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.

Summary We screened 124 genes that are amplified in human HCC using a mouse hepatoblast model and identified 18 tumor-promoting genes, including CCND1 and its neighbor on 11q13.3, FGF19. Although it is widely assumed that CCND1 is the main driving oncogene of this common amplicon (15% frequency in HCC), both forward-transformation assays and RNAi-mediated inhibition in human HCC cells established that FGF19 is an equally important driver gene in HCC. Furthermore, clonal growth and tumorigenicity of HCC cells harboring the 11q13.3 amplicon were selectively inhibited by RNAi-mediated knockdown of CCND1 or FGF19, as well as by an anti-FGF19 antibody. These results show that 11q13.3 amplification could be an effective biomarker for patients most likely to respond to anti-FGF19 therapy.

Dynamic regulation of the number and activity of AMPA receptors is believed to underlie many forms of synaptic plasticity and is presumably mediated by specific protein-protein interactions involving the C-terminal domain of the receptor. Several proteins interacting with the C-terminal tails of the glutamate receptor (GluR)-A and GluR-B subunits have been identified and implicated in the regulation of endocytosis and exocytosis, clustering, and anchoring of AMPA receptors to the cytoskeleton. In contrast, little is known of the molecular interactions of the GluR-D subunit, or of the mechanisms regulating the traffic of GluR-D-containing AMPA receptors. We analyzed the subcellular localization of homomeric GluR-D receptors carrying C-terminal deletions in transfected human embryonic kidney (HEK) 293 cells and in primary neurons by immunofluorescence microscopy and ELISA. A minimal requirement for a 14-residue cytoplasmic segment for the surface expression of homomeric GluR-D receptors was identified. Previously, a similar region in the GluR-A subunit was implicated in an interaction with 4.1 family proteins. Coimmunoprecipitation demonstrated that GluR-D associated with 4.1 protein(s) in both HEK293 cells and rat brain. Moreover, glutathione S-transferase pull-down experiments showed that the same 14-residue segment is critical for 4.1 binding to GluR-A and GluR-D. Point mutations within this segment dramatically decreased the surface expression of GluR-D in HEK293 cells, with a concomitant loss of the 4.1 interaction. Our findings demonstrate a novel molecular interaction for the GluR-D subunit and suggest that the association with the 4.1 family protein(s) plays an essential role in the transport to and stabilization of GluR-D-containing AMPA receptors at the cell surface.

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 vitro MAGUK-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.Synapse-associated protein 97 (SAP97) 1 and the closely related SAP90/PSD-95, SAP102, and PSD-93/chapsyn-110 form a family of membrane-associated guanylate kinase homologues (MAGUKs), characterized by the presence of three PDZ domains, an SH3 domain, and a C-terminal guanylate kinase homologous domain (1-5). Interactions of the PDZ domains of MAGUK proteins with type I C-terminal binding motifs present in ionotropic glutamate receptor subunits have been implicated in the regulation of the organization and functional activity of glutamatergic synapses (for review, see Refs. 6 and 7). Direct physical association of these MAGUK proteins with the subunits of N-methyl-D-aspartate (NMDA) (4, 8 -10), kainate (11), and ␣-amino-5-hydroxy-3-methyl-4-isoxazole propionate (AMPA)-selective glutamate receptors (12) has been demonstrated, but the regulation and detailed physiological functions of these interactions are still unclear.Interaction between the AMPA receptor subunit GluR-A (GluR1) and SAP97 is particularly interesting (12). First, an interaction between the C terminus of GluR-A and a type I PDZ domain protein has been implicated in several models of activitydependent regulation of synaptic strength (13). Second, it is to date the only PDZ domain interaction of AMPA receptors that does not involve the GluR-B subunit and, therefore, may be relevant for the synaptic organization of calcium-permeable AMPA receptors, which lack this subunit (14). Third, in contrast to the majority of synaptic MAGUKs, which generally share binding partners, GluR-A appears to bind only to SAP97. However, the true selectivity and its underlying mechanisms have yet to be defined. In the present study, we show that GluR-A binds to the...

No abstract