover, deploying an extremely simple peptide-based scanning approach, we found that short peptides (penta- to nonapeptides) derived from the protein/protein interaction region of RhoA could disrupt the RhoA/Net1 interaction and thereby diminish the rate of nucleotide exchange. The most inhibitory peptide, EVKHF, spanning residues 102–106 in the RhoA sequence, displayed an IC(50) of ∼100 μm without further modifications. The peptides identified here could be useful in further investigations of the RhoA/Net1 interaction region. We propose that our structural and functional insights might inform chemical approaches for transforming the pentapeptide into an optimized pseudopeptide that antagonizes Net1-mediated RhoA activation with therapeutic anticancer potential.

Peroxisome proliferator–activated receptor α (PPARα) is a transcriptional regulator of lipid metabolism. GW7647 is a potent PPARα agonist that must reach the nucleus to activate this receptor. In cells expressing human fatty acid–binding protein 1 (FABP1), GW7647 treatment increases FABP1's nuclear localization and potentiates GW7647-mediated PPARα activation; GW7647 is less effective in cells that do not express FABP1. To elucidate the underlying mechanism, here we substituted residues in FABP1 known to dictate lipid signaling by other intracellular lipid-binding proteins. Substitutions of Lys-20 and Lys-31 to Ala in the FABP1 helical cap affected neither its nuclear localization nor PPARα activation. In contrast, Ala substitution of Lys-57, Glu-77, and Lys-96, located in the loops adjacent to the ligand-binding portal region, abolished both FABP1 nuclear localization and GW7647-induced PPARα activation but had little effect on GW7647–FABP1 binding affinity. Using solution NMR spectroscopy, we determined the WT FABP1 structure and analyzed the dynamics in the apo and GW7647-bound structures of both the WT and the K57A/E77A/K96A triple mutant. We found that GW7647 binding causes little change in the FABP1 backbone, but solvent exposes several residues in the loops around the portal region, including Lys-57, Glu-77, and Lys-96. These residues also become more solvent-exposed upon binding of FABP1 with the endogenous PPARα agonist oleic acid. Together with previous observations, our findings suggest that GW7647 binding stabilizes a FABP1 conformation that promotes its interaction with PPARα. We conclude that full PPARα agonist activity of GW7647 requires FABP1-dependent transport and nuclear localization processes.

International audience; > De récentes avancées technologiques révo-lutionnent le domaine des biologistes structu-ralistes. Plus précisément, des progrès spec-taculaires liés au développement de nouvelles technologies de capture d'images de microscope électronique (la détection directe d'électrons) ainsi que la mise à disposition de nouveaux logiciels d'analyse d'images ont conduit à une percée en terme de résolution en cryo-micros-copie électronique à transmission. Il est ainsi possible de calculer relativement rapidement des structures à haute résolution de molécules biologiques dont l'étude résiste aux méthodes plus classiques comme la diffraction des rayons X ou la résonance magnétique nucléaire (RMN). Ces structures ainsi obtenues peuvent aussi venir en complément des informations structurales déjà décrites par d'autres méthodes. Certaines de ces nouvelles structures résolues grâce à la cryo-microscopie électronique révèlent pour la première fois le fonctionnement précis de méca-nismes essentiels au bon déroulement physiolo-gique d'une cellule. La capacité à résoudre ces structures à la résolution du détail atomique est une condition essentielle pour le développement de nouveaux médicaments ayant comme cible thérapeutique ces protéines d'intérêt. Grâce à ces avancées techniques que nous résumons ici, des questions biologiques et médicales sont maintenant devenues accessibles, ce qui était inconcevable il y a seulement cinq ans.

International audience; Here, we describe a plug-and-play microfluidic platform, suitable for protein crystallization. The droplet factory is designed to generate hundreds of droplets as small as a few nanoliters (2 to 10nL) for screening and optimization of crystallization conditions. Commercially-available microfluidic junctions and tubing are combined to create the appropriate geometry. In addition, a " chemical library " is produced in tubing. The microfluidic geometry for a " crystallization agent-based chemical library " is validated by screening crystallization conditions of lysozyme. The microfluidic geometry for a " ligand-based chemical library " is also explored to co-crystallize the protein QR2 with a ligand, for the purposes of structure-based drug design. This platform mixes aqueous phases (containing the protein and the crystallization agent) and organic phases (containing the ligand), during the droplet generation and circulation without using any surfactant. The droplet composition is controlled by the respective flow-rates of the different solutions, and checked by measuring on-line absorbance. The low volumes involved in the crystallization trials, the speed of execution and the absence of a microfabrication stage make our platform a cheap, easy-to-use and versatile tool for crystallization studies.

