Agrin: Soluble and Substrate -Attached Studies of a Synaptogenic Protein
Cornish, Toby Charles
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https://hdl.handle.net/2142/82497
Description
Title
Agrin: Soluble and Substrate -Attached Studies of a Synaptogenic Protein
Author(s)
Cornish, Toby Charles
Issue Date
2004
Doctoral Committee Chair(s)
James T. Campanelli
Department of Study
Neuroscience
Discipline
Neuroscience
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Language
eng
Abstract
During the development of the nervous system, environmental information affects neuronal phenotype, pathfinding, and synaptic connections. At the neuromuscular junction (NMJ), the extracellular matrix (ECM) protein agrin is secreted into the synaptic cleft by the presynaptic motor neuron. Agrin interacts with an unknown receptor to induce local aggregation of acetylcholine receptors (AChRs) and other postsynaptic molecules. It is known that agrin's AChR clustering activity resides wholly in its carboxyl-terminal half, which is composed of laminin-like globular domains (G1, G2 and G3) and EGF-like domains. Furthermore, this activity is modulated by tissue-specific alternative mRNA splicing in two of these G-domains. This study employs a two-fold approach to analyze agrin's structure-function relationship: (1) isolate and recombine agrin's structural domains to analyze their contribution to agrin's AChR clustering activity in the traditional soluble context and (2) examine agrin isoforms in a novel, substrate-attached assay that more closely mimics the ECM-attached nature of agrin in vivo. Briefly, the first portion of this study has revealed the following: (1) while only the G3(8) domain exhibits detectable activity by itself, all G-domains studied (G1, G2(0), G2(4), G3(0) and G3(8)) enhance G3(8) activity when physically linked to G3(8). This effect is most pronounced when G2(4) is linked to G3(8) and is independent of the order of the G-domains. In contrast, simple mixing of equimolar soluble G2(4) and G3(8) domains does not augment G3(8) activity. (2) The deletion of EGF-like repeats enhances activity. (3) Increasing the physical separation between linked G1 and G3(8) domains significantly increases activity; similar alterations to linked G2 and G3(8) domains are without effect. (4) AChR clusters induced by concatenated G3(8) domains are significantly smaller than those induced by all other agrin forms studied. The second portion of this study has demonstrated that: (1) microprinted agrin induces AChR colocalization in cultured muscle cells. (2) Both agrin(4,8) and agrin(4,0) are active when printed, while control proteins are not. (3) Clustering induced by agrin(4,8) extends beyond the printed pattern, while that induced by agrin (4,0) does not. Additionally, we have demonstrated that microcontact printing is a viable method for studying cellular biological phenomena such as synaptogenesis.
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