NMR assignments for the Ca2+-bound B0 isoform of the C-terminal globular domain of agrin (agrin-G3)

oligomeric state was determined by by analytical ultracentrifugation
B0 isoform of the 3rd(C-terminal) globular domain of agrin

domain acts as a signal to induce aggregation (clustering) of acetylcholine receptors on the postynaptic membrane (muscle) of neuromuscular junctions.

non-homologus predicted to belong to same fold family as agrin-G3 For the purposes of structural genomics lumping, the agrin G3 domain is predicted to belong to a b-jellyroll fold family that includes laminin G, thrombospondin N, and the pentraxin family. Although sequence homologies within this superfamily are below the 'twilight' threshold, the fold assignment is based on the alignment of hydrophobic consensus strings with conserved hydrophobic residues in the b-strands of the known structures of two pentraxins [1] The closest structural siblings to agrin-G3 are beleived to be the laminin G domains (PDB:1QU0, 1DYK) and the ligand binding domain of neurexin 1-beta (PDB:1C4R) [1] Beckmann,G., Hanke, J., Bork, P. & Reich, J.G. (1998). Merging extracellular domains: fold prediction for laminin G-like and amino-terminal thrombospondin-like modules based on homology to pentraxins. J. Mol. Biol. 275: 725-730.

All aliphatic proton chemical shifts were obtained from a 15N labeled sample. All other chemical shifts were obtained from a 2H/13C/15N labled sample and the values reported are not corrected for 2H isotope effects.

Information obtained from PDB's Chemical Component Dictionary at http://wwpdb-remediation.rutgers.edu/downloads.html Downloaded on Thu Jul 21 10:35:28 2011

1H, 13C and 15N backbone assignments for the C-terminal globular domain of agrin.

We describe the formation, maturation, elimination, maintenance, and regeneration of vertebrate neuromuscular junctions (NMJs), the best studied of all synapses. The NMJ forms in a series of steps that involve the exchange of signals among its three cellular components--nerve terminal, muscle fiber, and Schwann cell. Although essentially any motor axon can form NMJs with any muscle fiber, an additional set of cues biases synapse formation in favor of appropriate partners. The NMJ is functional at birth but undergoes numerous alterations postnatally. One step in maturation is the elimination of excess inputs, a competitive process in which the muscle is an intermediary. Once elimination is complete, the NMJ is maintained stably in a dynamic equilibrium that can be perturbed to initiate remodeling. NMJs regenerate following damage to nerve or muscle, but this process differs in fundamental ways from embryonic synaptogenesis. Finally, we consider the extent to which the NMJ is a suitable model for development of neuron-neuron synapses.

Sanes, J.R. & Lichtman, J.W. (1999)
Development of the vertebrate neuromuscular junction.
Annu. Rev. Neurosci 22: 389-442.

Development of the vertebrate neuromuscular junction.

Marx, J. (1992)
Getting it together at the synapse.
Science 258: 1304-1306.

Synapses are essential relay stations for the transmission of information between neurones and other cells. An ordered and tightly regulated formation of these structures is crucial for the functioning of the nervous system. The induction of the intensively studied synapse between nerve and muscle is initiated by the binding of neurone-specific isoforms of the basal membrane protein agrin to receptors on the surface of myotubes. Agrin activates a receptor complex that includes the muscle-specific kinase and most likely additional, yet to be identified, components. Receptor activation leads to the aggregation of acetylcholine receptors (AChR) and other proteins of the postsynaptic apparatus. This activation process has unique features which distinguish it from other receptor tyrosine kinases. In particular, the autophosphorylation of the kinase domain, which usually induces the recruitment of adaptor and signalling molecules, is not sufficient for AChR aggregation. Apparently, interactions of the extracellular domain with unknown components are also required for this process. Agrin binds to a second protein complex on the muscle surface known as the dystrophin-associated glycoprotein complex. This binding forms one end of a molecular link between the extracellular matrix and the cytoskeleton. While many components of the machinery triggering postsynaptic differentiation have now been identified, our picture of the molecular pathway causing the redistribution of synaptic proteins is still incomplete.

