The tumor suppressor protein p53 is known to undergo cytoplasmic dynein-dependent nuclear translocation in response to DNA damage. However, the molecular link between p53 and the minus end-directed microtubule motor dynein complex has not been described. We report here that the 8-kDa light chain (LC8) of dynein binds to p53-binding protein 1 (53BP1). The LC8-binding domain was mapped to a short peptide segment immediately N-terminal to the kinetochore localization region of 53BP1. The LC8-binding domain is completely separated from the p53-binding domain in 53BP1. Therefore, 53BP1 can potentially act as an adaptor to assemble p53 to the dynein complex. Unlike other known LC8-binding proteins, 53BP1 contains two distinct LC8-binding motifs that are arranged in tandem. We further showed that 53BP1 can directly associate with the dynein complex. Disruption of the interaction between LC8 and 53BP1 in vivo prevented DNA damage-induced nuclear accumulation of p53. These data illustrate that LC8 is able to function as a versatile acceptor to link a wide spectrum of molecular cargoes to the dynein motor.

Ionotropic glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system. Their modulation is believed to affect learning and memory, and their dysfunction has been implicated in the pathogenesis of neurological and psychiatric diseases. Despite a wealth of functional data, little is known about the intact, three-dimensional structure of these ligand-gated ion channels. Here, we present the structure of native AMPA receptors (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; AMPA-Rs) purified from rat brain, as determined by single-particle electron microscopy. Unlike the homotetrameric recombinant GluR2 (ref. 3), the native heterotetrameric AMPA-R adopted various conformations, which reflect primarily a variable separation of the two dimeric extracellular amino-terminal domains. Members of the stargazin/TARP family of transmembrane proteins co-purified with AMPA-Rs and contributed to the density representing the transmembrane region of the complex. Glutamate and cyclothiazide markedly altered the conformational equilibrium of the channel complex, suggesting that desensitization is related to separation of the N-terminal domains. These data provide a glimpse of the conformational changes of an important ligand-gated ion channel of the brain.

NMDA receptors (NMDARs) control bidirectional synaptic plasticity by regulating postsynaptic AMPA receptors (AMPARs). Here we show that NMDAR activation can have differential effects on AMPAR trafficking, depending on the subunit composition of NMDARs. In mature cultured neurons, NR2A-NMDARs promote, whereas NR2B-NMDARs inhibit, the surface expression of GluR1, primarily by regulating its surface insertion. In mature neurons, NR2B is coupled to inhibition rather than activation of the Ras-ERK pathway, which drives surface delivery of GluR1. Moreover, the synaptic Ras GTPase activating protein (GAP) SynGAP is selectively associated with NR2B-NMDARs in brain and is required for inhibition of NMDAR-dependent ERK activation. Preferential coupling of NR2B to SynGAP could explain the subtype-specific function of NR2B-NMDARs in inhibition of Ras-ERK, removal of synaptic AMPARs, and weakening of synaptic transmission.

Here, we use a cell surface thrombin cleavage assay to investigate directly the role of NSF in the surface delivery and synaptic accumulation of alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) receptors. In cultured hippocampal neurons, the GluR2 subunit (which specifically interacts with NSF) inserts rapidly into the plasma membrane from intracellular compartments and accumulates in synaptic sites. In contrast, surface accumulation of GluR3 (a subunit that does not interact with NSF) or a GluR2 mutant defective in NSF binding (DeltaA849-Q853) occurs initially at extrasynaptic sites and is kinetically slower than wild-type GluR2. Introducing a binding site for NSF into GluR3 (GluR3NSF) generates a subunit that behaves like GluR2 in terms of kinetics and site of surface insertion. These data suggest that the NSF interaction is necessary for rapid incorporation of AMPA receptor subunits into synapses and is sufficient to confer this property on GluR3.

Leukocyte common antigen-related (LAR) family receptor protein tyrosine phosphatases (LAR-RPTP) bind to liprin-alpha (SYD2) and are implicated in axon guidance. We report that LAR-RPTP is concentrated in mature synapses in cultured rat hippocampal neurons, and is important for the development and maintenance of excitatory synapses in hippocampal neurons. RNA interference (RNAi) knockdown of LAR or dominant-negative disruption of LAR function results in loss of excitatory synapses and dendritic spines, reduction of surface AMPA receptors, impairment of dendritic targeting of the cadherin-beta-catenin complex, and reduction in the amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). Cadherin, beta-catenin and GluR2/3 are tyrosine phosphoproteins that coimmunoprecipitate with liprin-alpha and GRIP from rat brain extracts. We propose that the cadherin-beta-catenin complex is cotransported with AMPA receptors to synapses and dendritic spines by a mechanism that involves binding of liprin-alpha to LAR-RPTP and tyrosine dephosphorylation by LAR-RPTP.

