Synaptic Adhesion-like Molecules Research Articles

Crystallization and low-resolution structure solution of the SALM3-PTPσ synaptic adhesion complex.

Synaptic adhesion molecules are major organizers of the neuronal network and play a crucial role in the regulation of synapse development and maintenance in the brain. Synaptic adhesion-like molecules (SALMs) and leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-PTPs) are adhesion protein families with established synaptic function. Dysfunction of several synaptic adhesion molecules has been linked to cognitive disorders such as autism spectrum disorders and schizophrenia. A recent study of the binding and complex structure of SALM3 and PTPσ using small-angle X-ray scattering revealed a 2:2 complex similar to that observed for the interaction of human SALM5 and PTPδ. However, the molecular structure of the SALM3-PTPσ complex remains to be determined beyond the small-angle X-ray scattering model. Here, the expression, purification, crystallization and initial 6.5 Å resolution structure of the mouse SALM3-PTPσ complex are reported, which further verifies the formation of a 2:2 trans-heterotetrameric complex similar to the crystal structure of human SALM5-PTPδ and validates the architecture of the previously reported small-angle scattering-based solution structure of the SALM3-PTPσ complex. Details of the protein expression and purification, crystal optimization trials, and the initial structure solution and data analysis are provided.

Structural basis of SALM3 dimerization and synaptic adhesion complex formation with PTP\u03c3

Synaptic adhesion molecules play an important role in the formation, maintenance and refinement of neuronal connectivity. Recently, several leucine rich repeat (LRR) domain containing neuronal adhesion molecules have been characterized including netrin G-ligands, SLITRKs and the synaptic adhesion-like molecules (SALMs). Dysregulation of these adhesion molecules have been genetically and functionally linked to various neurological disorders. Here we investigated the molecular structure and mechanism of ligand interactions for the postsynaptic SALM3 adhesion protein with its presynaptic ligand, receptor protein tyrosine phosphatase σ (PTPσ). We solved the crystal structure of the dimerized LRR domain of SALM3, revealing the conserved structural features and mechanism of dimerization. Furthermore, we determined the complex structure of SALM3 with PTPσ using small angle X-ray scattering, revealing a 2:2 complex similar to that observed for SALM5. Solution studies unraveled additional flexibility for the complex structure, but validated the uniform mode of action for SALM3 and SALM5 to promote synapse formation. The relevance of the key interface residues was further confirmed by mutational analysis with cellular binding assays and artificial synapse formation assays. Collectively, our results suggest that SALM3 dimerization is a pre-requisite for the SALM3-PTPσ complex to exert synaptogenic activity.

SALM/Lrfn Family Synaptic Adhesion Molecules.

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.

Structural basis of trans-synaptic interactions between PTP\u03b4 and SALMs for inducing synapse formation

Synapse formation is triggered by trans-synaptic interactions of cell adhesion molecules, termed synaptic organizers. Three members of type-II receptor protein tyrosine phosphatases (classified as type-IIa RPTPs; PTPδ, PTPσ and LAR) are known as presynaptic organizers. Synaptic adhesion-like molecules (SALMs) have recently emerged as a family of postsynaptic organizers. Although all five SALM isoforms can bind to the type-IIa RPTPs, only SALM3 and SALM5 reportedly have synaptogenic activities depending on their binding. Here, we report the crystal structures of apo-SALM5, and PTPδ–SALM2 and PTPδ–SALM5 complexes. The leucine-rich repeat (LRR) domains of SALMs interact with the second immunoglobulin-like (Ig) domain of PTPδ, whereas the Ig domains of SALMs interact with both the second and third Ig domains of PTPδ. Unexpectedly, the structures exhibit the LRR-mediated 2:2 complex. Our synaptogenic co-culture assay using site-directed SALM5 mutants demonstrates that presynaptic differentiation induced by PTPδ–SALM5 requires the dimeric property of SALM5.