Hoch, W. (1999)
Formation of the neuromuscular junction: agrin and its unusual receptors.
Eur. J. Biochem. 265: 1-10.

Formation of the neuromuscular junction. Agrin and its unusual receptors.

Agrin is thought to mediate the motor neuron-induced aggregation of synaptic proteins on the surface of muscle fibers at neuromuscular junctions. Recent experiments provide direct evidence in support of this hypothesis, reveal the nature of agrin immunoreactivity at sites other than neuromuscular junctions, and have resulted in findings that are consistent with the possibility that agrin plays a role in synaptogenesis throughout the nervous system.
McMahan, U.J.Horton, S.E., Werle, M.J., Honig, L.S., Kroger, S., Ruegg, M.A. & Escher, G. (1992) Agrin isoforms and their roles in synaptogenesis. Curr. Opin. Cell Biol. 4: 869-874.
Agrin isoforms and their role in synaptogenesis.

During neuromuscular synapse formation, motor axons induce clustering of acetylcholine receptors (AChRs) in the muscle fiber membrane. The protein agrin, originally isolated from the basal lamina of the synaptic cleft, is synthesized and secreted by motoneurons and triggers formation of AChR clusters on cultured myotubes. We show here postsynaptic AChR aggregates are markedly reduced in number, size, and density in muscles of agrin-deficient mutant mice. These results support the hypothesis that agrin is a critical organizer of postsynaptic differentiation does occur in the mutant, suggesting the existence of a second-nerve-derived synaptic organizing signal. In addition, we show that intramuscular nerve branching and presynaptic differentiation are abnormal in the mutant, phenotypes which may reflect either a distinct effect of agrin or impaired retrograde signaling from a defective postsynaptic apparatus.
Gautam, M., Noakes, P.G., Moscoso, L., Rupp, F., Scheller, R.H., & Hall, Z. (1996) Defective neuromuscular synaptogenesis in agrin-deficient mutant mice. Cell 85: 525-535.
Defective neuromuscular synaptogenesis in agrin-deficient mutant mice.

Agrin is an extracellular matrix protein that directs neuromuscular junction formation. Early signal transduction events in agrin-mediated postsynaptic differentiation include activation of a receptor tyrosine kinase and phosphorylation of acetylcholine receptors (AChRs), but later steps in this pathway are unknown. Here, we have investigated the role of intracellular calcium in agrin-induced AChR clustering on cultured myotubes. Clamping intracellular calcium levels by loading with the fast chelator BAPTA inhibited agrin-induced AChR aggregation. In addition, preexisting AChR aggregates dispersed under these conditions, indicating that the maintenance of AChR clusters is similarly dependent on intracellular calcium fluxes. The decrease in AChR clusters in BAPTA-loaded cells was dose-dependent and reversible, and no change in the number or mobility of AChRs was observed. Clamping intracellular calcium did not block agrin-induced tyrosine phosphorylation of the AChR beta-subunit, indicating that intracellular calcium fluxes are likely to act downstream from or parallel to AChR phosphorylation. Finally, the targets of the intracellular calcium are likely to be close to the calcium source, since agrin-induced AChR clustering was unaffected in cells loaded with EGTA, a slower-binding calcium chelator. These findings distinguish a novel step in the signal transduction mechanism of agrin and raise the possibility that the pathways mediating agrin- and activity-driven changes in synaptic architecture could intersect at the level of intracellular calcium fluxes.

Megeath, L.J. & Fallon, J.R. (1998)
Intracellular calcium regulates agrin-induced acetylcholine receptor clustering.
J. Neurosci. 18: 672-678.

The Journal of neuroscience : the official journal of the Society for Neuroscience
Intracellular calcium regulates agrin-induced acetylcholine receptor clustering.

