An amino-terminal extension is required for the secretion of chick agrin and its binding to extracellular matrix

Agrin is an extracellular matrix (ECM) protein with a calculated relative molecular mass of more than 200 kD that induces the aggregation of acetylcholine receptors (AChRs) at the neuromuscular junction. This activity has been mapped to its COOH terminus. In an attempt to identify the functions of the NH2-terminal end, we have now characterized full-length chick agrin. We show that chick agrin encoded by a previously described cDNA is not secreted from transfected cells. Secretion is achieved with a construct that includes an additional 350 bp derived from the 5' end of chick agrin mRNA. Recombinant agrin is a heparan sulfate proteoglycan (HSPG) of more than 400 kD with glycosaminoglycan side chains attached only to the NH2-terminal half. Endogenous agrin in tissue homogenates also has an apparent molecular mass of > 400 kD. While the amino acid sequence encoded by the 350-bp extension has no homology to published rat agrin, it includes a stretch of 15 amino acids that is 80% identical to a previously identified bovine HSPG. The extension is required for binding of agrin to ECM. AChR aggregates induced by recombinant agrin that includes the extension are considerably smaller than those induced by agrin fragments, suggesting that binding of agrin to ECM modulates the size of receptor clusters. In addition, we found a site encoding seven amino acids at the NH2-terminal end of agrin that is alternatively spliced. While motor neurons express the splice variant with the seven amino acid long insert, muscle cells mainly synthesize isoforms that lack this insert. In conclusion, the cDNAs described here code for chick agrin that has all the characteristics previously allocated to endogenous agrin.     

The role of agrin in synaptic development,plasticity and signaling in the central nervous system

Development of the neuromuscular junction (NMJ) requires secretion of specific isoforms of the proteoglycan agrin by motor neurons. Secreted agrin is widely expressed in the basal lamina of various tissues, whereas a transmembrane form is highly expressed in the brain. Expression in the brain is greatest during the period of synaptogenesis, but remains high in regions of the adult brain that show extensive synaptic plasticity. The well-established role of agrin in NMJ development and its presence in the brain elicited investigations of its possible role in synaptogenesis in the brain. Initial studies on the embryonic brain and neuronal cultures of agrin-null mice did not reveal any defects in synaptogenesis. However, subsequent studies in culture demonstrated inhibition of synaptogenesis by agrin antisense oligonucleotides or agrin siRNA. More recently, a substantial loss of excitatory synapses was found in the brains of transgenic adult mice that lacked agrin expression everywhere but in motor neurons. The mechanisms by which agrin influences synapse formation, maintenance and plasticity may include enhancement of excitatory synaptic signaling, activation of the “muscle-specific” receptor tyrosine kinase (MuSK) and positive regulation of dendritic filopodia. In this article I will review the evidence that agrin regulates synapse development, plasticity and signaling in the brain and discuss the evidence for the proposed mechanisms.     

Alternatively spliced isoforms of nerve- and muscle-derived agrin: their roles at the neuromuscular junction.

Agrin induces synaptic differentiation at the skeletal neuromuscular junction (NMJ); both pre- and postsynaptic differentiation are drastically impaired in its absence. Multiple alternatively spliced forms of agrin that differ in binding characteristics and bioactivity are synthesized by nerve and muscle cells. We used surgical chimeras, isoform-specific mutant mice, and nerve-muscle cocultures to determine the origins and nature of the agrin required for synaptogenesis. We show that agrin containing Z exons (Z+) is a critical nerve-derived inducer of postsynaptic differentiation, whereas neural isoforms containing a heparin binding site (Y+) and all muscle-derived isoforms are dispensable for major steps in synaptogenesis. Our results also suggest that the requirement of agrin for presynaptic differentiation is mediated indirectly by its ability to promote postsynaptic production or localization of appropriate retrograde signals.     

Expression and functional analysis of Apaf-1 isoforms. Extra Wd-40 repeat is required for cytochrome c binding and regulated activation of procaspase-9