International audience; We describe a universal, high-throughput droplet-based microfluidic platform for crystallization. It is suitable for a multitude of applications, due to its flexibility, ease of use, compatibility with all solvents and low cost. The platform offers four modular functions: droplet formation, on-line characterization, incubation and observation. We use it to generate droplet arrays with a concentration gradient in continuous long tubing, without using surfactant. We control droplet properties (size, frequency and spacing) in long tubing by using hydrodynamic empirical relations. We measure droplet chemical composition using both an off-line and a real-time on-line method. Applying this platform to a complicated chemical environment, membrane proteins, we successfully handle crystallization, suggesting that the platform is likely to perform well in other circumstances. We validate the platform for fine-gradient screening and optimization of crystallization conditions. Additional on-line detection methods may well be integrated into this platform in the future, for instance, an on-line diffraction technique. We believe this method could find applications in fields such as fluid interaction engineering, live cell study and enzyme kinetics.

The invention of the electron microscope has greatly enhanced the view scientists have of small structural details. Since its implementation, this technology has undergone considerable evolution and the resolution that can be obtained for biological objects has been extended. In addition, the latest generation of cryo-electron microscopes equipped with direct electron detectors and software for the automated collection of images, in combination with the use of advanced image-analysis methods, has dramatically improved the performance of this technique in terms of resolution. While calculating a sub-10 Å resolution structure was an accomplishment less than a decade ago, it is now common to generate structures at sub-5 Å resolution and even better. It is becoming possible to relatively quickly obtain high-resolution structures of biological molecules, in particular large ones (>500 kDa) which, in some cases, have resisted more conventional methods such as X-ray crystallography or nuclear magnetic resonance (NMR). Such newly resolved structures may, for the first time, shed light on the precise mechanisms that are essential for cellular physiological processes. The ability to attain atomic resolution may support the development of new drugs that target these proteins, allowing medicinal chemists to understand the intimacy of the relationship between their molecules and targets. In addition, recent developments in cryo-electron microscopy combined with image analysis can provide unique information on the conformational variability of macromolecular complexes. Conformational flexibility of macromolecular complexes can be investigated using cryo-electron microscopy and multiconformation reconstruction methods. However, the biochemical quality of the sample remains the major bottleneck to routine cryo-electron microscopy-based determination of structures at very high resolution.

International audience; Planar cell polarity (PCP) signaling is well known to play a critical role during prenatal brain development; whether it plays specific roles at postnatal stages remains rather unknown. Here, we investigated the role of a key PCP-associated gene scrib in CA1 hippocampal structure and function at postnatal stages. We found that Scrib is required for learning and memory consolidation in the Morris water maze as well as synaptic maturation and NMDAR-dependent bidirectional plasticity. Furthermore, we unveiled a direct molecular interaction between Scrib and PP1/PP2A phosphatases whose levels were decreased in postsynaptic density of conditional knockout mice. Remarkably, exposure to enriched environment (EE) preserved memory formation in CaMK-Scrib −/− mice by recovering synaptic plasticity and maturation. Thus, Scrib is required for synaptic function involved in memory formation and EE has beneficiary therapeutic effects. Our results demonstrate a distinct new role for a PCP-associated protein, beyond embryonic development, in cognitive functions during adulthood.

A microfluidic platform was used to address the problems of obtaining diffraction-quality crystals and crystal handling during transfer to the X-ray diffractometer. Crystallization conditions of a protein of pharmaceutical interest were optimized and X-ray data were collected both in situ and ex situ.

International audience; Synthetic biology (or chemical biology) is a growing field to which the chemical synthesis of proteins, particularly enzymes, makes a fundamental contribution. However, the chemical synthesis of catalytically active proteins (enzymes) remains poorly documented because it is difficult to obtain enough material for biochemical experiments. We chose calstabin, a 107-amino-acid proline isomerase, as a model. We synthesized the enzyme using the native chemical ligation approach and obtained several tens of milligrams. The polypeptide was refolded properly, and we characterized its biophysical properties, measured its catalytic activity, and then crystallized it in order to obtain its tridimensional structure after X-ray diffraction. The refolded enzyme was compared to the recombinant, wild-type enzyme. In addition, as a first step of validating the whole process, we incorporated exotic amino acids into the N-terminus. Surprisingly, none of the changes altered the catalytic activities of the corresponding mutants. Using this body of techniques, avenues are now open to further obtain enzymes modified with exotic amino acids in a way that is only barely accessible by molecular biology, obtaining detailed information on the structure-function relationship of enzymes reachable by complete chemical synthesis.

