During nerve regeneration, extracellular matrix (ECM) directs the rebuilding of the neuromuscular junction. Active zones for neurotransmitter release in the presynaptic plasma membrane appear to be rebuilt following cues of the synaptic ECM. Presumably, transmembrane nerve terminal ECM receptors (nerve terminal anchorage proteins) bind these synaptic ECM cues and form a link to the cytosolic, exocytotic machinery of the nerve terminal. We have identified a potential nerve terminal ECM receptor, a protein complex of 4 proteins and a transmembrane proteoglycan, SV2pg. We call this protein complex the SV2pg complex. The SV2pg complex is a component of the synaptic vesicle membrane and has the properties expected of an ECM receptor. 1) In addition to being part of the synaptic vesicle, this complex is found on the nerve terminal surface in the presynaptic plasma membrane. 2) Subcellular fractionation of electric organ demonstrates that, although most SV2pg is in synaptic vesicles, a significant amount is tightly bound to ECM. 3) We find that the purified SV2pg complex binds to a component of the electric organ ECM extracts. 4) The SV2pg complex binds to laminin. Laminin is a known component of the synaptic ECM and is abundant in electric organ ECM extracts. We propose to test the hypothesis that the SV2pg complex acts as an ECM receptor on the nerve terminal surface: l) We will fully characterize the SV2pg complex and determine how to isolate the components from one another. 2) We will determine whether the SV2pg becomes tightly associated with synaptic ECM during synaptogenesis at the neuromuscular junction. 3) We will determine whether we can block this association during synaptogenesis with antibodies to the SV2pg complex and look at the effects of this blockage on the resulting synaptic structure. 4) We will attempt to isolate the ECM ligand for the SV2pg complex and determine which component of the complex binds the ligand. As mentioned above, this ligand may be laminin. From successful completion of our research we hope to gain some understanding of the molecular events involved in building the synapse during synaptogenesis. In addition, since the SV2pg complex is a synaptic vesicle component and perhaps an ECM receptor, we may elucidate an activity-dependent adhesion mechanism of the synapse.
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