Apaf-1 is an important apoptotic signaling molecule that can activate procaspase-9 in a cytochrome c/dATP-dependent fashion. Alternative splicing can create an NH(2)-terminal 11-amino acid insert between the caspase recruitment domain and ATPase domains or an additional COOH-terminal WD-40 repeat. Recently, several Apaf-1 isoforms have been identified in tumor cell lines, but their expression in tissues and ability to activate procaspase-9 remain poorly characterized. We performed analysis of normal tissue mRNAs to examine the relative expression of the Apaf-1 forms and identified Apaf-1XL, containing both the NH(2)-terminal and COOH-terminal inserts, as the major RNA form expressed in all tissues tested. We also identified another expressed isoform, Apaf-1LN, containing the NH(2)-terminal insert, but lacking the additional WD-40 repeat. Functional analysis of all identified Apaf-1 isoforms demonstrated that only those with the additional WD-40 repeat activated procaspase 9 in vitro in response to cytochrome c and dATP, while the NH(2)-terminal insert was not required for this activity. Consistent with this result, in vitro binding assays demonstrated that the additional WD-40 repeat was also required for binding of cytochrome c, subsequent Apaf-1 self-association, binding to procaspase-9, and formation of active Apaf-1 oligomers. These experiments demonstrate the expression of multiple Apaf-1 isoforms and show that only those containing the additional WD-40 repeat bind and activate procaspase-9 in response to cytochrome c and dATP.     

Agrin Binds to the Nerve–Muscle Basal Lamina via Laminin

Agrin is a heparan sulfate proteoglycan that is required for the formation and maintenance of neuromuscular junctions. During development, agrin is secreted from motor neurons to trigger the local aggregation of acetylcholine receptors (AChRs) and other proteins in the muscle fiber, which together compose the postsynaptic apparatus. After release from the motor neuron, agrin binds to the developing muscle basal lamina and remains associated with the synaptic portion throughout adulthood. We have recently shown that full-length chick agrin binds to a basement membrane-like preparation called Matrigel™. The first 130 amino acids from the NH₂ terminus are necessary for the binding, and they are the reason why, on cultured chick myotubes, AChR clusters induced by full-length agrin are small. In the current report we show that an NH₂-terminal fragment of agrin containing these 130 amino acids is sufficient to bind to Matrigel™ and that the binding to this preparation is mediated by laminin-1. The fragment also binds to laminin-2 and -4, the predominant laminin isoforms of the muscle fiber basal lamina. On cultured myotubes, it colocalizes with laminin and is enriched in AChR aggregates. In addition, we show that the effect of full-length agrin on the size of AChR clusters is reversed in the presence of the NH₂-terminal agrin fragment. These data strongly suggest that binding of agrin to laminin provides the basis of its localization to synaptic basal lamina and other basement membranes.     

Mapping sites responsible for interactions of agrin with neurons

The multidomain proteoglycan agrin is a critical organizer of postsynaptic differentiation at the skeletal neuromuscular junction. Agrin is also abundant in the brain, but its roles there are unknown. As a step toward understanding these roles, we mapped sites responsible for interactions of neurons with agrin. First, we used a series of recombinant agrin fragments to show that at least four sites on agrin interact with chick ciliary neurons. Use of blocking antibodies and peptides indicated that neurons adhere to a site in the second of three G domains by means of alphaVbeta1 integrin, and to a site in the last of four epidermal growth factor (EGF) repeats via a distinct beta1 integrin. A third, integrin-independent adhesion site is near to but distinct from the site that induces postsynaptic differentiation in muscles. These domains are insufficient, however, to account for neurite outgrowth-inhibiting properties of full-length agrin, which are mediated by the N-terminal half of the molecule. We then used a second set of agrin mutants to demonstrate and map a transmembrane domain in the amino-terminus of the SN-isoform of agrin. The extracellular matrix-bound form of agrin, called LN, bears an amino-terminus required for secretion and binding to laminin. The SN form, which is selectively expressed by neurons, bears a variant amino terminus that converts agrin from a secreted, matrix-associated protein to a type-II transmembrane protein, providing a mechanism for presenting agrin in central, as opposed to neuromuscular, synaptic clefts. The SN-amino terminus can mediate externalization and membrane anchoring of heterologous proteins, but is insufficient to target them to the synapse. Together, these studies define sites that contribute to the subcellular localization of and signaling by neuronal agrin.     

Comparison of agrin-like proteins from the extracellular matrix of chicken kidney and muscle with neural agrin, a synapse organizing protein.

