The agrin gene codes for a family of basal lamina proteins that differ in function and distribution.

We isolated two cDNAs that encode isoforms of agrin, the basal lamina protein that mediates the motor neuron-induced aggregation of acetylcholine receptors on muscle fibers at the neuromuscular junction. Both proteins are the result of alternative splicing of the product of the agrin gene, but unlike agrin, they are inactive in standard acetylcholine receptor aggregation assays. They lack one (agrin-related protein 1) or two (agrin-related protein 2) regions in agrin that are required for its activity. Expression studies provide evidence that both proteins are present in the nervous system and muscle and that, in muscle, myofibers and Schwann cells synthesize the agrin-related proteins while the axon terminals of motor neurons are the sole source of agrin.     

Asparagine of z8 Insert Is Critical for the Affinity,Conformation, and Acetylcholine Receptor-clustering Activity of Neural Agrin

Agrin isoforms with different bioactivities are synthesized by the nerve and the muscle. Neural agrin containing an 8-amino acid insert (z8) introduced by alternative splicing is the active form that induces synaptic differentiation at the neuromuscular junction. In addition to alternative splicing, extracellular calcium is also required for the activity of neural agrin. To understand better how the activity of agrin is regulated by alternative splicing, we have applied alanine substitution mutagenesis to the z8 insert and the calcium binding site in the minimally functional AgG3z8 fragment. Single alanine substitutions in the 4th through the 7th amino acid of the z8 splice insert significantly reduced the function of agrin, in terms of acetylcholine receptor clustering activity and the affinity for binding to the muscle surface. Mutation of the asparagine at the 4th position drastically reduces bioactivity such that it is equivalent to that of muscle form AgG3z0. These reduced activity mutants also show reduced magnitudes of the calcium-induced CD spectrum change from that observed in AgG3z8 fragments, indicating that cross-talk between calcium and the z8 insert is critical for the normal activity of agrin. However, removal of Ca²⁺ binding via mutation of both aspartic acids in the calcium binding site did not totally eliminate the activity of AgG3z8. These results suggest a model wherein the z8 insert is a Ca²⁺-responsive allosteric element that is essential in forming an active conformation in neuronal agrin.     

Acetylcholine receptor-aggregating activity of agrin isoforms and mapping of the active site

Agrin is a basal lamina protein that induces aggregation of acetylcholine receptors (AChRs) and other molecules at the developing neuromuscular junction. Alternative splicing of chick agrin mRNA at two sites, A and B, gives rise to eight possible isoforms of which five are expressed in vivo. Motor neurons express high levels of isoforms with inserts at sites A and B, muscle cells synthesize isoforms that lack amino acids at the B-site. To obtain further insights into the mechanism of agrin-induced AChR aggregation, we have determined the EC50 (effective concentration to induce half-maximal AChR clustering) of each agrin isoform and of truncation mutants. On chick myotubes, EC50 of the COOH-terminal, 95-kD fragment of agrinA4B8 was approximately 35 pM, of agrinA4B19 approximately 110 pM and of agrinA4B11 approximately 5 nM. While some AChR clusters were observed with 64 nM of agrinA4B0, no activity was detected for agrinA0B0. Recombinant full-length chick agrin and a 100-kD fragment of ray agrin showed similar EC50 values. A 45-kD, COOH-terminal fragment of agrinA4B8 retained high activity (EC50 approximately equal to 130 pM) and a 21-kD fragment was still active, but required higher concentrations (EC50 approximately equal to 13 nM). Unlike the 45-kD fragment, the 21-kD fragment neither bound to heparin nor did heparin inhibit its capability to induce AChR aggregation. These data show quantitatively that agrinA4B8 and agrinA4B19, expressed in motor neurons, are most active, while no activity is detected in agrinA0B0, the dominant isoform synthesized by muscle cells. Furthermore, our results show that a fragment comprising site B8 and the most COOH- terminal G-like domain is sufficient for this activity, and that agrin domains required for binding to heparin and those for AChR aggregation are distinct from each other.     

A unique intronic splicing enhancer controls the inclusion of the agrin Y exon.

