Phosphoinositide 3-Kinase Acts Through Rac and Cdc42 During Agrin-Induced Acetylcholine Receptor Clustering Viktoria Nizhynska, Ralph Neumueller,* Ruth Herbst Center for Brain Research, Medical University of Vienna, Vienna, Austria Received 24 July 2006; revised 16 November 2006; accepted 20 November 2006 ABSTRACT: The formation of the neuromuscular junction (NMJ) is regulated by the nerve-derived hepa- ran sulfate proteoglycan agrin and the muscle-specific kinase MuSK. Agrin induces a signal transduction path- way via MuSK, which promotes the reorganization of the postsynaptic muscle membrane. Activation of MuSK leads to the phosphorylation and redistribution of ace- tylcholine receptors (AChRs) and other postsynaptic proteins to synaptic sites. The accumulation of high den- sities of AChRs at postsynaptic regions represents a hallmark of NMJ formation and is required for proper NMJ function. Here we show that phosphoinositide 3-ki- nase (PI3-K) represents a component of the agrin/ MuSK signaling pathway. Muscle cells treated with spe- cific PI3-K inhibitors are unable to form full-size AChR clusters in response to agrin and AChR phosphorylation is reduced. Moreover, agrin-induced activation of Rac and Cdc42 is impaired in the presence of PI3-K inhibi- tors. PI3-K is localized to the postsynaptic muscle mem- brane consistent with a role during agrin/MuSK signaling. These results put PI3-K downstream of MuSK as regulator of AChR phosphorylation and clustering. Its role during agrin-stimulated Rac and Cdc42 activation suggests a criti- cal function during cytoskeletal reorganizations, which lead to the redistribution of actin-anchored AChRs. ' 2007 Wiley Periodicals, Inc. Develop Neurobiol 67: 1047–1058, 2007 Keywords: acetylcholine receptors; neuromuscular junc- tion; phosphoinositide 3-kinase; MuSK; agrin INTRODUCTION The neuromuscular junction (NMJ) develops as a consequence of contact-mediated signal exchange between the growth cone of a motor neuron and the developing muscle fiber. One of the first events dur- ing NMJ formation is the concentration of acetylcho- line receptors (AChRs) on the muscle membrane at the site of contact. Subsequent changes at the cellular and molecular level induce pre- as well as postsynaptic differentiation (Sanes and Lichtman, 2001; Burden, 2002). Agrin, a large heparan sulfate proteoglycan, is synthesized by motor neurons and secreted by their nerve terminals, where it stably associates with the basal lamina (McMahan, 1990; Reist et al., 1992). Agrin activates the muscle-specific receptor tyrosine kinase (RTK) MuSK and thereby initiates signal transduction events that lead to the formation of a mature NMJ (Jennings et al., 1993; Valenzuela et al., 1995; Glass et al., 1996). Agrin and MuSK are the key players in NMJ formation since mice lacking ei- ther agrin or MuSK fail to form NMJs and conse- quently die shortly after birth (DeChiara et al., 1996; Gautam et al., 1996). Recombinant soluble agrin is able to induce MuSK activation and AChR clustering in muscle cells (Reist et al., 1992; Glass et al., 1996). This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/1932-8451/ suppmat. *Present address: Institute of Molecular Biotechnology, Dr. Bohr-Gasse 3, A-1030, Vienna, Austria. Correspondence to: R. Herbst (ruth.herbst@meduniwien.ac.at). Contract grant sponsor: Austrian Science Fund; contract grant number: R10-B02. Contract grant sponsor: Austrian National Bank; contract grant number: 10017. ' 2007 Wiley Periodicals, Inc. Published online 6 March 2007 in Wiley InterScience (www. interscience.wiley.com). DOI 10.1002/dneu.20371 1047