Agrin is a synapse-organizing protein that is concentrated in embryonic motor neurons and the synaptic basal lamina of the neuromuscular junction. Agrin or closely related proteins are also associated with most other basal laminae. Here I report that the major agrin-like proteins from the nervous system and other tissues of the chicken are immunochemically and biochemically similar. Four major agrin-like proteins of approximately 60, 72, 80, and 90 kDa were identified on immunoblots of agrin preparations from both neural and non-neural tissues. Agrin-like proteins from embryonic chicken brain and adult kidney were similar in amino acid composition. Rabbit antisera against each of the kidney proteins labeled basement membranes of several tissues, as well as spinal cord motor neurons. These antibodies specifically precipitated and inhibited acetylcholine receptor (AChR)-aggregating activity from the chicken nervous system and Torpedo electric organ. Thus, the agrin-like proteins of non-neural tissues in the chicken are closely related to agrin from the nervous system. However, the AChR-aggregating activity of chicken agrin preparations differed depending on the tissue of origin. Agrin enriched by immunoaffinity chromatography from the central nervous system induced large numbers of AChR aggregates on cultured myotubes. In contrast, agrin preparations from other chicken tissues induced dramatically fewer and smaller AChR aggregates. The difference in biological activity between these agrin preparations may reflect differential inactivation or the existence of tissue- or cell-specific isoforms of agrin.     

Synapse formation and agrin expression in stratospheroid cultures from embryonic chick retina

Stratospheroids are three-dimensional cellular spheres which develop in vitro through the proliferation and differentiation of retinal neuroepithelial precursor cells. We investigated synapse formation in stratospheroids by analyzing the development of aggregates of synapse-associated molecules and of electron microscopically identifiable synaptic specializations. Our results show that the first aggregates of the GABA(A) receptor, the glycine receptor, and gephyrin appear in the inner plexiform layer after 8 days in culture simultaneously with the development of the first active zones and postsynaptic densities. In contrast, presynaptic molecules including synaptophysin could be detected in the inner plexiform layer before synaptogenesis, suggesting functions for these molecules in addition to neurotransmitter exocytosis at mature synapses. Similar to the retina in vivo, synapses were not found in the nuclear layers of stratospheroids. We also analyzed the isoform pattern, expression, and distribution of the extracellular matrix molecule agrin, a key regulator during formation, maintenance, and regeneration of the neuromuscular junction. In stratospheroids, several agrin isoforms were expressed as highly glycosylated proteins with an apparent molecular weight of approximately 400 kDa, similar to the molecular weight of agrin in the retina in vivo. The expression specifically of the neuronal isoforms of agrin was concurrent with the onset of synaptogenesis. Moreover, the neuronal agrin isoforms were exclusively found in the synapse-containing inner plexiform layer, whereas other agrin isoforms were associated also with the inner limiting membrane and with Müller glial cells. These results show that synapse formation is very similar in stratospheroids and in the retina in vivo, and they suggest an important role for agrin during CNS development.     

Identification of the isoforms of Ca2+/calmodulin-dependent protein kinase II and expression of brain-derived neurotrophic factor mRNAs in the substantia nigra

Ca2+/calmodulin-dependent protein kinase (CaMK)II is highly expressed in the CNS and mediates activity-dependent neuronal plasticity. Four CaMKII isoforms, alpha, beta, gamma and delta, have a large number of splicing variants. Here we identified isoforms of CaMKII in the rat substantia nigra (SN). Northern blot and RT-PCR analyses revealed that the gamma and delta isoform mRNAs with several splicing variants were predominantly expressed in SN. Immunoblot analysis indicated that the major isoforms were gammaA, gammaC, delta1 and delta3. An immunohistochemical study also confirmed the preferential localization of gamma and delta isoforms in SN dopaminergic neurons. In dopaminergic neurons, immunoreactivity against anti-CaMKIIdelta1-4 antibody was detected in both nucleus and cytoplasm, in contrast to the predominant expression of gamma isoforms in the cytoplasm. Furthermore, we showed expression of brain-derived neurotrophic factor (BDNF) mRNAs with exons II and IV in SN. Taken together with our previous observations, the results suggest that the CaMKIIdelta3 isoform is involved in the expression of BDNF in the SN.     

A conserved domain in the NH2 terminus important for assembly and functional expression of pacemaker channels

Pacemaker channels are formed by co-assembly of hyperpolarization-activated cyclic nucleotide-gated (HCN) subunits. Previously, we suggested that the NH(2) termini of the mouse HCN2 isoform were important for subunit co-assembly and functional channel expression. Using an alignment strategy together with yeast two-hybrid assays, patch clamp electrophysiology, and confocal imaging, we have now identified a domain within the NH(2) terminus of the HCN2 subunit that is responsible for interactions between NH(2) termini and promoting the trafficking of functional channels to the plasma membrane. This domain is composed of 52 amino acids, is located adjacent to the putative first transmembrane segment, and is highly conserved among the mammalian HCN isoforms. This conserved domain, but not the remaining unconserved NH(2)-terminal regions of HCN2, specifically interacted with itself in yeast two-hybrid assays. Moreover, the conserved domain was important for expression of currents. Whereas relatively normal whole cell HCN2 currents were produced by channels containing only the conserved domain, further deletion of this region, leaving only a more polar and putative coiled-coil segment, eliminated HCN2 currents and resulted in proteins that localized predominantly in perinuclear compartments. Thus, we suggest that this conserved domain is the critical NH(2)-terminal determinant of subunit co-assembly and trafficking of pacemaker channels.     

