Biochemical characterization of a putative agrin receptor from Torpedo electric organ

摘要

Agrin, a synaptic basal lamina component, plays a pivotal role in organizing the postsynaptic apparatus at developing and regenerating neuromuscular junctions. A primary biological action of agrin is to induce the clustering of acetylcholine receptors on the surface of cultured myotubes. Our previous experiments showed that there is a plasma membrane receptor that binds agrin and may mediate agrin's clustering activity in cultured myotubes. However, little is known about the biochemical nature of the putative agrin receptor (PAR) and how it interacts with agrin. The topic of my thesis is the biochemical characterization of the PAR from Torpedo electric organ.;To perform this work, I first developed a radioimmunoassay (RIA) for the quantitative analysis of agrin binding to Torpedo electric organ postsynaptic membranes. Using this RIA, I then demonstrated a wide range of biochemical characteristics of the PAR. The PAR is selectively concentrated in postsynaptic membranes. Agrin binding to postsynaptic membranes requires calcium and is saturable. Enzymatic digestion suggests that the PAR is a proteinaceous. Agrin binding to postsynaptic membranes persists following alkaline stripping of peripheral membrane proteins, and is unaffected by pretreatment of membranes with phosphatidylinositol specific phospholipase C. Thus, the PAR is likely to be a transmembrane protein. Further, the PAR or a closely associated protein binds to a range of lectins, suggesting that it is a glycoprotein.;I developed methods to solubilize PAR in non-ionic detergent. The solubilized PAR fully retains ligand binding capacity and calcium dependence. The PAR has been purified $>$13-fold using membrane fractionation, alkaline extraction and solubilization methods. In addition, I established the optimal range of pH and salt concentration for agrin binding.;To determine the relationship between the PAR and AChR, I designed $alpha$-bungarotoxin and agrin affinity column methods. The PAR, but not the AChR, binds to agrin affinity columns. In contrast, the PAR does not bind to $alpha$-bungarotoxin affinity columns, while the AChR does. Thus, the PAR is distinct from the AChR.;The outcome of this work revealed many biochemical characteristics of the PAR that provide important insights into the function and mechanisms of action of the PAR in synaptogenesis.