Peptidoglycan recognition protein (PGRP) from eri-silkworm, Samia cynthia ricini; protein purification and induction of the gene expression

Peptidoglycan recognition protein (PGRP) was isolated from immunized hemolymph of the wild silkworm, Samia cynthia ricini, detecting the biding activity with (125)I-labeled peptidoglycan (PGN). The binding specificity of PGRP was tested by competitive inhibition of the binding to (125)I-labeled-PGN by a large excess amount of non-labeled-PGN or other glucans. The binding to labeled uncross-linked Lys-type PGN from Micrococcus luteus was strongly inhibited by non-labeled-PGN of the same structure and meso-diaminopimelic acid (DAP)-type cross-linked PGN from Bacillus cell wall, but only a little by cross-linked PGN from M. luteus cell wall. The PGRP cDNA encodes a 193 amino acid open reading frame. The deduced amino acid sequence had 62 to 91% identities to known lepidopteran PGRPs, but less than 40% to Drosophila PGRPs. The PGRP gene constitutively expressed at a low level in naive fat body, and strongly induced by an injection of DAP-type cross-linked and Lys-type uncross-linked PGNs, but only weakly by Lys-type cross-linked PGN from M. luteus. The silkworm possibly distinguish between PGNs based on the structure of cross-linking peptide, but has less if any preference for the diamino acid residue of the stem peptide.     

Elucidating the molecular interaction of Zebrafish (Danio rerio) peptidoglycan recognition protein 2 with diaminopimelic acid and lysine type peptidoglycans using in silico approaches

Abstract

Peptidoglycan recognition proteins (PGRPs) belong to the family of pattern recognition receptor, represent the major constituent of innate immunity. Although PGRPs are structurally conserved through evolution, their involvement in innate immunity is different in vertebrates and invertebrates. They are highly specific towards recognition of ligands and can hydrolyze bacterial peptidoglycans (PGNs). Zebrafish PGRPs (zPGRPs) have both peptidoglycans lytic amidase activity and broad-spectrum bactericidal activity, but far less is known about how these receptors recognize these microbial ligands. Such studies are hindered due to lack of structural and functional configuration of zPGRPs. Therefore, in this study, we predicted the three-dimensional structure of zPGRP2 through theoretical modeling, investigated the conformational and dynamic properties through molecular dynamics simulations. Molecular docking study revealed the microbial ligands, that is, muramyl pentapeptide–DAP , muramyl pentapeptide–LYS, muramyl tripeptide–DAP, muramyl tripeptide–Lys, muramyl tetrapeptide–DAP, muramyl tetrapeptide–LYS and tracheal cytotoxin interacts with the conserved amino acids of the ligand recognition site comprised of β1, α2, α4, β4 and loops connecting β1 ? α2, α2 ? β2, β3 ? β4 and α4 ? α5. Conserved His31, His32, Ala34, Ile35, Pro36, Lys38, Asp60, Trp61, Trp63, Ala89, His90, Asp106, His143 and Arg144 are predicted to essential for binding and provides stability to these zPGRP–PGN complexes. Our study provides basic molecular information for further research on the immune mechanisms of PGRP’s in Zebrafish. The plasticity of the zPGRP’s binding site revealed by these microbial ligands suggests an intrinsic capacity of the innate immune system to rapidly evolve specificities to meet new microbial challenges in the future.

Communicated by Ramaswamy H. Sarma     

INVOLVEMENT OF PEPTIDOGLYCAN RECOGNITION PROTEIN L6 IN ACTIVATION OF IMMUNE DEFICIENCY PATHWAY IN THE IMMUNE RESPONSIVE SILKWORM CELLS

