This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. A link to any extended data will be provided when the final version is posted online. Research Articles: Cellular/Molecular Calcium Activated Calpain Specifically Cleaves Glutamate Receptor IIA but not IIB at the Drosophila Neuromuscular Junction Elsayed Metwally1,2, Guoli Zhao1, Wenhua Li1, Qifu Wang1 and Yong Q. Zhang1,2 1State Key Laboratory for Molecular and Developmental Biology, CAS Center for Excellence in Brain Science and Intelligence Technology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. 2International College, University of Chinese Academy of Sciences, Beijing 10080, China. https://doi.org/10.1523/JNEUROSCI.2213-17.2019 Received: 31 July 2017 Revised: 21 December 2018 Accepted: 16 January 2019 Published: 31 January 2019 Author contributions: E.M., G.Z., and Y.Q.Z. designed research; E.M., G.Z., W.L., and Q.W. performed research; E.M., G.Z., and Y.Q.Z. analyzed data; E.M. and Y.Q.Z. wrote the paper. Conflict of Interest: The authors declare no competing financial interests. This work was supported by the Strategic Priority Research Program B of the Chinese Academy of Sciences (XDBS1020100) and the National Science Foundation of China (NSFC No 31110103907 and 31490590) to Y.Q. Zhang and NSFC grant No 31500824 to G. Zhao. We thank Drs. Endre Kókai and Helena Araujo for antibodies and flies. We thank Bloomington and Tsinghua stock centers for supplying stocks. We are grateful to Drs. Yingchun Wang and Cathrine Rein Carlson for identification of cleavage sites and Dr. Thomas Schwarz for discussion and advice. We thank the members of the Yong Q. Zhang lab for discussion and suggestions. Corresponding author: Yong Q. Zhang, PhD, Professor, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No. 1 Datun Road, Chao Yang District, Beijing 100101, China., Tel: 86 10 6480 7611; fax: 86 10 6480 7611, Email: [email protected], Webpage: http://english.genetics.cas.cn/ zhangyongqing Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.2213-17.2019 Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2019 the authors 1 Title Page 2 Calcium Activated Calpain Specifically Cleaves Glutamate Receptor IIA but not IIB at 3 the Drosophila Neuromuscular Junction 4 5 Abbreviated title: Calpain Cleaves GluRIIA at Synapses 6 7 Elsayed Metwally,1, 2, 3, 4 Guoli Zhao,1, 3, 5 Wenhua Li,1 Qifu Wang,1 and Yong Q. 8 Zhang1, 2, # 9 1State Key Laboratory for Molecular and Developmental Biology, CAS Center for 10 Excellence in Brain Science and Intelligence Technology, Institute of Genetics and 11 Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. 12 2International College, University of Chinese Academy of Sciences, Beijing 10080, China. 13 3These authors contributed equally to this work. 14 15 Present address: 4Department of Cytology and Histology, Faculty of Veterinary Medicine, 16 Suez Canal University, Ismailia 41522, Egypt. 5F. M. Kirby Neurobiology Center, Boston 17 Children’s Hospital, Boston MA 02115; Department of Neurobiology, Harvard Medical 18 School, Harvard University, Cambridge MA 02138,USA. 19 20 #Corresponding author: 21 Yong Q. Zhang, PhD, Professor, 22 Institute of Genetics and Developmental Biology, 23 Chinese Academy of Sciences, 24 No. 1 Datun Road, Chao Yang District, 25 Beijing 100101, China. 26 Tel: 86 10 6480 7611; fax: 86 10 6480 7611 27 Email: [email protected] 28 Webpage: http://english.genetics.cas.cn/zhangyongqing 29 30 Number of pages (33) 31 Number of Figures (10), tables (0), multimedia and 3D models (0) 32 Number of extended data figures (0), extended data tables (2) 33 Number of words for Abstract (160), Introduction (595), Discussion (1499) 34 The authors declare no competing financial interests. 1 35 This work was supported by the Strategic Priority Research Program B of the Chinese 36 Academy of Sciences (XDBS1020100) and the National Science Foundation of China (NSFC 37 No 31110103907 and 31490590) to Y.Q. Zhang and NSFC grant No 31500824 to G. Zhao. 38 We thank Drs. Endre Kókai and Helena Araujo for antibodies and flies. We thank 39 Bloomington and Tsinghua stock centers for supplying stocks. We are grateful to Drs. 40 Yingchun Wang and Cathrine Rein Carlson for identification of cleavage sites and Dr. 41 Thomas Schwarz for discussion and advice. We thank the members of the Yong Q. Zhang lab 42 for discussion and suggestions. 43 44 Author Contributions: E.M., G.Z. and Y.Q.Z. conceived and designed the experiments, E.M., 45 G.Z., W.L., and Q.W. performed the experiments, E.M., G.Z. and Y.Q.Z. analyzed the data 46 and wrote the manuscript. 