John P. Merlie

Defective Neuromuscular Synaptogenesis in Agrin-Deficient Mutant Mice

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.

Differential Expression of Voltage-Gated K+ Channel Subunits in Adult Rat Heart Relation to Functional K+ Channels?

Polyclonal antibodies against each of the K+ channel subunits (Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2) shown previously to be expressed in adult rat heart at the mRNA level were used to examine the distributions of these K+ channel subunits in adult rat atrial and ventricular membranes. Immunohistochemistry on isolated adult rat ventricular myocytes revealed strong labeling with the anti-Kv4.2 and anti-Kv1.2 antibodies. Although somewhat weaker (than with anti-Kv1.2 or anti-Kv4.2), positive staining was also observed with the anti-Kv1.5 and anti-Kv2.1 antibodies. Ventricular myocytes exposed to the anti-Kv1.4 antibody, in contrast, did not appear significantly different from background. Qualitatively similar results were obtained on isolated adult rat atrial myocytes. Western blots of atrial and ventricular membrane proteins confirmed the presence of Kv1.2, Kv1.5, Kv2.1, and Kv4.2 and revealed differences in the relative abundances of these subunits in the two membrane preparations. Kv4.2, for example, is more abundant in ventricular than in atrial membranes, whereas Kv1.2 and Kv2.1 are higher in atrial membranes; Kv1.5 levels are comparable in the two preparations. In contrast to these results, nothing was detected in Western blots of atrial or ventricular membrane proteins with the anti-Kv1.4 antibody at concentrations that revealed intense labeling of a 97-kD protein in adult rat brain membranes. A very faint band was detected at 97 kD in the atrial and ventricular preparations when the anti-Kv1.4 antibody was used at a 5- to 10-fold higher concentration. The simplest interpretation of these results is that Kv1.4 is not an abundant protein in adult rat atrial or ventricular myocytes. Therefore, it seems unlikely that Kv1.4 plays an important role in the formation of functional depolarization-activated K+ channels in these cells. The relation(s) between the (other four) K+ channel subunits and the depolarization-activated K+ channels identified electrophysiologically in adult rat atrial and ventricular myocytes is discussed in the present study.

Molecular basis of the two nonequivalent ligand binding sites of the muscle nicotinic acetylcholine receptor

We have stably expressed in fibroblasts different pairs of alpha and non-alpha subunits of the mouse muscle nicotinic acetylcholine receptor (AChR). The gamma and delta, but not the beta, subunits associated efficiently with the alpha subunit, and they extensively modified its binding characteristics. The alpha gamma and alpha delta complexes formed distinctly different high affinity binding sites for the competitive antagonist d-tubocurarine that, together, completely accounted for the two nonequivalent antagonist binding sites in native AChR. The alpha delta complex and native AChR had similar affinities for the agonist carbamylcholine. In contrast, although the alpha gamma complex contains the higher affinity competitive antagonist binding site, it had an affinity for carbamylcholine that was an order of magnitude less than that of the alpha delta complex or the AChR. The comparatively low agonist affinity of the alpha gamma complex may represent an allosterically regulated binding site in the native AChR. These data support a model of two nonequivalent binding sites within the AChR and imply that the basis for this nonequivalence is the association of the alpha subunit with the gamma or delta subunit.

Rapsyn may function as a link between the acetylcholine receptor and the agrin-binding dystrophin-associated glycoprotein complex.

The 43 kDa AChR-associated protein rapsyn is required for the clustering of nicotinic acetylcholine receptors (AChRs) at the developing neuromuscular junction, but the functions of other postsynaptic proteins colocalized with the AChR are less clear. Here we use a fibroblast expression system to investigate the role of the dystrophin-glycoprotein complex (DGC) in AChR clustering. The agrin-binding component of the DGC, dystroglycan, is found evenly distributed across the cell surface when expressed in fibroblasts. However, dystroglycan colocalizes with AChR-rapsyn clusters when these proteins are coexpressed. Furthermore, dystroglycan colocalizes with rapsyn clusters even in the absence of AChR, indicating that rapsyn can cluster dystroglycan and AChR independently. Immunofluorescence staining using a polyclonal antibody to utrophin reveals a lack of staining of clusters, suggesting that the immunoreactive species, like the AChR, does not mediate the observed rapsyndystroglycan interaction. Rapsyn may therefore be a molecular link connecting the AChR to the DGC. At the neuromuscular synapse, rapsyn-mediated linkage of the AChR to the cytoskeleton-anchored DGC may underlie AChR cluster stabilization.

ACh receptor-rich membrane domains organized in fibroblasts by recombinant 43-kildalton protein

Neurotransmitter receptors are generally clustered in the postsynaptic membrane. The mechanism of clustering was analyzed with fibroblast cell lines that were stably transfected with the four subunits for fetal (alpha, beta, gamma, delta) or adult (alpha, beta, epsilon, delta) type mouse muscle nicotinic acetylcholine receptors (AChRs). Immunofluorescent staining indicated that AChRs were dispersed on the surface of these cells. When transiently transfected with an expression construct encoding a 43-kilodalton protein that is normally concentrated under the postsynaptic membrane, AChRs expressed in these cells became aggregated in large cell-surface clusters, colocalized with the 43-kilodalton protein. This suggests that 43-kilodalton protein can induce AChR clustering and that cluster induction involves direct contact between AChR and 43-kilodalton protein.