The immune deficiency (Imd) signaling pathway is activated by Gram‐negative bacteria for producing antimicrobial peptides (AMPs). In Drosophila melanogaster, the activation of this pathway is initiated by the recognition of Gram‐negative bacteria by peptidoglycan (PGN) recognition proteins (PGRPs), PGRP‐LC and PGRP‐LE. In this study, we found that the Imd pathway is involved in enhancing the promoter activity of AMP gene in response to Gram‐negative bacteria or diaminopimelic (DAP) type PGNs derived from Gram‐negative bacteria in an immune responsive silkworm cell line, Bm‐NIAS‐aff3. Using gene knockdown experiments, we further demonstrated that silkworm PGRP L6 (BmPGRP‐L6) is involved in the activation of E. coli or E. coli‐PGN mediated AMP promoter activation. Domain analysis revealed that BmPGRP‐L6 contained a conserved PGRP domain, transmembrane domain, and RIP homotypic interaction motif like motif but lacked signal peptide sequences. BmPGRP‐L6 overexpression enhances AMP promoter activity through the Imd pathway. BmPGRP‐L6 binds to DAP‐type PGNs, although it also binds to lysine‐type PGNs that activate another immune signal pathway, the Toll pathway in Drosophila. These results indicate that BmPGRP‐L6 is a key PGRP for activating the Imd pathway in immune responsive silkworm cells.     

Crystal structure of human peptidoglycan recognition protein S (PGRP-S) at 1.70 A resolution

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind peptidoglycans (PGNs) of bacterial cell walls. These molecules, which are highly conserved from insects to mammals, contribute to host defense against infections by both Gram-positive and Gram-negative bacteria. Here, we present the crystal structure of human PGRP-S at 1.70A resolution. The overall structure of PGRP-S, which participates in intracellular killing of Gram-positive bacteria, is similar to that of other PGRPs, including Drosophila PGRP-LB and PGRP-SA and human PGRP-Ialpha. However, comparison with these PGRPs reveals important differences in both the PGN-binding site and a groove formed by the PGRP-specific segment on the opposite face of the molecule. This groove, which may constitute a binding site for effector or signaling proteins, is less hydrophobic and deeper in PGRP-S than in PGRP-IalphaC, whose PGRP-specific segments vary considerably in amino acid sequence. By docking a PGN ligand into the PGN-binding cleft of PGRP-S based on the known structure of a PGRP-Ialpha-PGN complex, we identified potential PGN-binding residues in PGRP-S. Differences in PGN-contacting residues and interactions suggest that, although PGRPs may engage PGNs in a similar mode, structural differences exist that likely regulate the affinity and fine specificity of PGN recognition.     

Activity Augmentation of Amphioxus Peptidoglycan Recognition Protein BbtPGRP3 via Fusion with a Chitin Binding Domain

Peptidoglycan recognition proteins (PGRPs), which have been identified in most animals, are pattern recognition molecules that involve antimicrobial defense. Resulting from extraordinary expansion of innate immune genes, the amphioxus encodes many PGRPs of diverse functions. For instance, three isoforms of PGRP encoded by Branchiostoma belcheri tsingtauense, termed BbtPGRP1~3, are fused with a chitin binding domain (CBD) at the N-terminus. Here we report the 2.7 Å crystal structure of BbtPGRP3, revealing an overall structure of an N-terminal hevein-like CBD followed by a catalytic PGRP domain. Activity assays combined with site-directed mutagenesis indicated that the individual PGRP domain exhibits amidase activity towards both DAP-type and Lys-type peptidoglycans (PGNs), the former of which is favored. The N-terminal CBD not only has the chitin-binding activity, but also enables BbtPGRP3 to gain a five-fold increase of amidase activity towards the Lys-type PGNs, leading to a significantly broadened substrate spectrum. Together, we propose that modular evolution via domain shuffling combined with gene horizontal transfer makes BbtPGRP1~3 novel PGRPs of augmented catalytic activity and broad recognition spectrum.     

Crystal structure of the Drosophila peptidoglycan recognition protein (PGRP)-SA at 1.56 A resolution

Peptidoglycan recognition proteins (PGRPs) form a recently discovered protein family, which is conserved from insect to mammals and is implicated in the innate immune system by interacting with/or degrading microbial peptidoglycans (PGNs). Drosophila PGRP-SA is a member of this family of pattern recognition receptors and is involved in insect Toll activation. We report here the crystal structure of PGRP-SA at 1.56 A resolution, which represents the first example of a "recognition" PGRP. Comparison with the catalytic Drosophila PGRP-LB reveals an overall structure conservation with an L-shaped hydrophilic groove that is likely the PGN carbohydrate core binding site, but further suggests some possible functional homology between recognition and catalytic PGRPs. Consistent with sequence analysis, PGRP-SA does not contain the canonical zinc-binding residues found in catalytic PGRPs. However, substitution of the zinc-binding cysteine residue by serine, along with an altered coordinating histidine residue, assembles a constellation of residues that resembles a modified catalytic triad. The serine/histidine juxtaposition to a threonine residue and a carbonyl oxygen atom, along with conservation of the catalytic water molecule found in PGRP-LB, tantalizingly suggests some hydrolytic function for this member of receptor PGRPs.     

