Chunlin Cai

KCC2 interacts with the dendritic cytoskeleton to promote spine development.

The neuron-specific K-Cl cotransporter, KCC2, induces a developmental shift to render GABAergic transmission from depolarizing to hyperpolarizing. Now we demonstrate that KCC2, independently of its Cl(-) transport function, is a key factor in the maturation of dendritic spines. This morphogenic role of KCC2 in the development of excitatory synapses is mediated by structural interactions between KCC2 and the spine cytoskeleton. Here, the binding of KCC2 C-terminal domain to the cytoskeleton-associated protein 4.1N may play an important role. A more general conclusion based on our data is that KCC2 acts as a synchronizing factor in the functional development of glutamatergic and GABAergic synapses in cortical neurons and networks.

Selective Binding of Synapse-associated Protein 97 to GluR-A α-Amino-5-hydroxy-3-methyl-4-isoxazole Propionate Receptor Subunit Is Determined by a Novel Sequence Motif

A family of four closely related PDZ domain-containing membrane-associated guanylate kinase homologues (MAGUKs) is involved in the regulation of the amount and functional state of ionotropic glutamate receptors in excitatory synapses. To understand the mechanisms that determine the specificity of these interactions, we examined the structural basis of the highly selective association between the ionotropic GluR subunit GluR-A and synapse-associated protein 97 (SAP97). The C terminus of GluR-A bound to the PDZ domains of SAP97, but not to those of three related MAGUKs, PSD-93, PSD-95, and SAP102. Experiments with single PDZ domains indicated that the strongest contribution was by the second PDZ domain. Unexpectedly, mutation analysis of the GluR-A C terminus revealed that a tripeptide sequence SSG at position −9 to −11 plays an essential role in this binding, in addition to a C-terminal type I PDZ binding motif (leucine at C terminus and threonine at the −2 position). Analysis of the in vitroMAGUK-binding properties of a GluR-D mutant with a one-residue deletion at the C terminus provides further support for the view that an SSG sequence located N-terminally from a type I PDZ binding motif can mediate selective binding to SAP97 and suggest the existence of a novel variation of the PDZ domain-peptide interaction.

Isoform-Specific Early Trafficking of AMPA Receptor Flip and Flop Variants

Flip and flop splice variants of AMPA receptor subunits are expressed in distinct but partly overlapping patterns and impart different desensitization kinetics to cognate receptor channels. In the absence of specific antibodies, isoform-specific differences in trafficking or localization of native flip and flop subunits remain uncharacterized. We report that in several transfected cell lines, transport of homomeric glutamate receptor (GluR)-Dflop receptors is largely blocked at the endoplasmic reticulum (ER) exit, whereas GluR-Dflip undergoes complex glycosylation and reaches the plasma membrane at >10× higher levels than GluR-Dflop, as determined by immunofluorescence, patch-clamp recordings and biochemical assays. The transport difference between flip and flop is independent of activity, is primarily determined by amino acid residue 780 (Leu in flop, Val in flip), and is manifested even in the secretion of the soluble ligand-binding domain, suggesting it is independent of oligomerization. Coexpression with stargazin or with the flip isoform rescues the surface expression of GluR-Dflop near to the level exhibited by GluR-Dflip. Our results demonstrate that the extracellular flip/flop region, via interactions with ER luminal splice form-specific protein(s), plays a hitherto unappreciated and important role in AMPA-receptor trafficking.

Interaction between SAP97 and PSD-95, two Maguk proteins involved in synaptic trafficking of AMPA receptors

Synapse-associated protein 97 (SAP97) and postsynaptic density 95 (PSD-95) are closely related membrane-associated guanylate kinase homologs (Maguks) implicated in the synaptic targeting and anchoring of α-amino-5-methyl-3-hydroxy-4-isoxazolepropionic acid (AMPA)-selective glutamate receptors. Prompted by accumulating evidence for an oligomeric nature of Maguks, we examined the potential of SAP97 and PSD-95 to form heteromeric complexes. SAP97 and PSD-95 coimmunoprecipitated from rat brain detergent extracts and subsequent glutathione S-transferase pull-down and immunoprecipitation experiments showed that the interaction is mediated by binding of the N-terminal segment of SAP97 (SAP97NTD) to the Src homology 3 domain of PSD-95 (PSD-95SH3). In cultured hippocampal neurons, expression of green fluorescent protein-tagged PSD-95 triggered accumulation of SAP97 in synaptic spines, which was totally inhibited by coexpression of PSD-95SH3. Furthermore, overexpression of green fluorescent protein-PSD-95 induced dendritic clustering of GluR-A subunit-containing AMPA receptors, which was strongly inhibited by cotransfection with SAP97NTD and PSD-95SH3 constructs. Our results demonstrated a direct interaction between SAP97 and PSD-95 and suggested that this association may play a functional role in the trafficking and clustering of AMPA receptors.