Recent technological advances have revolutionized the field of structural biologists. Specifically, dramatic progress related to the development of new electron microscopes and image capture (direct electron detection camera) and the provision of new image analysis software has led to a breakthrough in terms of resolution attained using cryo-electron transmission microscopy. It is thus possible to calculate relatively quickly high-resolution structures of biological molecules whom structural study still resists to more conventional methods such as X-ray diffraction or nuclear magnetic resonance (NMR). These structures thus obtained may also bring complementary structural information to those already described by other methods. Some of these new structures resolved through cryo-electron microscopy revealed for the first time the precise operation of essential mechanisms necessary for the good physiological process of a cell. The ability to solve these structures at atomic resolution detail is essential for the development of new drugs that target these proteins of therapeutic interest. Thanks to these advanced techniques that we summarize in this revew, biological and medical issues have now become accessible, whereas this approach was inconceivable only five yeras ago. ‡.

Glucokinase (GK) catalyses the formation of glucose 6-­phosphate from glucose and ATP. A specific feature of GK amongst hexokinases is that it can cycle between active and inactive conformations as a function of glucose concentration, resulting in a unique positive kinetic cooperativity with glucose, which turns GK into a unique key sensor of glucose metabolism, notably in the pancreas. GK is a target of antidiabetic drugs aimed at the activation of GK activity, leading to insulin secretion. Here, the first structures of a GK–glucose complex without activator, of GK–glucose–AMP-PNP and of GK–glucose–AMP-PNP with a bound activator are reported. All these structures are extremely similar, thus demonstrating that binding of GK activators does not result in conformational changes of the active protein but in stabilization of the active form of GK.

The first selenium containing non-detergent sulphobetaines are described. They have been prepared in multi-gram quantities from readily available pyridyl precursors. Incorporation of selenium was achieved using nucleophilic organoselenide anions. The resulting products were shown by 77 Se NMR spectroscopy to be homogeneous with respect to selenium oxidation state.

Some non-detergent sulfobetaines had been shown to prevent aggregation and improve the yield of active proteins when added to the buffer during in vitro protein renaturation. With the aim of designing more efficient folding helpers, a series of non-detergent sulfobetaines have been synthesized and their efficiency in improving the renaturation of a variety of proteins (E. coli tryptophan synthase and β- d -galactosidase, hen lysozyme, bovine serum albumin, a monoclonal antibody) have been investigated. Attempts to correlate the structure of each sulfobetaines with its effect on folding revealed some molecular features that appear important in helping renaturation. This enabled us to design and synthesize new non-detergent sulfobetaines that act as potent folding helpers.

The 1,024-amino-acid acylated hemolysin ofEscherichia colisubverts host cell functions and causes cell lysis. Both activities require insertion of the toxin into target mammalian cell membranes. To identify directly the principal toxin sequences dictating membrane binding and insertion, we assayed the lipid bilayer interaction of native protoxin, stably active toxin, and recombinant peptides. Binding was assessed by flotation of protein-liposome mixtures through density gradients, and insertion was assessed by labeling with a photoactivatable probe incorporated into the target lipid bilayer. Both the active acylated hemolysin and the inactive unacylated protoxin were able to bind and also insert. Ca2+binding, which is required for toxin activity, did not influence the in vitro interaction with liposomes. Three overlapping large peptides were expressed separately. A C-terminal peptide including residues 601 to 1024 did not interact in either assay. An internal peptide spanning residues 496 to 831, including the two acylation sites, bound to phospholipid vesicles and showed a low level of insertion-dependent labeling. In vitro acylation had no effect on the bilayer interaction of either this peptide or the full-length protoxin. An N-terminal peptide comprising residues 1 to 520 also bound to phospholipid vesicles and showed strong insertion-dependent labeling, ca. 5- to 25-fold that of the internal peptide. Generation of five smaller peptides from the N-terminal region identified the principal determinant of lipid insertion as the hydrophobic sequence encompassing residues 177 to 411, which is conserved among hemolysin-related toxins.

