, 1987; reviewed in Sanes and Lichtman, 2001) and by the Drosophila glypican Dally-like, a GPI-anchored HSPG ( Johnson et al., 2006). In the CNS, overexpression of the transmembrane HSPG syndecan-2 accelerates dendritic spine morphogenesis ( Ethell and Yamaguchi, 1999). Secreted forms of glypican

Selleck MK-1775 4 and 6 promote glutamate receptor clustering and excitatory synapse formation in retinal ganglion cells ( Allen et al., 2012), suggesting that glypican may have synaptic organizing activity. However, the molecular interactions that mediate the effects of glypican have not been identified. Here we used a mass spectrometric approach to compare LRRTM2 and LRRTM4 binding partners and find that these proteins have distinct receptor preferences: whereas LRRTM2 primarily binds to neurexins, the preferential binding partners for LRRTM4 are glypicans. We find that the glypican-LRRTM4 interaction requires HS and can occur in trans. LRRTM4 regulates excitatory synapse development in cultured neurons and in vivo, and the synaptogenic activity of LRRTM4, but not of LRRTM2, requires HS. Our data identify glypican as a receptor for LRRTM4 and indicate that a trans-synaptic glypican-LRRTM4 interaction regulates excitatory synapse development. The LRRTM genes are expressed in specific and partially

nonoverlapping expression patterns during synaptogenesis and in the adult brain ( de Wit et al., 2009 and Laurén et al., 2003). During the synaptogenic period from postnatal day (P) 7 to P14, Imatinib LRRTM2 and LRRTM4 show complementary expression patterns in cortex, with LRRTM2 expression restricted to layer 6 and LRRTM4 mainly expressed in layer 2/3 and layer 5 ( Figures 1A and 1B). In the hippocampus, LRRTM2 and LRRTM4 are coexpressed in dentate gyrus (DG) granule cells ( Figures 1A and 1B, arrowheads). Whether different LRRTM family members expressed in the same neuron signal through the same new presynaptic receptor, or whether various LRRTMs have different mechanisms of action, is unknown. Phylogenetic analysis indicates that human LRRTM2 and LRRTM4 share only 43% amino acid identity and that LRRTM4 and LRRTM3 are

more closely related to each other than to other LRRTMs ( Laurén et al., 2003). These observations raised the possibility that LRRTM4 may have a different presynaptic receptor than LRRTM2. To identify candidate LRRTM4 interactors, we took an unbiased, discovery-based approach. We purified recombinant ecto-Fc proteins for LRRTM2 and LRRTM4 (Figure 1C) and used these in a side-by-side comparison to identify interacting proteins in detergent-solubilized whole-brain homogenate from 3- to 4-week-old rats by affinity chromatography. Bound proteins were analyzed by tandem mass spectrometry. In agreement with previous results (de Wit et al., 2009 and Ko et al., 2009a), the most abundant proteins bound to LRRTM2-Fc were neurexins (Figure 1D).