At the mammalian neuromuscular junction (NMJ) acetylcholine receptors (AChRs) accumulate in dense aggregates on the muscle fiber's plasma membrane beneath the nerve terminal. Recent studies provide keen insights into molecules, such as the protein agrin, that are used by the nerve to induce AChR clusters at the developing NMJ. A second nerve-derived signal also contributes to the high concentration of AChRS at the synapse by selectively activating transcription of the AChR subunit genes in those muscle nuclei positioned underneath the nerve terminal. Although the molecular nature of this activator remains unknown, proteins of the neuregulin (NRG) family of growth factors are the leading candidates for this signal. There are four NRG genes: nrg-1. nrg-2, nrg-3, and nrg-4, which code for proteins with high amino acid homology. Although, the biological functions of NRG-2, NRG-3, and NRG-4 are currently unknown, their structural similarity with NRG-1 raises the possibility that they could account for some of the activities presently attributed to NRG-1. At the NMJ, NRG-1 is thought to locally activate AChR transcription in subsynaptic myonuclei. However, our preliminary data suggest that NRG-2, like NRG-1, is concentrated at the NMJ in vivo and can induce AChR expression in cultured muscle fibers. The experiments proposed here are aimed at addressing three major questions: (1) Is NRG-2's AChR-inducing activity controlled by alternative splicing of the nrg-2 gene? (2) What are the NRG-2 isoforms made by the cellular components of the NMJ? (3) Do NRG-2 and NRG-1 perform redundant functions at the NMJ? The hypothesis being tested is that specific NRG-2 isoforms made by motor neurons are involved in activating AChR expression at the NMJ, and that NRG-2 and NRG-1 regulate overlapping but different sets of postsynaptic genes. Results from the proposed research are expected to yield new insights into the molecular mechanisms that control neurotransmitter receptor density at the NMJ, which may bear on such control at synapses in the brain.
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