Agrin is a heparan sulfate proteoglycan that is widely expressed in neurons and microvascular basal lamina in the rodent and avian central nervous system. Agrin induces the differentiation of nerve-muscle synapses, but its function in either normal or diseased brains is not known. Alzheimer's disease (AD) is characterized by loss of synapses, changes in microvascular architecture, and formation of neurofibrillary tangles and senile plaques. Here we have asked whether AD causes changes in the distribution and biochemical properties of agrin. Immunostaining of normal, aged human central nervous system revealed that agrin is expressed in neurons in multiple brain areas. Robust agrin immunoreactivity was observed uniformly in the microvascular basal lamina. In AD brains, agrin is highly concentrated in both diffuse and neuritic plaques as well as neurofibrillary tangles; neuronal expression of agrin also was observed. Furthermore, patients with AD had microvascular alterations characterized by thinning and fragmentation of the basal lamina. Detergent extraction and Western blotting showed that virtually all the agrin in normal brain is soluble in 1% SDS. In contrast, a large fraction of the agrin in AD brains is insoluble under these conditions, suggesting that it is tightly associated with beta-amyloid. Together, these data indicate that the agrin abnormalities observed in AD are closely linked to beta-amyloid deposition. These observations suggest that altered agrin expression in the microvasculature and the brain parenchyma contribute to the pathogenesis of AD.
Donahue, J.E., Berzin, T.M., Rafii, M.S., Glass, D.J., Yancopoulos, G.D., Fallon, J.R., & Stopa, E.G. (1999) Agrin in alzheimer's disease: altered solubility and abnormal distribution within microvasculature and brain parenchyma. Proc. Natl. Acad. Sci. USA 96: 6468-6472.
Proceedings of the National Academy of Sciences of the United States of America
Agrin in Alzheimer's disease: altered solubility and abnormal distribution within microvasculature and brain parenchyma.

Agrin is an extracellular matrix heparan sulfate proteoglycan (HSPG) well known for its role in modulation of the neuromuscular junction during development. Although agrin is one of the major HSPGs of the brain, its function there remains elusive. Here we provide evidence suggesting a possible function for agrin in Alzheimer's disease brain. Agrin protein binds the amyloidogenic peptide Abeta (1-40) in its fibrillar state via a mechanism that involves the heparan sulfate glycosaminoglycan chains of agrin. Furthermore, agrin is able to accelerate Abeta fibril formation and protect Abeta (1-40) from proteolysis, in vitro. Supporting a biological significance for these in vitro data, immunocytochemical studies demonstrate agrin's presence within senile plaques and cerebrovascular amyloid deposits, and agrin immunostained capillaries exhibit pathological alterations in AD brain. These data therefore suggest that agrin may be an important factor in the progression of Abeta peptide aggregation and/or its persistence in Alzheimer's disease brain.
Cotman, S.L., Halfter, W., & Cole G.J. (2000) Agrin binds to b-amyloid (Ab), accelerates Ab fibril formation, and is localized to Ab deposits in Alzheimer's disease brain . Molec. Cell. Neurosci. 15: 183-198.
Agrin binds to beta-amyloid (Abeta), accelerates abeta fibril formation, and is localized to Abeta deposits in Alzheimer's disease brain.

Using a new method for construction and database searches of sequence consensus strings, we have identified a new superfamily of protein modules comprising laminin G, thrombospondin N and the pentraxin families. The conserved patterns correspond mainly to hydrophobic core residues located in central beta strands of the known three-dimensional structures of two pentraxins, the human C-reactive protein and the serum amyloid P-component. Thus, we predict a similar jellyroll fold for all members of this superfamily. In addition, the conservation of two exposed aspartate residues in the majority of superfamily members suggests hitherto unrecognised functional sites.

Beckmann,G., Hanke, J., Bork, P. & Reich, J.G. (1998).
Merging extracellular domains: fold prediction for laminin G-like
and amino-terminal thrombospondin-like modules based on homology to pentraxins.
J. Mol. Biol. 275: 725-730.

Merging extracellular domains: fold prediction for laminin G-like and amino-terminal thrombospondin-like modules based on homology to pentraxins.

