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.
Deyst
K A
KA
Department of Neuroscience, Brown University, Providence, RI 02912, USA.
McKechnie
B A
BA
Fallon
J R
JR
D018171:Agrin / Q000235:genetics* / Q000378:metabolism*; D017398:Alternative Splicing; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D002642:Chick Embryo; D003598:Cytoskeletal Proteins / Q000378:metabolism; D049030:Dystroglycans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D051381:Rats; D011950:Receptors, Cholinergic / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D018171:Agrin; D003598:Cytoskeletal Proteins; D008562:Membrane Glycoproteins; D020033:Protein Isoforms; D011950:Receptors, Cholinergic; D017978:Receptors, Growth Factor; D011994:Recombinant Proteins; D049030:Dystroglycans