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 2 69 Abstract 70 Calpains are calcium dependent, cytosolic proteinases active at neutral pH. They do not 71 degrade but cleave substrates at limited sites. Calpains are implicated in various pathologies 72 such as ischemia, injuries, muscular dystrophy, and neurodegeneration. Despite so, the 73 physiological function of calpains remains to be clearly defined. Using the neuromuscular 74 junction of Drosophila of both sexes as a model, we performed RNAi screening and 75 uncovered that calpains negatively regulated protein levels of the glutamate receptor GluRIIA 76 but not GluRIIB. We then showed that calpains enrich at the postsynaptic area, and the 77 calcium-dependent activation of calpains induced cleavage of GluRIIA at Q788 of its C- 78 terminus. Further genetic and biochemical experiments revealed that different calpains 79 genetically and physically interact to form a protein complex. The protein complex was 80 required for the proteinase activation to downregulate GluRIIA. Our data provide a novel 81 insight into the mechanisms by which different calpains act together as a complex to 82 specifically control GluRIIA levels and consequently synaptic function. 83 84 85 Key words: Calpain, Calcium, Glutamate receptor, Drosophila Neuromuscular junction, 86 GluRIIA, GluRIIB, GluR, Cytosolic proteinase, Cytoplasmic cysteine protease. 87 88 89 Significance Statement 90 Calpain has been implicated in neural insults and neurodegeneration. However, the 91 physiological function of calpains in the nervous system remains to be defined. Here, we 92 show that calpain enriches at the postsynaptic area and negatively and specifically regulates 93 GluRIIA but not IIB level during development. Calcium-dependent activation of calpain 94 cleaves GluRIIA at Q788 of its C-terminus. Different calpains constitute an active protease 95 complex to cleave its target. This study reveals a critical role of calpains during development 96 to specifically cleave GluRIIA at synapses and consequently regulate synaptic function. 97 98 99 100 101 102 3 103 Introduction 104 Calpains are a family of calcium-activated cytoplasmic cysteine proteases, which are 105 ubiquitously expressed in various mammalian tissues and are functionally active at neutral 106 pH (Franco and Huttenlocher, 2005; Hanna et al., 2008). Members of the calpain family act 107 in pathological processes associated with calcium overload such as ischemia and Alzheimer’s 108 disease (Bano et al., 2005; Lee et al., 2000; Vosler et al., 2011; Xu et al., 2007). For example, 109 calpains cleave multiple synaptic proteins including both inotropic and metabotropic 110 glutamate receptors in excitotoxic conditions, as well as in synaptic plasticity (Baudry and Bi, 111 2017; Chan and Mattson, 1999; Doshi and Lynch, 2009; Xu et al., 2007). However, little is 112 known of the precise physiological function of calpain-dependent cleavage of target proteins 113 at synapses during normal development. 114 Calpains exist in organisms ranging from bacteria to humans (Ono et al., 2016; Sorimachi 115 et al., 2011). To date, fifteen different calpains have been identified in mammals and four in 116 Drosophila (Friedrich et al., 2004; Sorimachi et al., 2011). Unlike most proteases, calpains do 117 not destroy but cleave their substrates at limited sites to modulate their function. Calpain 1 118 (μ-calpain) and calpain 2 (m-calpain) are the most ubiquitous and well-studied calpain family 119 members. They are activated in vitro by micromolar and millimolar concentrations of 120 calcium, respectively (Ono et al., 2016; Sorimachi et al., 2011). Conventional calpain 1 and 2 121 are heterodimers composed of a large catalytic subunit with four domains (dI-dIV) and a 122 small regulatory subunit with two domains (dV and dVI). Heterodimerization of the large and 123 small subunits occurs through a unique interaction between their C-terminal domains 124 (Sorimachi et al., 2011; Strobl et al., 2000). There are two calcium-binding sites within the 125 crystal structure of the enzymatically active domain II of μ-calpain (Moldoveanu et al., 2002). 126 These sites, together with domains IV and VI interact with calcium and are required for full 127 enzymatic activity. Calpastatin is an endogenous calpain inhibitor that binds and inhibits 128 calpains via its calpain-inhibitor domains when the proteases are activated by calcium (Hanna 129 et al., 2008). Unlike mammalian calpains, Drosophila calpains comprise a large subunit only 130 and calpastatin has not been identified in Drosophila (Friedrich et al., 2004). 131 The efficiency of neurotransmission is determined by the level of neurotransmitter 132 receptors at the postsynaptic densities (PSDs). However, the molecular mechanism by which 133 the level of neurotransmitter receptors at synapses is regulated is not well understood. At 134 Drosophila neuromuscular junction (NMJ) synapses, there are two subtypes of glutamate 135 receptors (GluR), GluRIIA and GluRIIB, which are developmentally, biophysically, and 136 pharmacologically distinct (DiAntonio et al., 1999; Marrus et al., 2004; Schmid et al., 2008).