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 Ca(2+) 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 Ca(2+)-responsive allosteric element that is essential in forming an active conformation in neuronal agrin.

Mouse RIC-3, an endoplasmic reticulum chaperone, promotes assembly of the alpha7 acetylcholine receptor through a cytoplasmic coiled-coil domain.

RIC-3 (resistant to inhibitor of cholinesterase) is a transmembrane protein, found in invertebrates and vertebrates, that modulates the surface expression of a variety of nicotinic acetylcholine receptors (nAChRs) in neurons and other cells. To understand its mechanism of action, we investigated the cellular location, transmembrane topology and cellular mechanism by which RIC-3 facilitates alpha7 assembly and surface expression in cultured mammalian cells. We show that the mouse protein is targeted to the ER by the first 31 aa which act as a cleavable signal sequence. The mature protein is a single-pass type I transmembrane protein whose N terminus resides in the lumen of the ER with the coiled-coil domain in the cytoplasm. RIC-3, which binds both unfolded and folded alpha7 subunits, facilitates the surface expression of receptor principally by promoting the folding and assembly of the alpha7 subunits in the ER into fully polymerized receptor. Functional analysis shows that facilitation of surface expression of alpha7 in mammalian cells is reduced in RIC-3 mutants lacking the signal peptide, the lumenal segment or the coiled-coil domain, but not in mutants lacking the long C-terminal region downstream of the coiled-coil domain. We show that the coiled-coil domain of mRIC-3 is not required for the interaction of mRIC-3 with alpha7, but does mediate a homotypic interaction between molecules of mRIC-3. We suggest that efficient assembly of the homomeric alpha7 nAChR may thus require mRIC-3 self-association through the cytoplasmic coiled-coil domain and suggest a model by which this may occur.

A mammalian homolog of Drosophila tumorous imaginal discs, Tid1, mediates agrin signaling at the neuromuscular junction.

Motoneuron-derived agrin clusters nicotinic acetylcholine receptors (AChRs) in mammalian muscle cells. We used two-hybrid screens to identify a protein, tumorous imaginal discs (Tid1), that binds to the cytoplasmic domain of muscle-specific kinase (MuSK), a major component of the agrin receptor. Like MuSK, Tid1 colocalizes with AChRs at developing, adult, and denervated motor endplates. Knockdown of Tid1 by short hairpin RNA (shRNA) in skeletal muscle fibers dispersed synaptic AChR clusters and impaired neuromuscular transmission. In cultured myotubes, Tid1 knockdown inhibited AChR clustering, as well as agrin-induced activation of the Rac and Rho small GTPases and tyrosine phosphorylation of the AChR, without affecting MuSK activation. Tid1 knockdown also decreased Dok-7-induced clustering of AChRs. Overexpression of the N-terminal half of Tid1 induced agrin- and MuSK-independent phosphorylation and clustering of AChRs. These results demonstrate that Tid1 is an essential component of the agrin signaling pathway, crucial for synaptic development.

Crystal structure of the extracellular domain of nAChR alpha1 bound to alpha-bungarotoxin at 1.94 A resolution.

We determined the crystal structure of the extracellular domain of the mouse nicotinic acetylcholine receptor (nAChR) alpha1 subunit bound to alpha-bungarotoxin at 1.94 A resolution. This structure is the first atomic-resolution view of a nAChR subunit extracellular domain, revealing receptor-specific features such as the main immunogenic region (MIR), the signature Cys-loop and the N-linked carbohydrate chain. The toxin binds to the receptor through extensive protein-protein and protein-sugar interactions. To our surprise, the structure showed a well-ordered water molecule and two hydrophilic residues deep in the core of the alpha1 subunit. The two hydrophilic core residues are highly conserved in nAChRs, but correspond to hydrophobic residues in the nonchannel homolog acetylcholine-binding proteins. We carried out site-directed mutagenesis and electrophysiology analyses to assess the functional role of the glycosylation and the hydrophilic core residues. Our structural and functional studies show essential features of the nAChR and provide new insights into the gating mechanism.

