FLRT2 and FLRT3 act as repulsive guidance cues for Unc5-positive neurons.

Netrin-1 induces repulsive axon guidance by binding to the mammalian Unc5 receptor family (Unc5A-Unc5D). Mouse genetic analysis of selected members of the Unc5 family, however, revealed essential functions independent of Netrin-1, suggesting the presence of other ligands. Unc5B was recently shown to bind fibronectin and leucine-rich transmembrane protein-3 (FLRT3), although the relevance of this interaction for nervous system development remained unclear. Here, we show that the related Unc5D receptor binds specifically to another FLRT protein, FLRT2. During development, FLRT2/3 ectodomains (ECDs) are shed from neurons and act as repulsive guidance molecules for axons and somata of Unc5-positive neurons. In the developing mammalian neocortex, Unc5D is expressed by neurons in the subventricular zone (SVZ), which display delayed migration to the FLRT2-expressing cortical plate (CP). Deletion of either FLRT2 or Unc5D causes a subset of SVZ-derived neurons to prematurely migrate towards the CP, whereas overexpression of Unc5D has opposite effects. Hence, the shed FLRT2 and FLRT3 ECDs represent a novel family of chemorepellents for Unc5-positive neurons and FLRT2/Unc5D signalling modulates cortical neuron migration.

Constitutive Gs-mediated, but not G12-mediated, activity of the 5-hydroxytryptamine 5-HT7(a) receptor is modulated by the palmitoylation of its C-terminal domain.

The 5-HT(7) receptor is the most recently described member of the serotonin receptor family. This receptor is mainly expressed in the thalamus, hypothalamus as well as in the hippocampus and cortex. In the present study, we demonstrate that the mouse 5-hydroxytryptamine 5-HT(7(a)) receptor undergoes post-translational modification by the palmitate, which is covalently attached to the protein through a thioester-type bond. Analysis of protein-bound fatty acids revealed that the 5-HT(7(a)) receptor predominantly contains palmitic acid. Labelling experiments performed in the presence of agonists show that the 5-HT(7(a)) receptor is dynamically palmitoylated in an agonist-dependent manner and that previously synthesized receptors may be subjected to repeated cycles of palmitoylation/depalmitoylation. Mutation analysis revealed that cysteine residues 404 and 438/441 located in the C-terminal receptor domain are the main palmitoylation sites responsible for the attachment of 90% of the receptor-bound palmitate. Analysis of acylation-deficient mutants revealed that non-palmitoylated 5-HT(7(a)) receptors were indistinguishable from the wild-type for their ability to interact with G(s)- and G(12)-proteins after agonist stimulation. However, mutation of the proximal palmitoylation site Cys404-Ser (either alone or in combination with Cys438/441-Ser) significantly increased the agonist-independent, G(s)-mediated constitutive 5-HT(7(a)) receptor activity, while the activation of Galpha(12)-protein was not affected. This demonstrates a functional importance of 5-HT(7(a)) dynamic palmitoylation for the fine tuning of receptor-mediated signaling.

5-HT7 receptor is coupled to G alpha subunits of heterotrimeric G12-protein to regulate gene transcription and neuronal morphology.

The neurotransmitter serotonin (5-HT) plays an important role in the regulation of multiple events in the CNS. We demonstrated recently a coupling between the 5-HT4 receptor and the heterotrimeric G13-protein resulting in RhoA-dependent neurite retraction and cell rounding (Ponimaskin et al., 2002). In the present study, we identified G12 as an additional G-protein that can be activated by another member of serotonin receptors, the 5-HT7 receptor. Expression of 5-HT7 receptor induced constitutive and agonist-dependent activation of a serum response element-mediated gene transcription through G12-mediated activation of small GTPases. In NIH3T3 cells, activation of the 5-HT7 receptor induced filopodia formation via a Cdc42-mediated pathway correlating with RhoA-dependent cell rounding. In mouse hippocampal neurons, activation of the endogenous 5-HT7 receptors significantly increased neurite length, whereas stimulation of 5-HT4 receptors led to a decrease in the length and number of neurites. These data demonstrate distinct roles for 5-HT7R/G12 and 5-HT4R/G13 signaling pathways in neurite outgrowth and retraction, suggesting that serotonin plays a prominent role in regulating the neuronal cytoarchitecture in addition to its classical role as neurotransmitter.

The GABA(B) receptor subunits R1 and R2 interact differentially with the activation transcription factor ATF4 in mouse brain during the postnatal development.

Gamma-aminobutyric acid type B receptors (GABA(B)R) belong to the family of G-protein-coupled receptors that mediate synaptic actions by modulation of different ion channels. Here, we demonstrate that the receptor subunits GABA(B)R1 and GABA(B)R2 interact directly with the soluble activating transcription factor 4 (ATF4) in different regions of the neonatal mouse brain. We found that about 5-12% of expressed ATF4 protein is involved in the complex formation with GABA(B) receptors. Confocal fluorescence microscopy showed that GABA(B)R and ATF4 are co-localized in several well-defined spots in neurons and in glial cells. Co-immunoprecipitation analysis also reveals that the interaction efficiency between GABA(B) receptors and ATF4 in the mouse brain markedly changed during postnatal development, and such changes in interaction were dependent on the GABA(B) receptor subtype.