Cloning and analysis of peptidoglycan recognition protein‐LC and immune deficiency from the diamondback moth,Plutella xylostella

Peptidoglycan (PGN) exists in both Gram‐negative and Gram‐positive bacteria as a component of the cell wall. PGN is an important target to be recognized by the innate immune system of animals. PGN recognition proteins (PGRP) are responsible for recognizing PGNs. In Drosophila melanogaster, PGRP‐LC and IMD (immune deficiency) are critical for activating the Imd pathway. Here, we report the cloning and analysis of PGRP‐LC and IMD (PxPGRP‐LC and PxIMD) from diamondback moth, Plutella xylostella (L.), the insect pest of cruciferous vegetables. PxPGRP‐LC gene consists of six exons encoding a polypeptide of 308 amino acid residues with a transmembrane region and a PGRP domain. PxIMD cDNA encodes a polypeptide of 251 amino acid residues with a death domain. Sequence comparisons indicate that they are characteristic of Drosophila PGRP‐LC and IMD homologs. PxPGRP‐LC and PxIMD were expressed in various tissues and developmental stages. Their mRNA levels were affected by bacterial challenges. The PGRP domain of PxPGRP‐LC lacks key residues for the amidase activity, but it can recognize two types of PGNs. Overexpression of full‐length and deletion mutants in Drosophila S2 cells induced expression of some antimicrobial peptide genes. These results indicate that PxPGRP‐LC and PxIMD may be involved in the immune signaling of P. xylostella. This study provides a foundation for further studies of the immune system of P. xylostella.     

Crystal structure of the C-terminal peptidoglycan-binding domain of human peptidoglycan recognition protein Ialpha

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind, and in some cases hydrolyze, peptidoglycans (PGNs) on bacterial cell walls. These molecules, which are highly conserved from insects to mammals, participate in host defense against both Gram-positive and Gram-negative bacteria. We report the crystal structure of the C-terminal PGN-binding domain of human PGRP-Ialpha in two oligomeric states, monomer and dimer, to resolutions of 2.80 and 1.65 A, respectively. In contrast to PGRPs with PGN-lytic amidase activity, no zinc ion is present in the PGN-binding site of human PGRP-Ialpha. The structure reveals that PGRPs exhibit extensive topological variability in a large hydrophobic groove, located opposite the PGN-binding site, which may recognize host effector proteins or microbial ligands other than PGN. We also show that full-length PGRP-Ialpha comprises two tandem PGN-binding domains. These domains differ at most potential PGN-contacting positions, implying different fine specificities. Dimerization of PGRP-Ialpha, which occurs through three-dimensional domain swapping, is mediated by specific binding of sodium ions to a flexible hinge loop, stabilizing the conformation found in the dimer. We further demonstrate sodium-dependent dimerization of PGRP-Ialpha in solution, suggesting a possible mechanism for modulating PGRP activity through the formation of multivalent adducts.     

Structural basis for preferential recognition of diaminopimelic acid-type peptidoglycan by a subset of peptidoglycan recognition proteins

Drosophila peptidoglycan recognition protein (PGRP)-LCx and -LCa are receptors that preferentially recognize meso-diaminopimelic acid (DAP)-type peptidoglycan (PGN) present in Gram-negative bacteria over lysine-type PGN of gram-positive bacteria and initiate the IMD signaling pathway, whereas PGRP-LE plays a synergistic role in this process of innate immune defense. How these receptors can distinguish the two types of PGN remains unclear. Here the structure of the PGRP domain of Drosophila PGRP-LE in complex with tracheal cytotoxin (TCT), the monomeric DAP-type PGN, reveals a buried ionic interaction between the unique carboxyl group of DAP and a previously unrecognized arginine residue. This arginine is conserved in the known DAP-type PGN-interacting PGRPs and contributes significantly to the affinity of the protein for the ligand. Unexpectedly, TCT induces infinite head-to-tail dimerization of PGRP-LE, in which the disaccharide moiety, but not the peptide stem, of TCT is positioned at the dimer interface. A sequence comparison suggests that TCT induces heterodimerization of the ectodomains of PGRP-LCx and -LCa in a closely analogous manner to prime the IMD signaling pathway, except that the heterodimer formation is nonperpetuating.     