Dileucine and PDZ-binding Motifs Mediate Synaptic Adhesion-like Molecule 1 (SALM1) Trafficking in Hippocampal Neurons

Synaptic adhesion-like molecules (SALMs) are a family of cell adhesion molecules involved in neurite outgrowth and synapse formation. Of the five family members, only SALM1, -2, and -3 contain a cytoplasmic C-terminal PDZ-binding motif. We have found that SALM1 is unique among the SALMs because deletion of its PDZ-binding motif (SALM1ΔPDZ) blocks its surface expression in heterologous cells. When expressed in hippocampal neurons, SALM1ΔPDZ had decreased surface expression in dendrites and the cell soma but not in axons, suggesting that the PDZ-binding domain may influence cellular trafficking of SALMs to specific neuronal locations. Endoglycosidase H digestion assays indicated that SALM1ΔPDZ is retained in the endoplasmic reticulum (ER) in heterologous cells. However, when the entire C-terminal tail of SALM1 was deleted, SALM1 was detected on the cell surface. Using serial deletions, we identified a region of SALM1 that contains a putative dileucine ER retention motif, which is not present in the other SALMs. Mutation of this DXXXLL motif allowed SALM1 to leave the ER and enhanced its surface expression in heterologous cells and neurons. An increase in the number of protrusions at the dendrites and cell body was observed when this SALM1 mutant was expressed in hippocampal neurons. With electron microscopy, these protrusions appeared to be irregular, enlarged spines and filopodia. Thus, enrichment of SALM1 on the cell surface affects dendritic arborization, and intracellular motifs regulate its dendritic versus axonal localization.

Flotillin-1 mediates neurite branching induced by synaptic adhesion-like molecule 4 in hippocampal neurons

Proper development of neurons in the hippocampus is essential for learning and memory. Our laboratory previously discovered a family of synaptic adhesion-like molecules (SALMs) which induce neurite outgrowth in this brain region (Wang et al., 2006). Here we establish flotillin-1 (flot-1) as a molecular mediator of neurite branching for SALM4. Knockdown of flot-1 alone in cultured hippocampal neurons using siRNA from 3–7 days in vitro (DIV) impaired neurite branching, whereas overexpression of flot-1 during the same time period increased the number of processes and branching. We show that induction of neurite outgrowth by flot-1 depends on amino acids 134–151 as well as lipid raft microdomains, SoHo proteins to regulate the actin cytoskeleton, and the exocyst complex to deliver new membrane proteins to growing neurites. When each of SALMs 1–5 was overexpressed, siRNA knockdown of flot-1 prevented neurite branching by SALM4. Overall, our data reveal a flot-1 signaling pathway for hippocampal neurite branching that is regulated by SALM4.

Selected SALM (Synaptic Adhesion-Like Molecule) Family Proteins Regulate Synapse Formation

Synaptic cell adhesion molecules regulate various steps of synapse formation. Despite the great diversity of neuronal synapses, relatively few adhesion molecules with synaptogenic activity have been identified. Synaptic adhesion-like molecules (SALMs) are members of a family of cell adhesion molecules known to regulate neurite outgrowth and synapse maturation; however, the role of SALMs in synapse formation remains unknown. We found that expression of the SALM family proteins SALM3 and SALM5 in nonneural and neural cells induces both excitatory and inhibitory presynaptic differentiation in contacting axons. SALM3 and SALM5 proteins are enriched in synaptic fractions, and form strong (SALM3) or weak (SALM5) complexes with postsynaptic density-95 (PSD-95), an abundant postsynaptic scaffolding protein at excitatory synapses. Aggregation of SALM3, but not SALM5, on dendritic surfaces induces clustering of PSD-95. Knockdown of SALM5 reduces the number and function of excitatory and inhibitory synapses. These results suggest that selected SALM family proteins regulate synapse formation, and that SALM3 and SALM5 may promote synapse formation through distinct mechanisms.

Reticulon 3 is an interacting partner of the SALM family of adhesion molecules

Synaptic adhesion-like molecules (SALMs) are a recently discovered family of adhesion molecules that is widely distributed in the central nervous system and has been implicated in neurite outgrowth and synapse formation. To identify proteins that interact with extracellular domains of SALMs, we carried out yeast two-hybrid screening using the extracellular domain of SALM1 as bait. A clone encoding full-length reticulon 3A1 was isolated. This interaction was shown to occur through the LRR domain, which is found on all SALMs. To determine whether this relationship also occurs in brain, we performed immunoprecipitation using antibodies to SALMs 1-4. A 19-kDa band, identified as reticulon 3C, bound to all four SALMs, whereas a 90-kDa band, which did not comigrate with any known reticulon 3 variant, bound to SALMs 2 and 3. These results show that reticulon 3 may play a role in the trafficking of the SALM family of adhesion molecules.

