THE ROLE OF ALTERNATIVE SPLICING IN REGULATING AGRIN BINDING TO MUSCLE-CELLS

The binding of agrin to the muscle cell surface can induce radical cha nges in the topography and physiology of the cell membrane, resulting in the organization of postsynaptic components opposite the nerve term inal. Alternative splicing of agrin mRNA yields several isoforms, whic h vary in their cellular expression, developmental profile, and acetyl choline receptor (AChR) clustering activity. Neurons and muscle cells express several of these agrin isoforms. To address the role of altern ative splicing in regulating agrin's function, we compared the effects of splicing at the y and z sites of agrin (denoted 'Ag y,z'). Agrin i soforms bound differently to the myotube surface: Ag0,0 and Ag4,0 show ed much higher levels of binding than Ag4,8. The artificial splice for m Ag0,8 showed binding levels similar to Ag4,8. Visualization of the b ound agrin after an acute incubation revealed that each isoform associ ated with the cell surface in a distinct pattern. These binding patter ns changed following stimulation of the myotubes with Ag4,8 for 4 h (w hich induces the clustering of AChRs). Ag4,8 binding sites were concen trated at > 90% of the induced AChR clusters, while those for Ag4,0, A g0,8, and Ag0,0 were enriched at 70%, 50% and 25%, respectively. Toget her, these observations indicate that alternatively spliced forms of a grin recognize at least partially non-overlapping populations of bindi ng 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. (C) 1998 Elsevier Science B.V. All ri ghts reserved.