Acetylcholine receptor-aggregating activity of agrin isoforms and mapping of the active site.
Agrin is a basal lamina protein that induces aggregation of acetylcholine receptors (AChRs) and other molecules at the developing neuromuscular junction. Alternative splicing of chick agrin mRNA at two sites, A and B, gives rise to eight possible isoforms of which five are expressed in vivo. Motor neurons express high levels of isoforms with inserts at sites A and B, muscle cells synthesize isoforms that lack amino acids at the B-site. To obtain further insights into the mechanism of agrin-induced AChR aggregation, we have determined the EC50 (effective concentration to induce half-maximal AChR clustering) of each agrin isoform and of truncation mutants. On chick myotubes, EC50 of the COOH-terminal, 95-kD fragment of agrinA4B8 was approximately 35 pM, of agrinA4B19 approximately 110 pM and of agrinA4B11 approximately 5 nM. While some AChR clusters were observed with 64 nM of agrinA4B0, no activity was detected for agrinA0B0. Recombinant full-length chick agrin and a 100-kD fragment of ray agrin showed similar EC50 values. A 45-kD, COOH-terminal fragment of agrinA4B8 retained high activity (EC50 approximately equal to 130 pM) and a 21-kD fragment was still active, but required higher concentrations (EC50 approximately equal to 13 nM). Unlike the 45-kD fragment, the 21-kD fragment neither bound to heparin nor did heparin inhibit its capability to induce AChR aggregation. These data show quantitatively that agrinA4B8 and agrinA4B19, expressed in motor neurons, are most active, while no activity is detected in agrinA0B0, the dominant isoform synthesized by muscle cells. Furthermore, our results show that a fragment comprising site B8 and the most COOH-terminal G-like domain is sufficient for this activity, and that agrin domains required for binding to heparin and those for AChR aggregation are distinct from each other.
Department of Pharmacology, Biozentrum, University of Basel, Switzerland.
M
M
Gesemann
A J
AJ
Denzer
M A
MA
Ruegg
D018171:Agrin / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002642:Chick Embryo; D018076:DNA, Complementary / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D014644:Genetic Variation; D051379:Mice; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology; D009469:Neuromuscular Junction / Q000254:growth & development; D009474:Neurons; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D011233:Precipitin Tests; D011487:Protein Conformation; D012326:RNA Splicing; D051381:Rats; D011940:Receptor Aggregation / Q000187:drug effects*; D011950:Receptors, Cholinergic / Q000187:drug effects*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D012860:Skates, Fish; D013045:Species Specificity; D013329:Structure-Activity Relationship
D018171:Agrin; D018076:DNA, Complementary; D010446:Peptide Fragments; D011950:Receptors, Cholinergic; D011994:Recombinant Proteins