Crystal structure of the second PDZ domain of SAP97 in complex with a GluR-A C-terminal peptide

Synaptic targeting of GluR‐A subunit‐containing glutamate receptors involves an interaction with synapse‐associated protein 97 (SAP97). The C‐terminus of GluR‐A, which contains a class I PDZ ligand motif (‐x‐Ser/Thr‐x‐φ‐COOH where φ is an aliphatic amino acid) associates preferentially with the second PDZ domain of SAP97 (SAP97PDZ2). To understand the structural basis of this interaction, we have determined the crystal structures of wild‐type and a SAP97PDZ2 variant in complex with an 18‐mer C‐terminal peptide (residues 890–907) of GluR‐A and of two variant PDZ2 domains in unliganded state at 1.8–2.44 Å resolutions. SAP97PDZ2 folds to a compact globular domain comprising six β‐strands and two α‐helices, a typical architecture for PDZ domains. In the structure of the peptide complex, only the last four C‐terminal residues of the GluR‐A are visible, and align as an antiparallel β‐strand in the binding groove of SAP97PDZ2. The free carboxylate group and the aliphatic side chain of the C‐terminal leucine (Leu907), and the hydroxyl group of Thr905 of the GluR‐A peptide are engaged in essential class I PDZ interactions. Comparison between the free and complexed structures reveals conformational changes which take place upon peptide binding. The βΑ−βΒ loop moves away from the C‐terminal end of αB leading to a slight opening of the binding groove, which may better accommodate the peptide ligand. The two conformational states are stabilized by alternative hydrogen bond and coulombic interactions of Lys324 in βΑ−βΒ loop with Asp396 or Thr394 in βΒ. Results of in vitro binding and immunoprecipitation experiments using a PDZ motif‐destroying L907A mutation as well as the insertion of an extra alanine residue between the C‐terminal Leu907 and the stop codon are also consistent with a ‘classical’ type I PDZ interaction between SAP97 and GluR‐A C‐terminus.

Interaction of the RhoA Exchange Factor Net1 with Discs Large Homolog 1 Protects It from Proteasome-mediated Degradation and Potentiates Net1 Activity

Net1 is a nuclear Rho guanine nucleotide exchange factor that is specific for the RhoA subfamily of small G proteins. Truncated forms of Net1 are transforming in NIH3T3 cells, and this activity requires cytoplasmic localization of Net1 as well as the presence of a COOH-terminal PDZ binding site. We have previously shown that Net1 interacts with PDZ domain-containing proteins within the Discs Large (Dlg) family and relocalizes them to the nucleus. In the present work, we demonstrate that Net1 binds directly to the first two PDZ domains of Dlg1 and that both PDZ domains are required for maximal interaction in cells. Furthermore, we show that Net1 is an unstable protein in MCF7 breast epithelial cells and that interaction with Dlg1 significantly enhances Net1 stability. Stabilization by Dlg1 significantly increases the ability of Net1 to stimulate RhoA activation in cells. The stability of endogenous Net1 is strongly enhanced by cell-cell contact, and this correlates with a dramatic increase in the interaction between Net1 and Dlg1. Importantly, disruption of E-cadherin-mediated cell contacts, either by depletion of external calcium or by treatment with transforming growth factor β, leads to a rapid loss of the interaction between Net1 and Dlg1 and a subsequent increase in the ubiquitylation of Net1. These results indicate that Net1 requires interaction with PDZ domain proteins, such as Dlg1, to protect it from proteasome-mediated degradation and to maximally stimulate RhoA and that this interaction is regulated by cell-cell contact.

Somatostatin receptor subtype 1 is a PDZ ligand for synapse-associated protein 97 and a potential regulator of growth cone dynamics.