Agrin is a large, multidomain heparan sulfate proteoglycan that is associated with basement membranes of several tissues. Particular splice variants of agrin are essential for the formation of synaptic structures at the neuromuscular junction. The binding of agrin to laminin appears to be required for its localization to synaptic basal lamina and other basement membranes. Here, electron microscopy was used to determine the structure of agrin and to localize its binding site in laminin-1. Agrin appears as an approximately 95 nm long particle that consists of a globular, N-terminal laminin-binding domain, a central rod predominantly formed by the follistatin-like domains and three globular, C-terminal laminin G-like domains. In a few cases, heparan sulfate glycosaminoglycan chains were seen emerging from the central portion of the core protein. Moreover, we show that agrin binds to the central region of the three-stranded, coiled-coil oligomerization domain in the long arm of laminin-1, which mediates subunit assembly of the native laminin molecule. In summary, our data show for the first time a protein-protein interaction of the extracellular matrix that involves a coiled-coil domain, and they assign a novel role to this domain of laminin-1. Based on this, we propose that agrin associates with basal lamina in a polarized way.
Denzer, A.J., Schulthess, T., Fauser, C., Schumacher, B., Kammerer, R.A., Engel, J. & Ruegg, M.A. (1998) Electron microscopic structure of agrin and mapping of its binding site in laminin-1. EMBO J 17: 335-343.
Electron microscopic structure of agrin and mapping of its binding site in laminin-1.

Agrin induces both phosphorylation and aggregation of nicotinic acetylcholine receptors (AChRs) when added to myotubes in culture, apparently by binding to a specific receptor on the myotube surface. One such agrin receptor is alpha-dystroglycan, although binding to alpha-dystroglycan appears not to mediate AChR aggregation. To determine whether agrin-induced AChR phosphorylation is mediated by alpha-dystroglycan or by a different agrin receptor, fragments of recombinant agrin that differ in affinity for alpha-dystroglycan were examined for their ability to induce AChR phosphorylation and aggregation in mouse C2 myotubes. The carboxy-terminal 95 kDa agrin fragment agrin-c95(A0B0), which binds to alpha-dystroglycan with high affinity, failed to induce AChR phosphorylation and aggregation. In contrast, agrin-c95(A4B8) which binds less strongly to alpha-dystroglycan, induced both phosphorylation and aggregation, as did a small 21 kDa fragment of agrin, agrin-c21(B8), that completely lacks the binding domain for alpha-dystroglycan. We conclude that agrin-induced AChR phosphorylation and aggregation are triggered by an agrin receptor that is distinct from alpha-dystroglycan.

Meier, T., Gessemann, M., Cavalli, V., Ruegg, M.A. & Wallace, B.G. (1996)
AChR phophorylation and aggregation induced by an agrin fragment that lacks
the binding domain for a-dystroglycan.
J. Cell Sci. 112: 1213-1223.

AChR phosphorylation and aggregation induced by an agrin fragment that lacks the binding domain for alpha-dystroglycan.

Laminin G-like (LG) modules in the extracellular matrix glycoproteins laminin, perlecan, and agrin mediate the binding to heparin and the cell surface receptor alpha-dystroglycan (alpha-DG). These interactions are crucial to basement membrane assembly, as well as muscle and nerve cell function. The crystal structure of the laminin alpha 2 chain LG5 module reveals a 14-stranded beta sandwich. A calcium ion is bound to one edge of the sandwich by conserved acidic residues and is surrounded by residues implicated in heparin and alpha-DG binding. A calcium-coordinated sulfate ion is suggested to mimic the binding of anionic oligosaccharides. The structure demonstrates a conserved function of the LG module in calcium-dependent lectin-like alpha-DG binding.
Hohenester, E., Tisi, D., Talts, J.F., & Timpl, R. (1999) The crystal structure of a laminin G-like module reveals the molecular basis of a-dystroglycan binding to laminins, perlecan, and agrin. Molecular Cell 4: 783-792.
The crystal structure of a laminin G-like module reveals the molecular basis of alpha-dystroglycan binding to laminins, perlecan, and agrin.