Synaptic adhesion-like molecules (SALMs) promote neurite outgrowth

SALMs are a family of five adhesion molecules whose expression is largely restricted to the CNS. Initial reports showed that SALM1 functions in neurite outgrowth while SALM2 is involved in synapse formation. To investigate the function of SALMs in detail, we asked if all five are involved in neurite outgrowth. Expression of epitope-tagged proteins in cultured hippocampal neurons showed that SALMs are distributed throughout neurons, including axons, dendrites, and growth cones. Over-expression of each SALM resulted in enhanced neurite outgrowth, but with different phenotypes. Neurite outgrowth could be reduced by applying antibodies targeting the extracellular leucine rich regions of SALMs and with RNAi. Through over-expression of deletion constructs, we found that the C-terminal PDZ binding domains of SALMs 1–3 are required for most aspects of neurite outgrowth. In addition, by using a chimera of SALMs 2 and 4, we found that the N-terminus is also involved in neurite outgrowth.

The SALM Family of Adhesion-like Molecules Forms Heteromeric and Homomeric Complexes

Synaptic adhesion-like molecules (SALMs) are a newly discovered family of adhesion molecules that play roles in synapse formation and neurite outgrowth. The SALM family is comprised of five homologous molecules that are expressed largely in the central nervous system. SALMs 1-3 contain PDZ-binding domains, whereas SALMs 4 and 5 do not. We are interested in characterizing the interactions of the SALMs both among the individual members and with other binding partners. In the present study, we focused on the interactions formed by the five SALM members in rat brain and heterologous cells. In brain, we found that SALMs 1-3 strongly co-immunoprecipitated with each other, whereas SALMs 4 and 5 did not, suggesting that SALMs 4 and 5 mainly form homomeric complexes. In heterologous cells transfected with SALMs, co-immunoprecipitation studies showed that all five SALMs form heteromeric and homomeric complexes. We also determined if SALMs could form trans-cellular associations between transfected heterologous cells. Both SALMs 4 and 5 formed homophilic, but not heterophilic associations, whereas no trans associations were formed by the other SALMs. The ability of SALM4 to form trans interactions is due to its extracellular N terminus because chimeras of SALM4 N terminus and SALM2 C terminus can form trans interactions, whereas chimeras of SALM2 N terminus and SALM4 C terminus cannot. Co-culture experiments using HeLa cells and rat hippocampal neurons expressing the SALMs showed that SALM4 is recruited to points of contact between the cells. In neurons, these points of contact were seen in both axons and dendrites.

A Novel Family of Adhesion-Like Molecules That Interacts with the NMDA Receptor

We have identified a novel family of synaptic adhesion-like molecules (SALMs). The family members, SALM1-SALM4, have a single transmembrane (TM) domain and contain extracellular leucine-rich repeats, an Ig C2 type domain, a fibronectin type III domain, and an intracellular postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1 (PDZ) binding domain, which is present on all members except SALM4. SALM1 interacts with PSD-95, synapse-associated protein 102 (SAP102), and SAP97 based on coimmunoprecipitation of detergent-solubilized brain. Distribution studies show that SALM1 is present in synaptic membrane and postsynaptic density fractions but is also distributed in axons and dendrites. Transfection of hippocampal neurons for 4 d in vitro (DIV) with SALM1 more than doubles the dendritic lengths of neurons after 48 h, whereas transfection of neurons 14 DIV has no significant effect on neurite outgrowth. Overexpression of SALM1 in 14 DIV neurons recruits NMDA receptors (NR) and PSD-95 to dendritic puncta. This effect is dependent on the PDZ-binding domain of SALM1. SALM1 also enhances surface expression of transfected NR2A subunit. Immunoprecipitation of detergent-solubilized brain membranes with anti-SALM1 antibodies shows coimmunoprecipitation of NR1 and NR2 subunits. After transfection of heterologous cells with NR1 and NR2 cDNAs, through coimmunoprecipitation analyses, we find that SALM1 also interacts with the NMDA receptor NR1 subunit through its extracellular or TM1 domains.