Synaptic adhesion molecules regulate various aspects of synapse development, function and plasticity. These functions mainly involve trans-synaptic interactions and positive regulations, whereas cis-interactions and negative regulation are less understood. Here we report that SALM4, a member of the SALM/Lrfn family of synaptic adhesion molecules, suppresses excitatory synapse development through cis inhibition of SALM3, another SALM family protein with synaptogenic activity. Salm4-mutant (Salm4−/−) mice show increased excitatory synapse numbers in the hippocampus. SALM4 cis-interacts with SALM3, inhibits trans-synaptic SALM3 interaction with presynaptic LAR family receptor tyrosine phosphatases and suppresses SALM3-dependent presynaptic differentiation. Importantly, deletion of Salm3 in Salm4−/− mice (Salm3−/−; Salm4−/−) normalizes the increased excitatory synapse number. These results suggest that SALM4 negatively regulates excitatory synapses via cis inhibition of the trans-synaptic SALM3–LAR adhesion.

Synaptic adhesion-like molecules (SALMs) are a family of cell adhesion molecules involved in regulating neuronal and synapse development that have also been implicated in diverse brain dysfunctions, including autism spectrum disorders (ASDs). SALMs, also known as leucine-rich repeat (LRR) and fibronectin III domain-containing (LRFN) proteins, were originally identified as a group of novel adhesion-like molecules that contain LRRs in the extracellular region as well as a PDZ domain-binding tail that couples to PSD-95, an abundant excitatory postsynaptic scaffolding protein. While studies over the last decade have steadily explored the basic properties and synaptic and neuronal functions of SALMs, a number of recent studies have provided novel insights into molecular, structural, functional and clinical aspects of SALMs. Here we summarize these findings and discuss how SALMs act in concert with other synaptic proteins to regulate synapse development and function.

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Angiogenesis depends on VEGF-mediated signaling. However, the regulatory mechanisms and functions of individual VEGF receptor 2 (VEGFR2) phosphorylation sites remain unclear. Here, we report that synaptic adhesion-like molecule 4 (SALM4) regulates a specific VEGFR2 phosphorylation site. SALM4 silencing in HUVECs and Salm4 knockout (KO) in lung endothelial cells (ECs) of Salm4 −/− mice suppressed phosphorylation of VEGFR2 tyrosine (Y) 1175 (Y1173 in mice) and downstream signaling upon VEGF-A stimulation. However, VEGFR2 phosphorylation at Y951 (Y949 in mice) and Y1214 (Y1212 in mice) remained unchanged. Knockdown and KO of SALM4 inhibited VEGF-A–induced angiogenic functions of ECs. SALM4 depletion reduced endothelial leakage, sprouting, and migratory activities. Furthermore, in an ischemia and reperfusion (I/R) model, brain injury was attenuated in Salm4 −/− mice compared with wild-type (WT) mice. In brain lysates after I/R, VEGFR2 phosphorylation at Y949, Y1173, and Y1212 were induced in WT brains, but only Y1173 phosphorylation of VEGFR2 was reduced in Salm4 −/− brains. Taken together, our results demonstrate that SALM4 specifically regulates VEGFR2 phosphorylation at Y1175 (Y1173 in mice), thereby fine-tuning VEGF signaling in ECs.—Kim, D. Y., Park, J. A., Kim, Y., Noh, M., Park, S., Lie, E., Kim, E., Kim, Y.-M., Kwon, Y.-G. SALM4 regulates angiogenic functions in endothelial cells through VEGFR2 phosphorylation at Tyr1175.

We report the first proteomic analysis of the SLP76 interactome in resting and activated primary mouse mast cells. This was made possible by a novel genetic approach used for the first time here. It consists in generating knock-in mice that express signaling molecules bearing a C-terminal tag that has a high affinity for a streptavidin analog. Tagged molecules can be used as molecular baits to affinity-purify the molecular complex in which they are engaged, which can then be studied by mass spectrometry. We examined first SLP76 because, although this cytosolic adapter is critical for both T cell and mast cell activation, its role is well known in T cells but not in mast cells. Tagged SLP76 was expressed in physiological amounts and fully functional in mast cells. We unexpectedly found that SLP76 is exquisitely sensitive to mast cell granular proteases, that Zn 2؉ -dependent metalloproteases are especially abundant in mast cells and that they were responsible for SLP76 degradation. Adding a Zn 2؉ chelator fully protected SLP76 in mast cell lysates, thereby enabling an efficient affinity-purification of this adapter with its partners. Label-free quantitative mass spectrometry analysis of affinity-purified SLP76 interactomes uncovered both 1 The abbreviations used are: FcRI, high-affinity receptors for the Fc portion of IgE; ADAP, adhesion-and degranulation-promoting adapter protein; Bcr, breakpoint cluster region protein; BMMC, Bone marrow-derived mast cells; DAG, diacyl-glycerol; DNP, Dinitrophenyl; ES, Embryonic stem; FDR, false discovery rate; Fyb, Fyn-binding protein; Grap2, Grb2-related adapter protein 2; Grb2, growth factor receptor-bound protein 2; HSA, human serum albumin; iBAQ, intensity-based absolute quantification; IP3, inositol tris-phosphate; ITAM, immunoreceptor tyrosine-based activation motif; Itk, IL-2-inducible T-cell kinase; KI, knock-in; LAT, linker of activation of T cells; LCP2, lymphocyte cytosolic protein 2, LM, laurylmaltoside; mAb, monoclonal antibody; MAP, mitogen-activated protein; MS/MS, tandem mass spectrometry; nanoLC, nano liquid chromatography; Nck, noncatalytic region of tyrosine kinase adapter protein; NTAL, non-T cell activation linker; OST, one-strep-tag; PAG, phosphoprotein associated with glycosphingolipid-enriched microdomains; PLC, phospholipase C; SH, Src homology; SHIP, SH2 domain-containing inositol 5Ј-phosphatase; SKAP, Src kinase-associated phopshoproteins; SLP76, SH2 domain-containing leukocyte protein of 76 kDa; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; TCR, T cell receptor; WT, wild-type; ZAP70, zeta-associated protein of 70 kDa. Research