Distribution of multiple peptidoglycan recognition proteins in the tissues of Pacific oyster, Crassostrea gigas

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors that specifically bind to peptidoglycans, a major component of bacterial cell wall. Generally, PGRPs are responsible for recognition of bacterial invasion in invertebrates. Full length cDNAs of PGRP, designated as CgPGRP-S1S, -S1L, -S2 and -S3, were identified from the Pacific oyster, Crassostrea gigas. Homology and domain searches classified these CgPGRPs as short-type PGRPs for extracellular PGN recognition. Amidase activity was predicted in all CgPGRPs, and defensin-like domains were found in CgPGRP-S1S and -S1L, suggesting that they may also function as antimicrobial proteins. Although phylogenetic analysis indicated that CgPGRPs are closely related to each other, they showed different tissue expression patterns; CgPGRP-S1S in the mantle and the gill, -S1L in the mantle, -S2 in the hemocytes and -S3 in the digestive diverticula. The CgPGRPs seem to survey bacterial invasion in their corresponding expression tissues. This is the first report of the possibility that bivalve mollusks have PGN recognition systems as suggested by the identification of multiple PGRPs distributed in various tissues.     

Bovine peptidoglycan recognition protein-S: antimicrobial activity, localization, secretion, and binding properties

Peptidoglycan (PGN) recognition proteins (PGRPs) are pattern recognition molecules of innate immunity that are conserved from insects to humans. Various PGRPs are reported to have diverse functions: they bind bacterial molecules, digest PGN, and are essential to the Toll pathway in Drosophila. One family member, bovine PGN recognition protein-S (bPGRP-S), has been found to bind and kill microorganisms in a PGN-independent manner, raising questions about the identity of the bPGRP-S ligand. Addressing this, we have determined the binding and microbicidal properties of bPGRP-S in a range of solutions approximating physiologic conditions. In this study we show that bPGRP-S interacts with other bacterial components, including LPS and lipoteichoic acid, with higher affinities than for PCP, as determined by their abilities to inhibit bPGRP-S-mediated killing of bacteria. Where and how PGRPs act in vivo is not yet clear. Using Immunogold electron microscopy, PGRP-S was localized to the dense/large granules of naive neutrophils, which contain the oxygen-independent bactericidal proteins of these cells, and to the neutrophil phagolysosome. In addition, Immunogold staining and secretion studies demonstrate that neutrophils secrete PGRP-S when exposed to bacteria. Bovine PGRP-S can mediate direct lysis of heat-killed bacteria; however, PGRP-S-mediated killing of bacteria is independent of this activity. Evidence that bPGRP-S has multiple activities and affinity to several bacterial molecules challenges the assumption that the PGRP family of proteins recapitulates the evolution of TLRs. Mammalian PGRPs do not have a single antimicrobial activity against a narrow range of target organisms; rather, they are generalists in their affinity and activity.     

Human peptidoglycan recognition protein-L is an N-acetylmuramoyl-L-alanine amidase

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules coded by up to 13 genes in insects and 4 genes in mammals. In insects PGRPs activate antimicrobial pathways in the hemolymph and cells, or are peptidoglycan (PGN)-lytic amidases. In mammals one PGRP is an antibacterial neutrophil protein. We report that human PGRP-L is a Zn2+-dependent N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28), an enzyme that hydrolyzes the amide bond between MurNAc and l-Ala of bacterial PGN. The minimum PGN fragment hydrolyzed by PGRP-L is MurNAc-tripeptide. PGRP-L has no direct bacteriolytic activity. The other members of the human PGRP family, PGRP-Ialpha, PGRP-Ibeta, and PGRP-S, do not have the amidase activity. The C-terminal region of PGRP-L, homologous to bacteriophage and bacterial amidases, is required and sufficient for the amidase activity of PGRP-L, although its activity (in the N-terminal delta1-343 deletion mutant) is reduced. The Zn2+ binding amino acids (conserved in PGRP-L and T7 amidase) and Cys-419 (not conserved in T7 amidase) are required for the amidase activity of PGRP-L, whereas three other amino acids, needed for the activity of T7 amidase, are not required for the activity of PGRP-L. These amino acids, although required, are not sufficient for the amidase activity, because changing them to the "active" configuration does not convert PGRP-S into an active amidase. In conclusion, human PGRP-L is an N-acetylmuramoyl-l-alanine amidase and this function is conserved in prokaryotes, insects, and mammals.     