Alternative splicing of the agrin mRNA controls the ability of agrin protein to induce the clustering of acetylcholine receptors at the neuromuscular junction. Using a transfectable reporter gene, we show that one agrin alternative exon, the Y exon, is controlled by a regulatory sequence in the downstream intron. Portions of this intronic sequence have the properties of a splicing enhancer that can activate splicing of a heterologous exon when placed in the intron downstream. The regulatory region is complex in structure, containing several different elements capable of activating splicing. Individual enhancing elements differ in their cell-type specificity, and are not apparently synergistic, as two elements together induce lower splicing than either does separately. Essential nucleotides within these regulatory elements were identified by scanning mutagenesis across the active region. Interestingly, the elements do not appear similar to known intronic splicing enhancer elements. This Y exon enhancer and its components take part in an apparent combinatorial system of control where multiple regulatory elements of varying activity combine to produce a precisely cell-specific exon inclusion. As a major contributor to the regulation of the Y exon, the enhancer ultimately controls the properties of the agrin protein.     

Edit, cut and paste in the nicotinic acetylcholine receptor gene family of Drosophila melanogaster

Nicotinic acetylcholine receptors (nAChRs) are important for fast synaptic cholinergic transmission. They are targets of drugs/chemicals for human and animal health as well as for pest control. With the advent of genome sequencing, entire nAChR gene families have now been described for vertebrates and invertebrates. Mostly, these are extensive with a large number of distinct subunits, making possible many nAChR subtypes differing in transmitter affinity, channel conductance, ion selectivity, desensitization, modulation and pharmacology. The smallest nAChR gene family to date is that of the fruit fly, Drosophila melanogaster, with only 10 members. This apparently compact family belies its true diversity as 4 of the 10 subunits show alternative splicing. Also, using Drosophila, A-to-I pre-mRNA editing has been demonstrated for the first time in nAChRs. Such is the extent of this variation, that one subunit alone (Dalpha6) can potentially generate far more isoforms than seen in entire gene families from other species. We present here three-dimensional models constructed for insect nAChRs, which show that many variations introduced by alternative splicing and RNA editing may influence receptor function.     

Host-cell specific effects of the nicotinic acetylcholine receptor chaperone RIC-3 revealed by a comparison of human and Drosophila RIC-3 homologues

RIC-3 is a transmembrane protein which enhances maturation (folding and assembly) of neuronal nicotinic acetylcholine receptors (nAChRs). In this study, we report the cloning and characterisation of 11 alternatively spliced isoforms of Drosophila melanogaster RIC-3 (DmRIC-3). Heterologous expression studies of alternatively spliced DmRIC-3 isoforms demonstrate that nAChR chaperone activity does not require a predicted coiled-coil domain which is located entirely within exon 7. In contrast, isoforms containing an additional exon (exon 2), which is located within a proline-rich N-terminal region, have a greatly reduced ability to enhance nAChR maturation. The ability of DmRIC-3 to influence nAChR maturation was examined in co-expression studies with human α7 nAChRs and with hybrid nAChRs containing both Drosophila and rat nAChR subunits. When expressed in a Drosophila cell line, several of the DmRIC-3 splice variants enhanced nAChR maturation to a significantly greater extent than observed with human RIC-3. In contrast, when expressed in a human cell line, human RIC-3 enhanced nAChR maturation more efficiently than DmRIC-3. The cloning and characterisation of 11 alternatively spliced DmRIC-3 isoforms has helped to identify domains influencing RIC-3 chaperone activity. In addition, studies conducted in different expression systems suggest that additional host cell factors may modulate the chaperone activity of RIC-3.     

Characterization of mouse DAF on transfectant cells using monoclonal antibodies which recognize different epitopes

Several membrane proteins prevent host cells from homologous complement attack. In humans, one such protein, decay-accelerating factor (DAF), exists as two isoforms, a GPI anchored form and a secreted form, which are generated by alternative splicing. DAF in mouse is also expressed as two isoforms, a GPI anchored form (GPI-DAF) and a transmembrane form (TM-DAF), which are produced from two separate genes. In this study, we transfected cDNA of mouse GPI-DAF or TM-DAF into Chinese hamster ovary (CHO) cells. Both isoforms of DAF on CHO cells were shown to regulate mouse complement C3 deposition mediated by the classical and alternative pathways and the inhibitory activity of both isoforms was species restricted. The two mouse DAF isoforms were effective against rat complement but not against human and guinea pig complement. Furthermore, we produced hamster mAbs to mouse DAF using GPI-DAF transfectant cells and established seven unique mAbs (RIKO-1-7). Western blotting analysis using RIKO-3, which reacts with both GPI-DAF and TM-DAF, and RIKO-4, which is an anti-GPI-DAF specific mAb, indicated that GPI-DAF was expressed on erythrocytes, spleen and testis, and that TM-DAF was expressed only in testis.     

