Abstract
Neural agrin, an extracellular matrix protein secreted by motor neurons, plays a key role in clustering of nicotinic acetylcholine receptors (AChR) on postsynaptic membranes of the neuromuscular junction. The action of agrin is critically dependent on an eight-amino acid insert (z8 insert) in the third of three consecutive laminin-like globular (G3) domains near the C terminus of neural agrin. Alternatively spliced agrin isoforms in non-neural tissue including muscle lack the z8 insert and are biologically inactive. Extracellular calcium has been shown to be imperative for the AChR-clustering activity of neural agrin. It is unclear, however, whether calcium preferentially interacts with the neural isoform or whether it acts solely as an intracellular messenger that mediates agrin signaling. Here, we report the G3 domain of rat neural agrin (AgG3z8) expressed in Pichia pastoris promoted AChR clustering on surface of C2C12 myotubes in a calcium-dependent manner. Direct binding of calcium to AgG3z8 was demonstrated by trypsin digestion and thermal denaturation experiments. Moreover, calcium induced a significant change in the conformation of AgG3z8, and the effect was correlated with an enhanced binding affinity of the protein to muscle receptor. Mutation of calcium-binding residues in the G3 domain diminished the conformational change of neural agrin, reduced its binding affinity to muscle membrane, and inhibited AChR-clustering activity. Conversely, the G3 domain of muscle agrin (AgG3z0) displayed little structural change in the presence of calcium, bound poorly to muscle surface, and was inactive in AChR-clustering assays. We conclude that distinct interactions of the G3 domain with calcium determine the biological activities of alternatively spliced agrin isoforms during synapse formation.
Highlights
Agrin, a heparin sulfate proteoglycan [1, 2], is the major signaling molecule that triggers the formation and development of the neuromuscular junction [3,4,5]
We report that the G3 domain of rat neural agrin (AgG3z8) expressed in Pichia pastoris promoted acetylcholine receptors (AChR) clustering on the surface of C2C12 myotubes in a calcium-dependent manner
Characterization of Agrin G3 Domain—Using truncated fragments of agrin expressed by mammalian cell lines, several previous studies have shown that the C-terminal 21-kDa sequence of agrin, which contains only the G3 domain with the z8 insert, is the minimal domain sufficient for inducing AChR clustering in cultured myotubes [22, 39, 40]
Summary
CDNAs, Expression Vectors, and Strains—cDNA clones encoding the C-terminal portion of the rat neural (CAg4,8) and muscle agrin (CAg0,0) were kindly provided by Dr Michael Ferns (McGill University, Montreal, Quebec, Canada). A single colony was used to inoculate a 50-ml overnight culture in buffered glycerol complex medium (1% yeast extract, 2% peptone, 3.4 g/liter yeast nitrogen base, 0.1 M potassium phosphate, pH 6.0, 0.4 mg/liter biotin, and 1% glycerol). The initial culture was expanded to 1 liter in buffered methanol complex medium (BMMY; 1% yeast extract, 2% peptone, 3.4 g/liter yeast nitrogen base, 0.1 M potassium phosphate, pH 6.0, 0.4 mg/liter biotin). Methanol was added daily to the BMMY medium at a final concentration of 0.75% to induce and maintain protein expression. Cell Culture and AChR-clustering Assay—C2C12 myoblast cultures were maintained in the growth medium (DMEM containing 20% fetal calf serum, 2 mM glutamine, 0.5% chick embryo extract (Invitrogen), and penicillin/streptomycin). AChR clusters were induced by adding AgG3 proteins to fully differentiated myotube cultures. The results presented in this study were averaged values from
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