Chemically synthesized pathogen-associated molecular patterns increase the expression of peptidoglycan recognition proteins via toll-like receptors, NOD1 and NOD2 in human oral epithelial cells

Peptidoglycan recognition proteins (PGRPs), a novel family of pattern recognition molecules (PRMs) in innate immunity conserved from insects to mammals, recognize bacterial cell wall peptidoglycan (PGN) and are suggested to act as anti-bacterial factors. In humans, four kinds of PGRPs (PGRP-L, -Ialpha, -Ibeta and -S) have been cloned and all four human PGRPs bind PGN. In this study, we examined the possible regulation of the expression of PGRPs in oral epithelial cells upon stimulation with chemically synthesized pathogen-associated molecular patterns (PAMPs) in bacterial cell surface components: Escherichia coli-type tryacyl lipopeptide (Pam3CSSNA), E. coli-type lipid A (LA-15-PP), diaminopimelic acid containing desmuramyl peptide (gamma-D-glutamyl-meso-DAP; iE-DAP), and muramyldipeptide (MDP). These synthetic PAMPs markedly upregulated the mRNA expression of the four PGRPs and cell surface expression of PGRP-Ialpha and -Ibeta, but did not induce either mRNA expression or secretion of inflammatory cytokines, in oral epithelial cells. Suppression of the expression of Toll-like receptor (TLR)2, TLR4, nucleotide-binding oligomerization domain (NOD)1 and NOD2 by RNA interference specifically inhibited the upregulation of PGRP mRNA expression induced by Pam3CSSNA, LA-15-PP, iE-DAP and MDP respectively. These PAMPs definitely activated nuclear factor (NF)-kappaB in the epithelial cells, and suppression of NF-kappaB activation clearly prevented the induction of PGRP mRNA expression induced by these PAMPs in the cells. These findings suggested that bacterial PAMPs induced the expression of PGRPs, but not proinflammatory cytokines, in oral epithelial cells, and the PGRPs might be involved in host defence against bacterial invasion without accompanying inflammatory responses.     

Molecular cloning and mRNA expression of two peptidoglycan recognition protein (PGRP) genes from mollusk Solen grandis

Peptidoglycan recognition proteins (PGRPs) play crucial role in innate immunity for both invertebrates and vertebrates, owing to their prominent ability in detecting and eliminating invading bacteria. In the present study, two short PGRPs from mollusk Solen grandis (designated as SgPGRP-S1 and SgPGRP-S2) were identified, and their expression patterns, both in tissues and toward three PAMPs stimulation, were then characterized. The full-length cDNA of SgPGRP-S1 and SgPGRP-S2 was 1672 and 1285 bp, containing an open reading frame (ORF) of 813 and 426 bp, respectively, and deduced amino acid sequences showed high similarity to other members of PGRP superfamily. Both SgPGRP-S1 and SgPGRP-S2 encoded a PGRP domain. The motif of Zn²⁺ binding sites and amidase catalytic sites were well conserved in SgPGRP-S1, but partially conserved in SgPGRP-S2. The two PGRPs exhibited different tissue expression pattern. SgPGRP-S1 was highly expressed in muscle and hepatopancreas, while SgPGRP-S2 was highly in gill and mantle. The mRNA expression of SgPGRP-S1 could be induced acutely by stimulation of PGN, and also moderately by β-1,3-glucan, but not by LPS, while expression of SgPGRP-S2 was significantly up-regulated (P < 0.01) when S. grandis was stimulated by all the three PAMPs, though the expression levels were relatively lower than SgPGRP-S1. Our results suggested SgPGRP-S1 and SgPGRP-S2 could serve as pattern recognition receptors (PRRs) involved in the immune recognition of S. grandis, and they might perform different functions in the immune defense against invaders.     