Non-detergent sulfobetaines (NDSB) are a family of solubilizing and stabilizing agents for proteins. In a previous study [Goldberg, M. E., Expert-Bezancon, N., Vuillard, L. & Rabilloud, T. (1996) Folding & Design 1, 21-27] we showed that the amount of active protein recovered in in vitro folding experiments could be significantly increased by some NDSBS. In this work we investigated the mechanisms by which these molecules facilitate protein renaturation. Stopped-flow and manual-mixing fluorescence and enzyme activity measurements were used to compare the kinetics of protein folding in the presence and absence of N-phenyl-methyl-N,N-dimethylammonium-propane-sulfonate (NDSB 256). Hen lysozyme and the beta2 subunit of Escherichia coli tryptophan synthase were chosen as model systems since their folding pathways had been previously investigated in detail. It is shown that, massive aggregation of tryptophan synthase occurs within less than 2.5 s after dilution in the renaturation buffer, but can be prevented by NDSB 256; only very early folding phases (such as the formation of a loosely packed hydrophobic core able to bind 8-anilino-1-naphthalenesulphonic acid, and the initial burying of tryptophan 177) are significantly altered by NDSB 256; none of the later phases is affected. Furthermore, NDSB 256 did not significantly affect any of the kinetic phases observed during the refolding of denatured lysozyme retaining intact disulphide bonds. This shows that NDSB 256 only interferes with very early steps in the folding process and acts by limiting the abortive interactions that could lead to the formation of inactive aggregates.

The Ku antigen consists of two subunits of 70 and 83 kDa and is endowed with both duplex DNA end-binding capacity and helicase activity (human DNA helicase II). HeLa Ku can be isolated from in vitro cultured human cells uniquely as a heterodimer, and the subunits can be separated by electrophoresis only under denaturing conditions. To dissect the molecular functions of the two subunits of the heterodimer, we have cloned and expressed their cDNAs separately in Escherichia coli. The two activities of Ku (DNA binding and unwinding) were reconstituted by mixing and refolding both subunits in equimolar amounts (Tuteja, N., Tuteja, R., Ochem, A., Taneja, P., Huang, N-W., Simoncsits, A., Susic, S., Rahman, K., Marusic, L., Chen, J., Zang, J., Wang, S., Pongor, S., and Falaschi, A. (1994) EMBO J. 13, 4991-5001). Renaturation of the separate subunits can be achieved in the presence of a synthetic solubilizing and stabilizing agent, dimethyl ethylammonium propane sulfonate (NDSB 195). The helicase activity of the Ku protein resides uniquely in the 70-kDa subunit, whereas the DNA end-binding activity can be reconstituted only through renaturation of the two subunits in the heterodimeric form and is practically absent in the separate subunits. The 83-kDa subunit, when refolded in the absence of the 70-kDa subunit, forms homodimers unable to unwind DNA and bind duplex ends. The three separate species (heterodimer, 70-kDa subunit, and 83-kDa subunit homodimer) all have ssDNA-dependent ATPase activity.

The use in protein crystal growth of the newly described non-detergent solubilizers/stabilizers sulfobetaines (NDSB) has been investigated with crystallographic methods. In the classic model of egg white lysozyme crystallization by NaCl, NDSB increased the protein solubility and the crystal growth rate without influencing the quality of crystals as shown by Wilson plots. Sulphur anomalous X-ray scattering was used to show the absence of any bound NDSB in the crystals. A practical example of the use of NDSB is given for Desulfovibrio Gigas type II ferrodoxin, where NDSB contributed to reduction in crystal twinning and resulted in the growth of a new crystal form. With their unique combination of solubilizing and stabilizing properties, the NDSB molecules might prove to be valuable additives for protein crystal growth.

