Targeting and clustering citron to synapses.

Citron-N (citron) binds activated Rho and concentrates at excitatory postsynaptic densities on the smooth dendritic shafts of hippocampal GABAergic neurons. Since little is known about how cytoplasmic proteins become targeted and retained at synapses, we asked how citron attains this discrete distribution. We first sought to determine whether the synaptic localization machinery is unique to GABAergic interneurons, but in neurons cultured from either cerebral cortex or hippocampus, low levels of citron are found in a small population of glutamatergic neurons concentrated at the tips of dendritic spines in addition to shaft synapses. These data suggest that the targeting domains in citron can be utilized appropriately by any neuron, and consistent with this idea, tagged, exogenous, full-length citron becomes concentrated at postsynaptic sites in all hippocampal neurons in which it is introduced. Citron contains multiple sites for protein-protein interaction, including coiled-coil, pleckstrin homology, cysteine-rich zinc binding and CMG domains, as well as a proline-rich region and a PDZ binding tail that is known to interact with PSD-95/SAP90. Using citron deletion mutants, we find that the coiled-coil (CC) domain, located in the N-terminal half of the protein, mediates somatodendritic targeting, self-oligomerization and puncta formation independent of the Rho binding domain, while regions in the C-terminal half of citron are responsible for precise alignment of postsynaptic citron clusters at sites apposing presynaptic boutons. Rho binding and activation regulate membrane targeting and the number of synapses containing citron. Thus, citron's clustering at postsynaptic membranes is mediated by multiple domains, and its synaptic distribution is modified in response to Rho activation.