Crystal structure of human peptidoglycan recognition protein I alpha bound to a muramyl pentapeptide from Gram-positive bacteria

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind bacterial peptidoglycans (PGNs). We determined the crystal structure, to 2.1 A resolution, of the C-terminal PGN-binding domain of human PGRP-I alpha in complex with a muramyl pentapeptide (MPP) from Gram-positive bacteria containing a complete peptide stem (L-Ala-D-isoGln-L-Lys-D-Ala-D-Ala). The structure reveals important features not observed previously in the complex between PGRP-I alpha and a muramyl tripeptide lacking D-Ala at stem positions 4 and 5. Most notable are ligand-induced structural rearrangements in the PGN-binding site that are essential for entry of the C-terminal portion of the peptide stem and for locking MPP in the binding groove. We propose that similar structural rearrangements to accommodate the PGN stem likely characterize many PGRPs, both mammalian and insect.     

Peptidoglycan recognition proteins: a novel family of four human innate immunity pattern recognition molecules.

The innate immune system recognizes microorganisms through a series of pattern recognition receptors that are highly conserved in evolution. Insects have a family of 12 peptidoglycan recognition proteins (PGRPs) that recognize peptidoglycan, a ubiquitous component of bacterial cell walls. We report cloning of three novel human PGRPs (PGRP-L, PGRP-Ialpha, and PGRP-Ibeta) that together with the previously cloned PGRP-S, define a new family of human pattern recognition molecules. PGRP-L, PGRP-Ialpha, and PGRP-Ibeta have 576, 341, and 373 amino acids coded by five, seven, and eight exons on chromosomes 19 and 1, and they all have two predicted transmembrane domains. All mammalian and insect PGRPs have at least three highly conserved C-terminal PGRP domains located either in the extracellular or in the cytoplasmic (or in both) portions of the molecules. PGRP-L is expressed in liver, PGRP-Ialpha and PGRP-Ibeta in esophagus (and to a lesser extent in tonsils and thymus), and PGRP-S in bone marrow (and to a lesser extent in neutrophils and fetal liver). All four human PGRPs bind peptidoglycan and Gram-positive bacteria. Thus, these PGRPs may play a role in recognition of bacteria in these organs.     

The differentially spliced mouse tagL gene,homolog of tag7/PGRP gene family in mammals and Drosophila,can recognize Gram-positive and Gram-negative bacterial cell wall independently of T phage lysozyme homology domain

Tag7/PGRP, a recently characterized antimicrobial protein, is conserved from insects to mammals. Recently its involvement in Toll signalling in Drosophila was demonstrated. A number of genes representing a new family homologous to PGRP were identified in Drosophila and human. Here we describe a splicing pattern of the tagL gene, mouse member of tag7/PGRP family. Some of the identified splice variants lacked characteristics for the family T phage lysozyme homology domain (also known as PGRP domain). Accordingly to the predicted transmembrane domains, mouse TagL may be secreted as inducible proteins or retained on intracellular membranes. All detected splice variant isoforms of TagL bound Gram-positive, Gram-negative bacteria and peptidoglycan. This binding did not depend on the presence of T phage lysozyme homology domain but was associated with the C-terminal portion of the polypeptides. Thus, this variety of isoforms of a single gene may play a role in circulating bacteria recognition in mammals.     

SAM68 regulates neuronal activity-dependent alternative splicing of neurexin-1

The assembly of synapses and neuronal circuits relies on an array of molecular recognition events and their modification by neuronal activity. Neurexins are a highly polymorphic family of synaptic receptors diversified by extensive alternative splicing. Neurexin variants exhibit distinct isoform-specific biochemical interactions and synapse assembly functions, but the mechanisms governing splice isoform choice are not understood. We demonstrate that Nrxn1 alternative splicing is temporally and spatially controlled in the mouse brain. Neuronal activity triggers a shift in Nrxn1 splice isoform choice via calcium/calmodulin-dependent kinase IV signaling. Activity-dependent alternative splicing of Nrxn1 requires the KH-domain RNA-binding protein SAM68 that associates with RNA response elements in the Nrxn1 pre-mRNA. Our findings uncover SAM68 as a key regulator of dynamic control of Nrxn1 molecular diversity and activity-dependent alternative splicing in the central nervous system.