The related small GTPases Ras and Rap1 are important for signaling synaptic AMPA receptor (-R) trafficking during long-term potentiation (LTP) and long-term depression (LTD), respectively. Rap2, which shares 60% identity to Rap1, is present at excitatory synapses, but its functional role is unknown. Here, we report that Rap2 activity, stimulated by NR2A-containing NMDA-R activation, depresses AMPA-R-mediated synaptic transmission via activation of JNK rather than Erk1/2 or p38 MAPK. Moreover, Rap2 controls synaptic removal of AMPA-Rs with long cytoplasmic termini during depotentiation. Thus, Rap2-JNK pathway, which opposes the action of the NR2A-containing NMDA-R-stimulated Ras-ERK1/2 signaling and complements the NR2B-containing NMDA-R-stimulated Rap1-p38 MAPK signaling, channels the specific signaling for depotentiating central synapses.

The total molecular mass of individual postsynaptic densities (PSDs) isolated from rat forebrain was measured by scanning transmission EM. PSDs had a mean diameter of 360 nm and molecular mass of 1.10 +/- 0.36 GDa. Because the mass represents the sum of the molecular masses of all of the molecules comprising a PSD, it becomes possible to derive the number of copies of each protein, once its relative mass contribution is known. Mass contributions of PSD-95, synapse-associated protein (SAP)97, and alpha-Ca2+/calmodulin-dependent protein kinase II (CaMKII) were determined by quantitative gel electrophoresis of PSD fractions. The number of PSD-95 molecules per average PSD, contributing 2.3% of the mass of the PSD, was calculated to be 300, whereas the number of SAP97 molecules, contributing 0.9% of the mass of the PSD, was 90. The alpha-CaMKII holoenzymes, which contribute 6% of the mass when brains are homogenized within 2 min of interrupting blood flow, have 80 holoenzymes associated with a typical PSD. When blood flow is interrupted 15 min before homogenization, the average mass of PSDs increases by approximately 40%. The additional alpha-CaMKII associated with PSDs accounts for up to 20% of this mass increase, representing the addition of 100-200 alpha-CaMKII holoenzymes.

The function of the multi-PDZ domain scaffold protein GRIP1 (glutamate receptor interacting protein 1) in neurons is unclear. To explore the function of GRIP1 in hippocampal neurons, we used RNA interference (RNAi) to knock down the expression of GRIP1. Knockdown of GRIP1 by small interfering RNA (siRNA) in cultured hippocampal neurons caused a loss of dendrites, associated with mislocalization of the GRIP-interacting proteins GIuR2 (AMPA receptor subunit), EphB2 (receptor tyrosine kinase) and KIF5 (also known as kinesin 1; microtubule motor). The loss of dendrites by GRIP1-siRNA was rescued by overexpression of the extracellular domain of EphB2, and was phenocopied by overexpression of the intracellular domain of EphB2 and extracellular application of ephrinB-Fc fusion proteins. Neurons from EphB1-EphB2-EphB3 triple knockout mice showed abnormal dendrite morphogenesis. Disruption of the KIF5-GRIP1 interaction inhibited EphB2 trafficking and strongly impaired dendritic growth. These results indicate an important role for GRIP1 in dendrite morphogenesis by serving as an adaptor protein for kinesin-dependent transport of EphB receptors to dendrites.

QZ1, 2-[2-chloro-6-hydroxy-3-oxo-5-(quinolin-8-ylaminomethyl)-3H-xanthen-9-yl]benzoic acid, and QZ2, 2-[6-hydroxy-3-oxo-4,5-bis-(quinolin-8-ylaminomethyl)-3H-xanthen-9-yl]benzoic acid, two fluorescein-based dyes derivatized with 8-aminoquinoline, have been prepared and their photophysical, thermodynamic, and zinc-binding kinetic properties determined. Because of their low background fluorescence and highly emissive Zn(II) complexes, QZ1 and QZ2 have a large dynamic range, with approximately 42- and approximately 150-fold fluorescence enhancements upon Zn(II) coordination, respectively. These dyes have micromolar K(d) values for Zn(II) and are selective for Zn(II) over biologically relevant concentrations of the alkali and alkaline earth metals. The Zn(II) complexes also fluoresce brightly in the presence of excess Mn(II), Fe(II), Co(II), Cd(II), and Hg(II), offering improved specificity for Zn(II) over di(2-picolyl)amine-based Zn(II) sensors. Stopped-flow kinetic investigations indicate that QZ1 and QZ2 bind Zn(II) with k(on) values of (3-4) x 10(6) M(-1) s(-1), compared to (6-8) x 10(5) M(-1) s(-1) for select ZP (Zinpyr) dyes, at 4.3 degrees C. Dissociation of Zn(II) from QZ1 and QZ2 occurs with k(off) values of 150 and 160 s(-1), over 5 orders of magnitude larger than those for ZP probes, achieving reversibility on the biological (millisecond) time scale. Laser scanning confocal and two-photon microscopy studies reveal that QZ2 is cell-permeable and Zn(II)-responsive in vivo. Because of its weaker affinity for Zn(II), QZ2 responds to higher concentrations of intracellular Zn(II) than members of the ZP family, illustrating that binding affinity is an important parameter for Zn(II) detection in vivo.