We present an interdisciplinary approach that, by incorporating a range of experimental and computational techniques, allows the identification and characterization of functional/immunogenic domains. This approach has been applied to ArtJ, an arginine-binding protein whose orthologs in Chlamydiae trachomatis (CT ArtJ) and pneumoniae (CPn ArtJ) are shown to have different immunogenic properties despite a high sequence similarity (60% identity). We have solved the crystallographic structures of CT ArtJ and CPn ArtJ, which are found to display a type II transporter fold organized in two α-β domains with the arginine-binding region at their interface. Although ArtJ is considered to belong to the periplasm, we found that both domains contain regions exposed on the bacterial surface. Moreover, we show that recombinant ArtJ binds to epithelial cells in vitro, suggesting a role for ArtJ in host-cell adhesion during Chlamydia infection. Experimental epitope mapping and computational analysis of physicochemical determinants of antibody recognition revealed that immunogenic epitopes reside mainly in the terminal (D1) domain of both CPn and CT ArtJ, whereas the surface properties of the respective binding-prone regions appear sufficiently different to assume divergent immunogenic behavior. Neutralization assays revealed that sera raised against CPn ArtJ D1 partially reduce both CPn and CT infectivity in vitro, suggesting that functional antibodies directed against this domain may potentially impair chlamydial infectivity. These findings suggest that the approach presented here, combining functional and structure-based analyses of evolutionary-related antigens can be a valuable tool for the identification of cross-species immunogenic epitopes for vaccine development.

Although the conformation of fibronectin has been widely investigated by various techniques, there has not yet been any determination of its rotational diffusion coefficient. We report here this determination by the transient electric birefringence study of solutions of bovine plasma fibronectin at physiological ionic strength. The solutions showed a positive birefringence. A linear relationship was observed between the intensity of the birefringence at equilibrium and the square of the electric field within the range of fields applied (up to 12.5 kV.cm-1). The field-independent decay of the induced birefringence was described by a single exponential with a relaxation time of 0.76 (+/- 0.08) microsecond at 23 degrees C. This establishes fibronectin in solution as a globally rigid structure with a rotational diffusion coefficient, at 20 degrees C, of 202,000 s-1. This result allows the first rigorous determination of the low-resolution structure of fibronectin. It is important to notice that the analysis combines only results obtained in physiological conditions on native molecules and follows a strict hydrodynamic interpretation. The conclusion of this work is that a hollow sphere of about 20 nm external diameter can be proposed as a model for the three-dimensional structure of the fibronectin molecule in solution. This new model suggests the fibronectin could have the structure of a carrier protein.

Two mutant strains of Escherichia coli BL21(DE3), called C41(DE3) and C43(DE3) and originally described by Miroux and Walker, are frequently used to overcome the toxicity associated with overexpressing recombinant proteins using the bacteriophage T7 RNA polymerase expression system. Even when the toxicity of the plasmids is so high that it prevents transformation in the strain BL21(DE3), the toxic proteins can often be expressed successfully in C41(DE3) and/or C43(DE3). In this work, using a range of plasmids coding for several types of proteins, we investigated in BL21(DE3), C41(DE3), and C43(DE3) their ability to undergo transformation and to express. While transformation was always possible in C41(DE3) and C43(DE3), we could not obtain transformants in BL21(DE3) for 62% of the expression vectors tested. Moreover, after induction, the expression of heterologous proteins in both mutant strains is generally better than in BL21(DE3). In this study, we also enhanced the stability of plasmids in culture during the expression of proteins by adding the par locus from the plasmid pSC101 to the vector backbone. The stability of a subset of the plasmids (measured 3 h after induction) was determined in C41(DE3) and C43(DE3) and varies from 62 to 92% for C43(DE3) and from 10 to 90% for C41(DE3). This study demonstrates the usefulness of these strains C41(DE3) and C43(DE3) in solving the problem of plasmid instability during the expression of toxic recombinant proteins.

The potential usefulness of the new zwitterionic solubilizing agent, dimethyl ethylammonium propane sulfonate (NDSB195), in protein crystallization was shown using hen egg-white lysozyme. In the presence of this agent, highly diffracting crystals were obtained using ammonium sulphate as a precipitant, whereas in its absence only amorphous precipitates were obtained. The crystals possess a triclinic unit cell not previously described and diffract to a resolution of 2 Å. To ascertain that the new reagent had not produced significant changes in the protein fold the structure was determined to a resolution of 2.6 Å. Only minor differences were observed (notably in regions of crystal contacts) with the known tetragonal lysozyme structure (Brookhaven Protein Data Bank entry 1HEL).