Structural determinants for alpha-neurotoxin sensitivity in muscle nAChR and their implications for the gating mechanism.

Neurotoxins from snake venoms act as potent antagonists on the nicotinic acetylcholine receptors (nAChRs). Alpha-neurotoxins such as alpha-bungarotoxin (alpha-Btx) selectively bind to the skeletal muscle nAChRs among other subtypes, causing failure of the neuromuscular transmission. Through evolution, some species including snakes and mongoose have developed resistance to alpha-neurotoxins via specific amino acid substitutions in their muscle-type nAChR alpha1 subunit, which constitutes most of the toxin-binding site. Here we analyze these sequence variations in the context of our recent crystal structure of the extracellular domain of the mouse nAChR alpha1 bound to alpha-Btx. Our structure suggests that alpha-Btx has evolved as an extremely potent antagonist of muscle nAChR by binding the receptor tightly, blocking its ligand site, and locking its conformation in a closed state. Conversely, most toxin-resistant mutations occur at the alpha-Btx binding interface on nAChR alpha1 but away from the agonist binding site. These mutations can interfere with the binding of alpha-Btx without having deleterious effect on the gating function. These analyses not only help understand the structural determinants for neurotoxin sensitivity in muscle-type nAChR, but also shed light on its gating mechanism.

Regulation of acetylcholine receptor clustering by the tumor suppressor APC.

At the developing neuromuscular junction, motor neuron-derived agrin triggers the differentiation of postsynaptic membrane into a highly specialized structure, where the nicotinic acetylcholine receptors (AChRs) are aggregated into high-density clusters. Agrin acts by activating the muscle-specific kinase MuSK and inducing coaggregation of the 43-kDa protein rapsyn with AChRs on muscle cell membrane. The signaling mechanism downstream of MuSK is poorly defined. We report here that the mouse tumor suppressor protein adenomatous polyposis coli (APC) has a role in AChR clustering and that the Wnt/beta-catenin pathway may crosstalk with agrin signaling cascade during synapse formation.

Aberrant development of motor axons and neuromuscular synapses in MyoD-null mice.

Myogenic regulatory factors (MRFs), muscle-specific transcription factors, are implicated in the activity-dependent regulation of nicotinic acetylcholine receptor (AChR) subunit genes. Here we show, with immunohistochemistry, Western blotting, and electron microscopy that MyoD, a member of the MRF family, also plays a role in fetal synapse formation. In the diaphragm of 14.5 d gestation (E14.5) wild-type and MyoD-/- mice, AChR clusters (the formation of which is under a muscle intrinsic program) are confined to a centrally located endplate zone. This distribution persists in wild-type adult muscles. However, beginning at E15.5 and extending to the adult, innervated AChR clusters are distributed all over the diaphragm of MyoD-/- mice, extending as far as the insertion of the diaphragm into the ribs. In wild-type muscle, motor axons terminate on clusters adjacent to the main intramuscular nerve; in MyoD-/- muscle, axonal bundles form extensive secondary branches that terminate on the widely distributed clusters. The number of AChR clusters on adult MyoD-/- and wild-type diaphram muscles is similar. Junctional fold density is reduced at MyoD-/- endplates, and the transition from the fetal (alpha, beta, gamma, delta) to adult-type (alpha, beta, delta, epsilon) AChRs is markedly delayed. However, MyoD-/- mice assemble a complex postsynaptic apparatus that includes muscle-specific kinase (MuSK), rapsyn, erbB, and utrophin.

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.

A transmembrane motif governs the surface trafficking of nicotinic acetylcholine receptors.