We report that somatostatin receptor subtype 1 (sst1) associates in vivo and in vitro with synapse-associated protein SAP) 97, a membrane-associated guanylate kinase homolog implicated as a scaffolding protein in the structural organization of specialized membrane complexes in various tissues, including the CNS. SAP97 and sst1 were coimmuno-precipitated from rodent brain and from transfected human embryonic kidney (HEK) 293 cells, and pull-down experiments demonstrated that the interaction is dependent on the class I PDZ binding motif in sst1 carboxyterminus. Calorimetric titration indicated that the postsynaptic density-95/discs large/zona occludens-1 (PDZ) 2 domain of SAP97 provides the main contribution to the interaction. We noticed substantial sst1 immunoreactivity in differentiating cortical neurons in culture which declined as the cultures matured. The sst1 immunoreactivity extended, together with SAP97 to neuronal growth cones. Somatostatin (1 microM) triggered retraction of the filopodia and lamellipodia in the growth cones. This growth cone collapse was enhanced by overexpression of green fluorescent protein-tagged sst1, whereas sst1 mutant lacking the PDZ binding motif had no effect. These findings suggest a role for somatostatin signaling in the regulation of growth cone stability, which may involve PDZ domain proteins interacting with sst1 and/or other somatostatin receptors. Consistent with a developmental role, sst1 immunoreactivity was present transiently in the developing mouse cortex, peaking at postnatal day 5 and declining thereafter to low levels in the adult cortex.

Analysis of the Potential Role of GluA4 Carboxyl-Terminus in PDZ Interactions

Background Specific delivery to synapses of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors with long-tailed subunits is believed to be a key event in many forms of activity-dependent changes in synaptic strength. GluA1, the best characterized long-tailed AMPA receptor subunit, contains a C-terminal class I PDZ binding motif, which mediates its interaction with scaffold and trafficking proteins, including synapse-associated protein 97 (SAP97). In GluA4, another long-tailed subunit implicated in synaptic plasticity, the PDZ motif is blocked by a single proline residue. This feature is highly conserved in vertebrates, whereas the closest invertebrate homologs of GluA4 have a canonical class I PDZ binding motif. In this work, we have examined the role of GluA4 in PDZ interactions. Methodology/Principal Findings Deletion of the carboxy-terminal proline residue of recombinant GluA4 conferred avid binding to SAP97 in cultured cells as shown by coimmunoprecipitation, whereas wild-type GluA4 did not associate with SAP97. Native GluA4 and SAP97 coimmunoprecipitated from mouse brain independently of the GluA1 subunit, supporting the possibility of in vivo PDZ interaction. To obtain evidence for or against the exposure of the PDZ motif by carboxyterminal processing of native GluA4 receptors, we generated an antibody reagent specific for proline-deleted GluA4 C-terminus. Immunoprecipitation and mass spectrometric analyses indicated that the carboxyl-terminus of native GluA4 AMPA receptors is intact and that the postulated single-residue cleavage does not occur to any significant extent. Conclusion/Significance We conclude that native GluA4 receptors are not capable of canonical PDZ interactions and that their association with SAP97 is likely to be indirect.

Molecular mechanisms controlling synaptic recruitment of GluA4 subunit-containing AMPA-receptors critical for functional maturation of CA1 glutamatergic synapses.

ABSTRACT Synaptic recruitment of AMPA receptors (AMPARs) represents a key postsynaptic mechanism driving functional development and maturation of glutamatergic synapses. At immature hippocampal synapses, PKA‐driven synaptic insertion of GluA4 is the predominant mechanism for synaptic reinforcement. However, the physiological significance and molecular determinants of this developmentally restricted form of plasticity are not known. Here we show that PKA activation leads to insertion of GluA4 to synaptic sites with initially weak or silent AMPAR‐mediated transmission. This effect depends on a novel mechanism involving the extreme C‐terminal end of GluA4, which interacts with the membrane proximal region of the C‐terminal domain to control GluA4 trafficking. In the absence of GluA4, strengthening of AMPAR‐mediated transmission during postnatal development was significantly delayed. These data suggest that the GluA4‐mediated activation of silent synapses is a critical mechanism facilitating the functional maturation of glutamatergic circuitry during the critical period of experience‐dependent fine‐tuning. This article is part of the Special Issue entitled ‘Ionotropic glutamate receptors’. HIGHLIGHTSPKA activation leads to synaptic unsilencing via insertion of GluA4.This depends on a novel mechanism involving the extreme C‐terminal end of GluA4.Absence of GluA4 causes delayed postnatal maturation of AMPA transmission.