Insect nicotinic acetylcholine receptor gene families: from genetic model organism to vector,pest and beneficial species

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in the insect nervous system and are targets of a major group of insecticides, the neonicotinoids. Analyses of genome sequences have shown that nAChR gene families remain compact in diverse insect species, when compared to their mammalian counterparts. Thus, Drosophila melanogaster and Anopheles gambiae each possess 10 nAChR genes while Apis mellifera has 11. Although these are among the smallest nAChR gene families known, receptor diversity can be considerably increased by alternative splicing and mRNA A-to-I editing, thereby generating species-specific subunit isoforms. In addition, each insect possesses at least one highly divergent nAChR subunit. Species-specific subunit diversification may offer promising targets for future rational design of insecticides that act on particular pests while sparing beneficial insects. Electrophysiological studies on cultured Drosophila cholinergic neurons show partial agonist actions of the neonicotinoid imidacloprid and super-agonist actions of another neonicotinoid, clothianidin, on native nAChRs. Recombinant hybrid heteromeric nAChRs comprising Drosophila Dα2 and a vertebrate β2 subunit have been instructive in mimicking such actions of imidacloprid and clothianidin. Unitary conductance measurements on native nAChRs indicate that more frequent openings of the largest conductance state may offer an explanation for the superagonist actions of clothianidin.     

Nitric oxide and cyclic GMP regulate early events in agrin signaling in skeletal muscle cells

Agrin released from motor nerve terminals directs differentiation of the vertebrate neuromuscular junction (NMJ). Activity of nitric oxide synthase (NOS), guanylate cyclase (GC), and cyclic GMP-dependent protein kinase (PKG) contributes to agrin signaling in embryonic frog and chick muscle cells. Stimulation of the NO/cyclic GMP (cGMP) pathway in embryos potentiates agrin's ability to aggregate acetylcholine receptors (AChRs) at NMJs. Here we investigated the timing and mechanism of NO and cGMP action. Agrin increased NO levels in mouse C2C12 myotubes. NO donors potentiated agrin-induced AChR aggregation during the first 20 min of agrin treatment, but overnight treatment with NO donors inhibited agrin activity. Adenoviruses encoding siRNAs against each of three NOS isoforms reduced agrin activity, indicating that these isoforms all contribute to agrin signaling. Inhibitors of NOS, GC, or PKG reduced agrin-induced AChR aggregation in mouse muscle cells by ∼ 50%. However, increased activation of the GTPase Rac1, an early step in agrin signaling, was dependent on NOS activity and was mimicked by NO donors and a cGMP analog. Our results indicate that stimulation of the NO/cGMP pathway is important during the first few minutes of agrin signaling and is required for agrin-induced Rac1 activation, a key step leading to reorganization of the actin cytoskeleton and subsequent aggregation of AChRs on the surface of skeletal muscle cells.     

Calcium plays a critical role in determining the acetylcholine receptor-clustering activities of alternatively spliced isoforms of Agrin

Neural agrin, an extracellular matrix protein secreted by motor neurons, plays a key role in clustering of nicotinic acetylcholine receptors (AChR) on postsynaptic membranes of the neuromuscular junction. The action of agrin is critically dependent on an eight-amino acid insert (z8 insert) in the third of three consecutive laminin-like globular (G3) domains near the C terminus of neural agrin. Alternatively spliced agrin isoforms in non-neural tissue including muscle lack the z8 insert and are biologically inactive. Extracellular calcium has been shown to be imperative for the AChR-clustering activity of neural agrin. It is unclear, however, whether calcium preferentially interacts with the neural isoform or whether it acts solely as an intracellular messenger that mediates agrin signaling. Here, we report the G3 domain of rat neural agrin (AgG3z8) expressed in Pichia pastoris promoted AChR clustering on surface of C2C12 myotubes in a calcium-dependent manner. Direct binding of calcium to AgG3z8 was demonstrated by trypsin digestion and thermal denaturation experiments. Moreover, calcium induced a significant change in the conformation of AgG3z8, and the effect was correlated with an enhanced binding affinity of the protein to muscle receptor. Mutation of calcium-binding residues in the G3 domain diminished the conformational change of neural agrin, reduced its binding affinity to muscle membrane, and inhibited AChR-clustering activity. Conversely, the G3 domain of muscle agrin (AgG3z0) displayed little structural change in the presence of calcium, bound poorly to muscle surface, and was inactive in AChR-clustering assays. We conclude that distinct interactions of the G3 domain with calcium determine the biological activities of alternatively spliced agrin isoforms during synapse formation.     