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.     

The developing avian retina expresses agrin isoforms during synaptogenesis

The localization, isoform pattern, and mRNA distribution of the synapse-organizing molecule agrin was investigated in the developing avian retina. Injection of anti-agrin Fab fragments into the vitreous humor of chick eyes of embryonic days 3 to 20, a procedure that labels only extracellular agrin, reveals staining in the inner and outer plexiform layers before, during, and after the period of synapse formation. The labeling in these layers changes from a diffuse to a punctate pattern at the time when synapses form. At all stages investigated, the inner limiting membrane (a basal lamina that separates vitreous from neural retina) is intensely labeled, as are the axonal processes of retinal ganglion cells in the optic fiber layer and in the optic nerve, although the staining intensity declines after embryonic day 10 in both retina and optic nerve. In culture, axons of retinal ganglion cells also express agrin-like immunoreactivity on their surfaces. Polymerase chain reaction analysis reveals that several different agrin isoforms are expressed in the developing neural retina. In situ hybridization studies show that agrin isoforms are expressed in the ganglion cell and inner nuclear layers, correlating well with the staining for agrin protein in the optic fiber and plexiform layers. The expression of mRNA coding for several agrin isoforms and the presence of extracellular agrin in the synapse-containing layers during the period of synapse formation is consistent with the idea that agrin isoforms might play a role during synapse formation in the central nervous system. © 1996 John Wiley & Sons, Inc.     

Molecular cloning and characterization of two new isoforms of the protein kinase A catalytic subunit from the human parasite Leishmania

Leishmania are protozoan parasites that cause extensive morbidity and mortality in humans. Genes for two new isoforms of the protein kinase A catalytic subunit (PKAC) in Leishmania, Lmpkac2a and Lmpkac2b, were cloned and characterized. The predicted open reading frames for these isoforms are 93.4% identical over 338 amino acids (aa). The conserved PK catalytic cores (subdomains I-XI) are identical, while the carboxy-terminal extensions differ by only two aa. However, LmPKAC2 shares only 62% identity over the 255 aa catalytic core region with the previously described LmPKAC1 (c-lpk2). Unlike LmPKAC1, the location of the FXXF motif at the carboxy-terminus is conserved in both LmPKAC2 isoforms; however, the aa sequence, LXXF, in isoform-2a is unusual. The leishmanial isoforms can be distinguished by their NH(2)-terminal extensions, which show minimal similarity at the primary sequence level. Structural analysis of the three enzymes based on the crystal structure of mammalian PKAs predicts that both LmPKAC2 isoforms, unlike LmPKAC1, have identical alpha-helix structures in the NH(2)-terminal extension. Lmpkac2 genes are located on chromosome 35 just downstream from the leishmanial prp8 gene. This genomic organization is conserved in two species of Leishmania and Crithidia fasciculata and allowed for the partial analysis of Cfpkac2a. Phylogenetic analysis groups the two LmPKAC2 isoforms together and separately from LmPKAC1, which is more similar to the Euglena gracilis PKAC, EPK2. These findings provide the basis for additional studies on the role of the PKA family in parasite differentiation and virulence.     

Identification of developmentally regulated expression of MuSK in astrocytes of the rodent retina

One of the master regulators of postsynaptic neuromuscular synaptogenesis is the muscle-specific receptor tyrosine kinase (MuSK). In mammals prominent MuSK expression is believed to be restricted to skeletal muscle. Upon activation by nerve-derived agrin MuSK-dependent signalling participates in both the induction of genes encoding postsynaptic components and aggregation of nicotinic acetylcholine receptors (AChR) in the subsynaptic muscle membrane. Strikingly, expression of certain isoforms of nerve-derived agrin can also be detected in the CNS. In this study, we examined the expression of MuSK in the brain and eye of rodents. In the retina MuSK was expressed in astrocytes between postnatal days 7 and 14, i.e. at the time when the eyes open. We found that agrin was localized adjacent to MuSK-expressing astrocytes which in turn were detected close to the inner limiting membrane of the rodent retina. In summary, the presence of MuSK on retinal astrocytes suggests a novel role of MuSK signalling pathways in the CNS.     