Primary sequence of a motor neuron-selective adhesive site in the synaptic basal lamina protein s-laminin

S-laminin, a novel homolog of laminin, is concentrated in a subset of basal laminae including the basal lamina that passes between motor nerve terminals and muscle fibers at the neuromuscular junction. Here we used recombinant fragments to localize a neuronal attachment site to the C-terminal 10% of s-laminin. We then used synthetic peptides spanning the active fragment to identify the primary sequence of the adhesive site as Leu-Arg-Glu (LRE): neurons attach to an immobilized LRE-containing peptide, and soluble LRE blocks attachment of neurons to the s-laminin fragment. Whereas ciliary ganglion neurons (which normally innervate muscle fibers) adhered well both to laminin and to an s-laminin fragment, sensory and central neurons and several neuronal cell lines all adhered well to laminin but poorly to the s-laminin fragment. Together, these results define a motor neuron-selective attachment site on s-laminin.

Assembly in vivo of mouse muscle acetylcholine receptor: Identification of an α subunit species that may be an assembly intermediate

We have studied assembly of acetylcholine receptor in vivo using subunit-specific monoclonal antibodies and immunoprecipitation with alpha-bungarotoxin and antitoxin. We have identified three distinct forms of the alpha subunit. The newly synthesized alpha subunit species has a sedimentation coefficient of 5S and is recognized only by antibody specific for SDS-denatured alpha subunit. We have called this species alpha 61. The 5S alpha Tx species is not associated with beta subunits and is probably monomeric. alpha Tx is formed from alpha 61 with a half-time of 15 min and an efficiency of approximately equal to 30%. Formation of alpha Tx involves a conformational change, and we suggest that this conformation is dependent upon or stabilized by disulfide bond formation. The assembly of alpha Tx with beta subunits (and probably gamma and delta) into a 9S complex appears to be an efficient but slow process requiring more than 90 min. Unassembled alpha 61 subunits are degraded rapidly. However, subunit degradation is a result of failure to assemble, rather than its cause.

Regulation of the acetylcholine receptor ϵ subunit gene by recombinant ARIA: An in vitro model for transynaptic gene regulation

Structural specialization of the postsynaptic skeletal muscle membrane is in part mediated by the motor neuron-induced transcriptional regulation of synaptic muscle nuclei. ARIA, a factor that stimulates production of acetylcholine receptors (AChRs), is a candidate signaling molecule for such regulation. Here we examine the transynaptic inducing potential of this polypeptide factor. ARIA immunoreactivity is detectable at synaptic sites in vivo. In vitro, recombinant heregulin beta 1 (rHRG beta 1), the human homolog of ARIA, induces expression of the AChR epsilon gene, the subunit most sensitive to synaptic input. The inducing property of rHRG beta 1 is demonstrated most dramatically in primary muscle cultures from transgenic mice bearing an epsilon promoter-nuclear lacZ reporter transgene. Transient transfection experiments using the Sol 8 muscle cell line indicate that sequences that confer responsiveness to ARIA are located within a 150 bp epsilon subunit promoter region and are E box-independent. These results suggest that ARIA performs a vital role by directing spatially restricted gene expression at the neuromuscular junction.

Interaction of the 43 kd postsynaptic protein with all subunits of the muscle nicotinic acetylcholine receptor

The 43 kd postsynaptic protein (43K) plays a key role in the aggregation of muscle nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane of the neuromuscular junction. By transiently coexpressing 43K and a single AChR subunit (alpha, beta, gamma, or delta) in the quail fibroblast cell line, QT-6, we show that 43K interacts with each subunit to form cell surface clusters in which 43K and receptor subunit are precisely colocalized. Although the level of cell surface expression of single subunits is much lower than that of fully assembled receptor, the clustering of both single subunits and fully assembled AChR occurs efficiently. In addition, 43K-induced clustering is specific for AChR subunits. From these results, we conclude that each pentameric AChR has five potential sites for interacting with 43K during cluster formation.

The Eph kinase ligand AL-1 is expressed by rostral muscles and inhibits outgrowth from caudal neurons

In the peripheral nervous system, neurons derived from specific rostrocaudal levels of the neuraxis selectively synapse on targets that arise from corresponding body positions. To identify molecules involved in such position-dependent connectivity, we used subtractive hybridization to isolate genes selectively expressed in rostral or caudal skeletal muscle. One mRNA that was more abundant in neck than in hindlimb muscles encoded the mouse ortholog of human AL-1 and chick RAGS, membrane-associated ligands of Eph tyrosine kinases that have recently been implicated in cortical axon fasciculation and retinotectal connectivity, respectively. We show here that mouse AL-1 is expressed in discrete regions of the central and peripheral nervous systems and in a subset of developing skeletal muscles. The abundance of AL-1 RNA in immortalized myogenic cell lines derived from rostral muscles is higher than in caudally derived lines, suggesting that levels are heritably maintained. Growth of neurites from cultured sensory ganglia and spinal cords is specifically inhibited by cells expressing AL-1, suggesting that this molecule could serve to guide peripheral axons. The inhibitory effects of AL-1 are position dependent, such that axons derived from caudal (lumbar) ganglia are more affected than those derived from rostral (cervical) ganglia. Together, these results support the notion that Eph kinases and their ligands regulate topographically appropriate neural connectivity in the peripheral nervous system, as well as in the central nervous system.