The role of alternative splicing in regulating agrin binding to muscle cells.

The binding of agrin to the muscle cell surface can induce radical changes in the topography and physiology of the cell membrane, resulting in the organization of postsynaptic components opposite the nerve terminal. Alternative splicing of agrin mRNA yields several isoforms, which vary in their cellular expression, developmental profile, and acetylcholine receptor (AChR) clustering activity. Neurons and muscle cells express several of these agrin isoforms. To address the role of alternative splicing in regulating agrin's function, we compared the effects of splicing at the y and z sites of agrin (denoted 'Agy,z'). Agrin isoforms bound differently to the myotube surface: Ag0,0 and Ag4,0 showed much higher levels of binding than Ag4,8. The artificial splice form Ag0,8 showed binding levels similar to Ag4,8. Visualization of the bound agrin after an acute incubation revealed that each isoform associated with the cell surface in a distinct pattern. These binding patterns changed following stimulation of the myotubes with Ag4,8 for 4 h (which induces the clustering of AChRs). Ag4,8 binding sites were concentrated at >90% of the induced AChR clusters, while those for Ag4,0, Ag0,8, and Ag0,0 were enriched at 70%, 50% and 25%, respectively. Together, these observations indicate that alternatively spliced forms of agrin recognize at least partially non-overlapping populations of binding sites on the cell surface, and that the eight amino acid insert is the dominant factor influencing the level of the agrin binding to the cell surface. Further, some of these populations redistribute to AChR clusters upon agrin stimulation.

Alternative splicing of agrin regulates its binding to heparin alpha-dystroglycan, and the cell surface.

Agrin is a basal lamina molecule that directs key events in postsynaptic differentiation, most notably the aggregation of acetylcholine receptors (AChRs) on the muscle cell surface. Agrin's AChR clustering activity is regulated by alternative mRNA splicing. Agrin splice forms having inserts at two sites (y and z) in the C-terminal region are highly active, but isoforms lacking these inserts are weakly active. The biochemical consequences of this alternative splicing are unknown. Here, the binding of four recombinant agrin isoforms to heparin, to alpha-dystroglycan (a component of an agrin receptor), and to myoblasts was tested. The presence of a four-amino acid insert at the y site is necessary and sufficient to confer heparin binding ability to agrin. Moreover, the binding of agrin to alpha-dystroglycan is inhibited by heparin when this insert is present. Agrin binding to the cell surface showed analogous properties: heparin inhibits the binding of only those agrin isoforms containing this four-amino acid insert. The results show that alternative splicing of agrin regulates its binding to heparin and suggest that agrin's interaction with alpha-dystroglycan may be modulated by cell surface glycosaminoglycans in an isoform-dependent manner.

The alpha-dystroglycan-beta-dystroglycan complex. Membrane organization and relationship to an agrin receptor.

Aberrant expression of the dystrophin-associated protein complex is thought to underlie the pathogenesis of Duchenne dystrophy, Becker muscular dystrophy, and severe childhood autosomal recessive muscular dystrophy. Recently, our laboratory identified an agrin receptor from Torpedo electric organ postsynaptic membranes. It is a heteromer of 190- and 50-kDa subunits with similarity to two components of the dystrophin-associated protein complex of alpha- and beta-dystroglycan. We now confirm the relationship between the Torpedo agrin receptor and mammalian dystroglycans and provide further information about the structure of the alpha-dystroglycan-beta-dystroglycan complex. The sequences of three peptides from each Torpedo subunit were 69% identical to mammalian dystroglycans. An antiserum to mammalian beta-dystroglycan recognizes the Torpedo 50-kDa polypeptide. Additionally, like alpha-dystroglycan, the 190-kDa agrin receptor subunit binds laminin. Previous studies have indicated that alpha- and beta-dystroglycan arise by cleavage of a precursor protein. Tryptic peptide mapping of both subunits and amino-terminal sequencing of Torpedo beta-dystroglycan indicate a single cleavage site, corresponding to serine 654 of the mammalian dystroglycan precursor. Gel electrophoresis analysis indicates there is at least one intrachain disulfide bond in beta-dystroglycan. These results provide precise primary structures for alpha- and beta-dystroglycan.

Production of hyaluronan-dependent pericellular matrix by embryonic rat glial cells.

The extracellular matrix of brain is largely composed of aggregates formed by assembly of many proteoglycan and link protein molecules along a hyaluronan polymer backbone. Some cell types construct large, highly hydrated, pericellular matrices or 'coats' from these hyaluronan-mediated aggregates. We show here that embryonic glial cells produce such hyaluronan-dependent pericellular matrices in response to addition of serum or basic fibroblast growth factor plus transforming growth factor-beta. It is proposed that such a matrix is a significant component of the extracellular milieu of the brain, especially during morphogenesis within the developing brain, and that basic fibroblast growth factor and transforming growth factor-beta regulate its production.

Agrin: toward a molecular understanding of synapse regeneration.

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.

Identification and purification of an agrin receptor from Torpedo postsynaptic membranes: a heteromeric complex related to the dystroglycans.

The selective concentration of neurotransmitter receptors at the postsynaptic membrane is an essential aspect of synaptic differentiation and function. Agrin is an extracellular matrix protein that is likely to direct the accumulation of acetylcholine receptors and several other postsynaptic elements at developing and regenerating neuromuscular junctions. How agrin interacts with the membrane to bring about these changes is unknown. We now report the identification and purification of a protein complex from Torpedo electric organ postsynaptic membranes that is likely to serve as an agrin receptor. The native receptor is a heteromeric complex of two membrane glycoproteins of 190 kDa and 50 kDa. The 190 kDa subunit is sufficient to bind ligand. Peptide sequence analysis revealed that the 190 kDa and 50 kDa subunits are related to the dystrophin-associated glycoproteins alpha- and beta-dystroglycan, respectively. No other candidate agrin receptors were detected. The identification of the agrin receptor opens new avenues toward a mechanistic understanding of synapse differentiation.

The muscular basis of aerial ventilation of the primitive lung of Amia calva.

Anatomical analysis, electromyography, pressure recordings, high-speed X-ray and light movies of the mechanism of air ventilation in Amia calva reveal that aerial ventilation proceeds by the action of a specialized pulse pump. The interhyoideus muscle is the dominant muscle being active during both the preparatory phase and the final, prolonged compressive phase during which new air is forced into the lung. Amia retains a relatively large residual volume in the lung and does not repeat inhalation. It often expels excess air from the buccal cavity after the lung has been fully reinflated. The pressure, kinematic and air flow patterns during air ventilation in Amia closely resemble those of the air breath in the lungfish Protopterus. We hypothesize that the basically similar electromyographic profiles of homologous muscles so characteristic for the air ventilation mechanism of Protopterus and Amia reflect a homologous anatomical as well as functional neuromuscular pattern, which has had a common and early evolutionary origin among the Teleostomi.