Modulation of Agrin Function by Alternative Splicing and Ca2+ Binding

Abstract

The aggregation of acetylcholine receptors on postsynaptic membranes is a key step in neuromuscular junction development. This process depends on alternatively spliced forms of the proteoglycan agrin with "B-inserts" of 8, 11, or 19 residues in the protein's globular C-terminal domain, G3. Structures of the neural B8 and B11 forms of agrin-G3 were determined by X-ray crystallography. The structure of G3-B0, which lacks inserts, was determined by NMR. The agrin-G3 domain adopts a beta jellyroll fold. The B insert site is flanked by four loops on one edge of the beta sandwich. The loops form a surface that corresponds to a versatile interaction interface in the family of structurally related LNS proteins. NMR and X-ray data indicate that this interaction interface is flexible in agrin-G3 and that flexibility is reduced by Ca(2+) binding. The plasticity of the interaction interface could enable different splice forms of agrin to select between multiple binding partners.

Activation of Muscle-specific Receptor Tyrosine Kinase and Binding to Dystroglycan Are Regulated by Alternative mRNA Splicing of Agrin

“…Amino acids were selected based on the difference between the structure of the inactive LG3 B0 and active LG3 B11 or LG3 B8 (38). The differences begin at histidine of position 1778 (His 1778 ), include the B-site and end at serine in position 1790 (Ser 1790 (38).…”

“…The molecular details that are important for the different functions of agrin are not known but structural insights obtained by x-ray crystallography and solution NMR of the LG3 domain from different agrin splice variants (38) have led to the following conclusions: 1) the overall fold of the agrin-LG3 domain is very similar to that of LG domains of other proteins (39 -42); 2) the amino acid insert at the B-site is highly flexible indicative of an "induced-fit" mechanism of docking to its putative receptor; 3) the structure of different splice forms of the LG3 domain diverge already seven amino acids before and six amino acids after the B-site; and 4) depletion of Ca 2ϩ from LG3 B8 induces structural changes at the same amino acid residues where the different protein isoforms diverge. Ca 2ϩ depletion also induces a high flexibility in LG3 B0 and the most dynamic residues are those whose orientations differ between LG3 B0 and LG3 B8 .…”

Crystal Structure of the Second LNS/LG Domain from Neurexin 1α

“…Splice inserts at the agrin G3 B-site do not provide ligands to the Ca 2ϩ ion and are flexible in the presence or absence of Ca 2ϩ , indicating little communication between the Ca 2ϩ ion and the splice inserts (60). Strikingly, unlike apparently n1␣_LNS#2, the agrin G3 affinity for Ca 2ϩ is not radically affected by the presence of splice inserts (K d ϳ 0.6 mM for G3-B0 with no insert, versus K d ϳ 1 mM for G3-B8 with an 8-aa insert) (60). Furthermore, in the case of neurexin LNS/LG domains, all three splice insertion sites arrange around the Ca 2ϩ -binding site, with loop ␤6 -␤7 and loop ␤10 -␤11 housing not only a splice insert but also providing a ligand to the Ca 2ϩ -binding site as well.…”

“…These domains are all now known to bind Ca 2ϩ (Fig. 4) (59,60) at the rim of the ␤-sandwich, all three proteins use LNS/LG domains to bind ␣-dystroglycan in a Ca 2ϩ -dependent way (22,(61)(62)(63)(64), and LNS/LG domains in agrin undergo alternative splicing (regulating their ability in neural cells to promote postsynaptic development at neuromuscular junctions, reviewed recently in Ref. 65).…”

“…The agrin G3 B-site (also called site Z) incorporates splice inserts in a loop equivalent to loop ␤2-␤3 in n1␣_LNS#2 (a loop that is not modified by splicing in neurexin LNS/LG domains but does localize close to the hyper-variable surface) (34,60). Splice inserts at the agrin G3 B-site do not provide ligands to the Ca 2ϩ ion and are flexible in the presence or absence of Ca 2ϩ , indicating little communication between the Ca 2ϩ ion and the splice inserts (60).…”