Agrin, a protein extracted from the electric organ of Torpedo californica, induces the formation of specializations on cultured chick myotubes that resemble the postsynaptic apparatus at the neuromuscular junction. The aim of the studies reported here was to characterize the effects of agrin on the distribution of acetylcholine receptors (AChRs) and cholinesterase as a step toward determining agrin's mechanism of action. When agrin was added to the medium bathing chick myotubes small (less than 4 micron 2) aggregates of AChRs began to appear within 2 h and increased rapidly in number until 4 h. Over the next 12-20 h the number of aggregates per myotube decreased as the mean size of each aggregate increased to approximately 15 micron 2. The accumulation of AChRs into agrin-induced aggregates occurred primarily by lateral migration of AChRs already in the myotube plasma membrane at the time agrin was added to the cultures. Aggregates of AChRs and cholinesterase remained as long as agrin was present in the medium; if agrin was removed the number of aggregates declined slowly. The formation and maintenance of agrin-induced AChR aggregates required Ca++, Co++ and Mn++ inhibited agrin-induced AChR aggregation and increased the rate of aggregate dispersal. Mg++ and Sr++ could not substitute for Ca++. Agrin-induced receptor aggregation also was inhibited by phorbol 12-myristate 13-acetate, an activator of protein kinase C, and by inhibitors of energy metabolism. The similarities between agrin's effects on cultured myotubes and events that occur during formation of neuromuscular junctions support the hypothesis that axon terminals release molecules similar to agrin that induce the differentiation of the postsynaptic apparatus.

Wallace, B.G. (1988)
Regulation of agrin-induced acetylcholine receptor aggregation by Ca++ and phorbol ester,
J. Cell Biol. 107: 267-278.

Regulation of agrin-induced acetylcholine receptor aggregation by Ca++ and phorbol ester.

One of the foremost challenges to repairing damage after stroke, trauma, or disease is the regeneration of synaptic connections between neurons. Here, we consider recent strides in our understanding of the molecular basis of synapse formation and regeneration. We will focus on the protein agrin, a key player in synaptogenesis at neuromuscular junctions and perhaps at central nervous system synapses as well. Insights into agrin and its receptor could guide the development of rational therapies to combat neuronal degeneration. We will also consider recent surprising and provocative data linking the mechanisms of synapse formation and the cellular pathology in Duchenne muscular dystrophy.

Deyst, K.A., Ma, J. & Fallon, J.R. (1995)
Agrin: toward a molecular understanding of synapse regeneration.
Neurosurgery 37: 71-77.

Agrin: toward a molecular understanding of synapse regeneration.

Nmr Structure Of The B0 Isoform Of The Agrin G3 Domain In Its Ca2+ Bound State

The recombinant agrin-G3 domain corresponds to residues 1748-1940 of the B0 mRNA alternative splice variant of the C-terminal globular domain of chicken agrin. The sequence is given in SWISS-PROT under accession number P31696. This sequence corresponds to the "B11" isoform of the agrin G3 domain. To obtain the sequence of the BO isoform of agrin-G3 for which NMR assignments are reported -> follow the hyperlink "Agrin related protein 2" under "VARSPLIC 1783 1793" Residues 1748-1940 of the sequence you arrive at from this hyperlink correspond to residues 3-195 of the agrin-G3 domain studied by NMR. The first two residues of the agrin-G3 domain for which NMR assignments are reported, GS, are a cloning artifact (part of a thrombin cleavage site used to separate agrin-G3 from the GST carrier protein used in the recombinant fusion protein).
pGSTHis: a derivative of pGEX-1 (Amersham Pharmacia Biotech) that contains a 6xHis tag followed by a thrombin cleavage site between the gluthathione S-transferase (GST) carrier protein and the agrin-G3 domain.

NMR spectrometer of UCONN Health Center @ Farmington