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Functional roles of P2Y2 nucleotide receptor in the formation and maintenance of vertebrate neuromuscular junctions

Authors Tung, Kwok Kwan
Issue Date 2003
Summary In vertebrate neuromuscular junctions (nmjs), adenosine 5’-triphosphate (ATP) is known to stabilize acetylcholine (ACh) in the synaptic vesicles. ATP is co-stored and co-released with ACh during neural stimulation. Previous results indicated that the synaptic ATP can act as a synapse-organizing factor to induce the expression of post-synaptic genes such as acetylcholinesterase (AChE) and acetylcholine receptor (AChR) in muscles. This function was mediated by a metabotropic P2Y1 receptor, a G protein-coupled receptor. In this project, I demonstrated that another P2Y subtype P2Y2 receptor was localized together with P2Y1 receptor at the nmjs in responding to the synaptic ATP. Transcripts encoding P2Y2 receptor at ~3.2 kb in rat muscle was low before birth but increased in the adult. The P2Y2 receptor protein was shown to be restricted to the nmjs and co-localized with AChRs in adult rat, chicken and Xenopus muscles. In rat, the degree of co-localization of P2Y2 receptor with AChR was around 50% in El9 and was increased to almost 100% in adult. With the use of mouse and chick myotubes expressing promoter-reporter constructs from genes of AChE or of AChR subunits, P2Y2 receptor agonists were shown to stimulate the transcription of each of those genes. The pathway to activation of the AChE gene was shown to involve inositol trisphosphate, intracellular Ca2+ and the mitogen-activated protein (MAP) kinase signaling. The P2Y2 receptor-mediated gene activation was via some of the known intermediates of extraceIIuIar signal-reguIated kinase (ERK) phosphorylation. In both mouse and chick myotubes, this culminates in activation of the transcription factor Elk-1. Two Elk-1 binding site sequences located in the AChE gene promoter were sufficient to drive P2Y2 receptor-induced reporter gene transcription. The same two Elk-1 sites are responsible for P2Y1 receptor-mediated gene activation. These results provide evidence for the co-existence of, at least, P2Y1 and P2Y2 receptors in the post-synaptic muscle, which could act as synapse-organizing effectors during the formation and maintenance of vertebrate nmjs via a MAP kinase signaling cascades.
Note Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2003
Language English
Format Thesis
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