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 P...[ Read more ]

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.