Acetylcholine receptors are required for agrin-induced clustering of postsynaptic proteins.

We have investigated the role of acetylcholine receptors (AChRs) in an early step of postsynaptic assembly at the neuromuscular synapse, the clustering of postsynaptic proteins induced by nerve-released agrin. To achieve this, we used two variants of C2 myotubes virtually lacking AChRs and C2 cells in which surface AChRs were down-regulated by AChR antibodies. In all cases, agrin caused normal clustering of the agrin receptor component MuSK, alpha-dystrobrevin and utrophin, but failed to aggregate AChRs, alpha- and beta-dystroglycan, syntrophin isoforms and rapsyn, an AChR-anchoring protein necessary for postsynaptic assembly and AChR clustering. In C2 variants, the stability of rapsyn was decreased, whereas in antibody-treated cells, rapsyn efficiently co-localized with remaining AChRs in microaggregates. Upon ectopic injection into myofibers in vivo, rapsyn did not form clusters in the absence of AChRs. These results show that AChRs and rapsyn are interdependent components of a pre-assembled protein complex that is required for agrin-induced clustering of a full set of postsynaptic proteins, thus providing evidence for an active role of AChRs in postsynaptic assembly.     

Role of rapsyn tetratricopeptide repeat and coiled-coil domains in self-association and nicotinic acetylcholine receptor clustering

Rapsyn, a 43-kDa peripheral membrane protein of skeletal muscle, is essential for clustering nicotinic acetylcholine receptors (nAChR) in the postsynaptic membrane. Previous studies with rapsyn NH(2)-terminal fragments fused to green fluorescent protein, expressed in 293T cells along with nAChRs, establish the following: Rapsyn-(1-90), containing the myristoylated amino terminus and two tetratricopeptide repeats (TPRs), was sufficient for self-association at the plasma membrane; rapsyn-(1-287), containing seven TPRs, did not cluster nAChRs; whereas rapsyn-(1-360)(,) containing a coiled-coil domain (rapsyn-(298-331)), clustered nAChRs. To further analyze the role of rapsyn structural domains in self-association and nAChR clustering, we have characterized the clustering properties of additional rapsyn mutants containing deletions and substitutions within the TPR and coiled-coil domains. A mutant lacking the coiled-coil domain alone (rapsyn-(black triangle288-348)), failed to cluster nAChRs. Within the coiled-coil domain neutralization of the charged side chains was tolerated, while alanine substitutions of large hydrophobic residues resulted in the loss of nAChR clustering. Rapsyn self-association requires at least two TPRs, as a single TPR (TPR1 or TPR2 alone) was not sufficient. While TPRs 1 and 2 are sufficient for self-association, they are not necessary, as TPRs 3-7 also formed clusters similar to wild-type rapsyn. Fragments containing TPRs co-localized with full-length rapsyn, while the expressed coiled-coil or RING-H2 domain did not. These results are discussed in terms of a homology model of rapsyn, based on the three-dimensional structure of the TPR domain of protein phosphatase 5.     

A Functional Role for Specific Spliced Variants of the α7β1 Integrin in Acetylcholine Receptor Clustering

The clustering of acetylcholine receptors (AChR) on skeletal muscle fibers is an early event in the formation of neuromuscular junctions. Recent studies show that laminin as well as agrin can induce AChR clustering. Since the α7β1 integrin is a major laminin receptor in skeletal muscle, we determined if this integrin participates in laminin and/or agrin-induced AChR clustering. The alternative cytoplasmic domain variants, α7A and α7B, and the extracellular spliced forms, α7X1 and α7X2, were studied for their ability to engage in AChR clustering. Immunofluorescence microscopy of C2C12 myofibers shows that the α7β1 integrin colocalizes with laminin-induced AChR clusters and to a much lesser extent with agrin-induced AChR clusters. However, together laminin and agrin promote a synergistic response and all AChR colocalize with the integrin. Laminin also induces the physical association of the integrin and AChR. High concentrations of anti-α7 antibodies inhibit colocalization of the integrin with AChR clusters as well as the enhanced response promoted by both laminin and agrin. Engaging the integrin with low concentrations of anti-α7 antibody initiates cluster formation in the absence of agrin or laminin. Whereas both the α7A and α7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the α7X2 extracellular domain were active. These results demonstrate that the α7β1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the α7 chain, and that laminin, agrin, and the α7β1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.     