Reaction–diffusion processes can lead to a macroscopic concentration pattern from an initially homogeneous solution, and thus provide a physical–chemical mechanism for biological pattern formation and morphogenesis. The central prediction of reaction–diffusion theory is that the patterns contain periodic concentration variations in some of the reactives. Microtubules assembled in vitro spontaneously self-organise and form stationary striped macroscopic structures. In agreement with reaction–diffusion theory. Here we show, in agreement with reaction–diffusion theory, that these preparations contain substantial microtubule concentration variations. Similar striped microtubule patterns arise during Drosophila embryogenesis. A characteristic of these patterns is their dependence on sample dimensions. In Drosophila eggs shortened by ligation, we found that the microtubule pattern varied with egg fragment length in the same way as the in vitro microtubule pattern varied with sample length, and as expected from theory. This is evidence that reaction–diffusion structures occur during Drosophila morphogenesis.

International audience; A better understanding of the causative agent's biology and the definition of new targets for the development of drugs and/or specific immune responses is necessary to face the spred of drug-resistant malaria in developing countries and the absence of an efficient vaccine against this most important infectious disease. Non-detergent sulphobetaines enhance the recovery and isoelectric focussing of active Plasmodium falciparum proteases, cytoskeleton-associated proteins and Maurer's cleft-associated proteins. This is a significant advantage for the purification of such proteins and might help pinpoint their role for red blood cell rupture and merozoite release.

Backgound: Attempts to renature proteins often yield aggregates rather than native protein. To minimize aggregation, low protein concentrations and/or solubilizing agents are used. Here, we test new solubilizing molecules, non-detergent sulphobetaines, to improve the renaturation of two very different enzymes, hen egg white lysozyme and bacterial β -D-galactosidase. Results: The renaturation was conducted in the presence of five different sulphobetaines and the yield of active enzyme was measured. The five sulphobetaines improved the yield of native lysozyme up to 12-fold. Some sulphobetaines improved the yield of galactosidase up to 80-fold, but one reduced it 100-fold. Conclusion: Non-detergent sulphobetaines strongly affect the balance between aggregation and folding. Their effect depends on their structure and on their interactions with folding intermediates. These results should serve as a basis for designing more efficient sulphobetaines; for designing improved renaturation protocols using existing sulphobetaines; and for characterizing folding intermediates that interact with sulphobetaines.

A significant increase in the yield of protein extraction by the addition of sulfobetaine-type mild solubilization agents is shown for microsomal membrane proteins and proteins from lyophilized platelets, with a maximum increase in yield of up to 100%. These agents are also applied to native isoelectric focusing in immobilized pH gradients. The absence of significant denaturation induced by these solubilizers is shown for creatine kinase.

Activation of transcription factor NF-κB is accomplished by degradation of its inhibitor IκBα. Signal induced phosphorylation of IκBα on serine 32 and 36 targets the protein for ubiquitination on lysine 21 and 22. Here we use a phosphorylated peptide substrate representing residues 20–43 of IκBα to investigate requirements for ubiquitination of IκBα. Phosphorylation dependent polyubiquitination is carried out by a multiprotein complex containing βTrCP, Skp1 and Cdc53 (Cul1). In the presence of ubiquitin activating enzyme and the protein complex containing βTrCP, polyubiquitination of IκBα peptide was dependent on the presence of Cdc34, while Ubc5 only stimulated mono- and di-ubiquitination.

Plasma fibronectin purified from the plasma of metastatic breast cancer patients has been studied by light scattering. It clearly shows abnormal self-aggregation properties; the possible significance of these findings to the in vivo situation is discussed.

International audience; The appropriate trafficking of glutamate receptors to synapses is crucial for basic synaptic function and synaptic plasticity. It is now accepted that NMDA receptors (NMDARs) internalize and are recycled at the plasma membrane but also exchange between synaptic and extrasynaptic pools; these NMDAR properties are also key to governing synaptic plasticity. Scribble1 is a large PDZ protein required for synaptogenesis and synaptic plasticity. Herein, we show that the level of Scribble1 is regulated in an activity-dependent manner and that Scribble1 controls the number of NMDARs at the plasma membrane. Notably, Scribble1 prevents GluN2A subunits from undergoing lysosomal trafficking and degradation by increasing their recycling to the plasma membrane following NMDAR activation. Finally, we show that a specific YxxR motif on Scribble1 controls these mechanisms through a direct interaction with AP2. Altogether, our findings define a molecular mechanism to control the levels of synaptic NMDARs via Scribble1 complex signaling.