Surface expression of the nicotinic acetylcholine receptor (AChR) requires the assembly of multiple subunits in the endoplasmic reticulum (ER). Little is known, however, about the mechanism by which assembled receptor pentamers are transported to the cell membrane while unassembled subunits are retained in the ER. Here we report that a motif conserved in the transmembrane domain of AChR subunits is critically involved in this process. In COS cells, mutation within this signal allowed surface expression of unassembled subunits. Conversely, insertion of the sequence to unrelated proteins that are normally transported to the surface resulted in ER retention. The signal is buried in AChR pentamers, but is exposed on unassembled subunits in the ER, where it promotes protein degradation. We therefore conclude that this signal ensures surface trafficking of only functional AChRs.

Yeast expression and NMR analysis of the extracellular domain of muscle nicotinic acetylcholine receptor alpha subunit.

The alpha subunit of the nicotinic acetylcholine receptor (AChR) from Torpedo electric organ and mammalian muscle contains high affinity binding sites for alpha-bungarotoxin and for autoimmune antibodies in sera of patients with myasthenia gravis. To obtain sufficient materials for structural studies of the receptor-ligand complexes, we have expressed part of the mouse muscle alpha subunit as a soluble, secretory protein using the yeast Pichia pastoris. By testing a series of truncated fragments of the receptor protein, we show that alpha211, the entire amino-terminal extracellular domain of AChR alpha subunit (amino acids 1-211), is the minimal segment that could fold properly in yeast. The alpha211 protein was secreted into the culture medium at a concentration of >3 mg/liter. It migrated as a 31-kDa polypeptide with N-linked glycosylation on SDS-polyacrylamide gel. The protein was purified to homogeneity by isoelectric focusing electrophoresis (pI 5.8), and it appeared as a 4.5 S monomer on sucrose gradient at concentrations up to 1 mm ( approximately 30 mg/ml). The receptor domain bound monoclonal antibody mAb35, a conformation-specific antibody against the main immunogenic region of the AChR. In addition, it formed a high affinity complex with alpha-bungarotoxin (k(D) 0.2 nm) but showed relatively low affinity to the small cholinergic ligand acetylcholine. Circular dichroism spectroscopy of alpha211 revealed a composition of secondary structure corresponding to a folded protein. Furthermore, the receptor fragment was efficiently (15)N-labeled in P. pastoris, and proton cross-peaks were well dispersed in nuclear Overhauser effect and heteronuclear single quantum coherence spectra as measured by NMR spectroscopy. We conclude that the soluble AChR protein is useful for high resolution structural studies.

Intrastriatal grafting of glomus cells ameliorates behavioral defects of Parkinsonian rats.

Parkinson's disease is associated with severe motor dysfunctions due to a progressive loss of dopaminergic neurons in substantia nigra. Transplantation of midbrain neurons from human fetuses to the striatum of patients provides effective treatment for the disease. This type of approach, however, could not be adopted widely due to insufficient supply of fetal materials and the controversial ethical and legal issues. The carotid body is a chemoreceptive organ containing chromaffin-like glomus cells that secrete dopamine (DA) as the neurotransmitter. Here, we report the generation of a clonal dopaminergic cell line of the carotid body using the H-2K(b)-tsA58 transgenic mouse. Cells from the carotid body were immortalized at the permissive temperatures and in the presence of gamma-interferon. The glomus cells were isolated by flow cytometry, and purified to homogeneity by a limited dilution procedure. Upon switching the culture to a nonpermissive condition, the immortal cells ceased to divide, became terminally differentiated and secreted high levels of DA. In rats rendered hemi-Parkinsonian by injection of 6-hydroxydopamine (6-OHDA) into the substantial nigra, intrastriatal grafting of the glomus cells resulted in significant recovery of motor asymmetries and sensorimotor dysfunction. The effects were apparent approximately 10 days after transplantation and remained throughout the 4 months of the study. The recovery of behavioral defects was correlated with the ability of cell grafts to release DA in the brain. As none of the existing treatments for Parkinson's disease is completely satisfactory, establishment of a clonal cell line that secretes DA opens a new avenue for the effective control of this neurological disorder.