Conjugation of LG domains of agrins and perlecan to polymerizing laminin-2 promotes acetylcholine receptor clustering

Neuromuscular junction (NMJ) assembly is characterized by the clustering and neuronal alignment of acetylcholine receptors (AChRs). In this study we have addressed post-synaptic contributions to assembly that may arise from the NMJ basement membrane with cultured myotubes. We show that the cell surface-binding LG domains of non-neural (muscle) agrin and perlecan promote AChR clustering in the presence of laminin-2. This type of AChR clustering occurs with a several hour lag, requires muscle-specific kinase (MuSK), and is accompanied by tyrosine phosphorylation of MuSK and betaAChR. It also requires conjugation of the agrin or perlecan to laminin together with laminin polymerization. Furthermore, AChR clustering can be mimicked with antibody binding to non-neural agrin, supporting a mechanism of ligand aggregation. Neural agrin, in addition to its unique ability to cluster AChRs through its B/z sequence insert, also exhibits laminin-dependent AChR clustering, the latter enhancing and stabilizing its activity. Finally, we show that type IV collagen, which lacks clustering activity on its own, stabilizes laminin-dependent AChR clusters. These findings provide evidence for cooperative and partially redundant MuSK-dependent functions of basement membrane in AChR assembly that can enhance neural agrin activity yet operate in its absence. Such interactions may contribute to the assembly of aneural AChR clusters that precede neural agrin release as well as affect later NMJ development.     

Use of cyclic peptide phage display library for the identification of a CD45RC epitope expressed on murine B cells and their precursors

The alternative splicing and variable expression of the exons near to the N-terminus of the leukocyte common antigen (L-CA, CD45) result in distinct extracellular isoforms expressed by cells with different functional and developmental properties. Here we report the tissue reactivity pattern and epitope specificity of a novel rat monoclonal antibody (IBL-8) against a restricted epitope of mouse CD45. We found that this mAb reacts with an epitope displayed by B cells and their precursors (both in newborn spleen and adult bone marrow). Moreover, peripheral CD8-positive T cells were also recognised at an intermediate intensity, whereas the CD4 T cell subset was weakly reactive. The epitope of this mAb was determined with M13 filamentous phages that display cysteine constrained nonapeptides on their coat proteins. The isolated bacteriophages expressing the putative epitope showed an isoform-specific inhibition of the binding of exon-specific mAbs. Deduced amino acid sequence data of these phages indicate that the epitope recognised by the IBL-8 mAb lies at the 136-144 region of the mouse CD45 molecule within its C exon, with a TAFP consensus sequence at its centre.     

Regulation of alternative splicing by SRrp86 and its interacting proteins

SRrp86 is a unique member of the SR protein superfamily containing one RNA recognition motif and two serine-arginine (SR)-rich domains separated by an unusual glutamic acid-lysine (EK)-rich region. Previously, we showed that SRrp86 could regulate alternative splicing by both positively and negatively modulating the activity of other SR proteins and that the unique EK domain could inhibit both constitutive and alternative splicing. These functions were most consistent with the model in which SRrp86 functions by interacting with and thereby modulating the activity of target proteins. To identify the specific proteins that interact with SRrp86, we used a yeast two-hybrid library screen and immunoprecipitation coupled to mass spectrometry. We show that SRrp86 interacts with all of the core SR proteins, as well as a subset of other splicing regulatory proteins, including SAF-B, hnRNP G, YB-1, and p72. In contrast to previous results that showed activation of SRp20 by SRrp86, we now show that SAF-B, hnRNP G, and 9G8 all antagonize the activity of SRrp86. Overall, we conclude that not only does SRrp86 regulate SR protein activity but that it is, in turn, regulated by other splicing factors to control alternative splice site selection.