Agrin-like molecules at synaptic sites in normal, denervated, and damaged skeletal muscles

Several lines of evidence have led to the hypothesis that agrin, a protein extracted from the electric organ of Torpedo, is similar to the molecules in the synaptic cleft basal lamina at the neuromuscular junction that direct the formation of acetylcholine receptor and acetylcholinesterase aggregates on regenerating myofibers. One such finding is that monoclonal antibodies against agrin stain molecules concentrated in the synaptic cleft of neuromuscular junctions in rays. In the studies described here we made additional monoclonal antibodies against agrin and used them to extend our knowledge of agrin-like molecules at the neuromuscular junction. We found that anti-agrin antibodies intensely stained the synaptic cleft of frog and chicken as well as that of rays, that denervation of frog muscle resulted in a reduction in staining at the neuromuscular junction, and that the synaptic basal lamina in frog could be stained weeks after degeneration of all cellular components of the neuromuscular junction. We also describe anti-agrin staining in nonjunctional regions of muscle. We conclude the following: (a) agrin-like molecules are likely to be common to all vertebrate neuromuscular junctions; (b) the long-term maintenance of such molecules at the junction is nerve dependent; (c) the molecules are, indeed, a component of the synaptic basal lamina; and (d) they, like the molecules that direct the formation of receptor and esterase aggregates on regenerating myofibers, remain associated with the synaptic basal lamina after muscle damage.     

Matrix metalloproteinase-3 removes agrin from synaptic basal lamina

Agrin, a heparin sulfate proteoglycan, is an integral member of the synaptic basal lamina and plays a critical role in the formation and maintenance of the neuromuscular junction. The N-terminal region of agrin binds tightly to basal lamina, while the C-terminal region interacts with a muscle-specific tyrosine kinase (MuSK) to induce the formation of the postsynaptic apparatus. Although the binding of agrin to basal lamina is tight, the binding of agrin to MuSK has yet to be shown; therefore, basal lamina binding is critical for maintaining the presentation of agrin to MuSK. Here we report evidence that supports our hypothesis that matrix metalloproteinase-3 (MMP-3) is responsible for the removal of agrin from synaptic basal lamina. Antibodies to the hinge region of human MMP-3 recognize molecules concentrated at the frog neuromuscular junction in both cross sections and whole mounts. Electron microscopy of neuromuscular junctions stained with antibodies to MMP-3 reveals that staining is found in the extracellular matrix surrounding the Schwann cell. Treatment of sections from frog anterior tibialis muscle with MMP-3 results in a clear and reproducible removal of agrin immunoreactivity from synaptic basal lamina. The same MMP-3 treatment does not alter anti-laminin staining. These results support our hypothesis that synaptic activity results in the activation of MMP-3 at the neuromuscular junction and that MMP-3 specifically removes agrin from synaptic basal lamina.     

Anti-agrin staining is absent at abandoned synaptic sites of frog neuromuscular junctions

The neuromuscular junction is a plastic structure and is constantly undergoing changes as the nerve terminals that innervate the muscle fiber extend and retract their processes. In vivo observations on developing mouse neuromuscular junctions revealed that prior to the retraction of a nerve terminal the acetylcholine receptors (AChRs) under that nerve terminal disperse. Agrin is a protein released by nerve terminals that binds to synaptic basal lamina and directs the aggregation of AChRs and acetylcholinesterase (AChE) in and on the surface of the myotube. Thus, if the AChRs under a nerve terminal disperse, then the cellular signaling mechanism by which agrin maintains the aggregation of those AChRs must have been disrupted. Two possibilities that could lead to the disruption of the agrin induced aggregation are that agrin is present at the synaptic basal lamina but is unable to direct the aggregation of AChRs, or that agrin has been removed from the synaptic basal lamina. Thus, if agrin were blocked, one would expect to see anti-agrin staining at abandoned synaptic sites; whereas if agrin were removed, anti-agrin staining would be absent at abandoned synaptic sites. We find that anti-agrin staining and α-bungarotoxin staining are absent at abandoned synaptic sites. Further, in vivo observations of retracting nerve terminals confirm that agrin is removed from the synaptic basal lamina within 7 days. Thus, while agrin will remain bound to synaptic basal lamina for months following denervation, it is removed within days following synaptic retraction. © 1996 John Wiley & Sons, Inc.