{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T20:18:50Z","timestamp":1768508330976,"version":"3.49.0"},"posted":{"date-parts":[[2023,8,30]]},"group-title":"Biophysics","reference-count":54,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2023,8,30]]},"abstract":"Abstract<\/jats:title>\n \n PLC\u03b2<\/jats:italic>\n enzymes cleave\n PIP2<\/jats:italic>\n producing IP3 and DAG.\n PIP2<\/jats:italic>\n modulates the function of many ion channels, while IP3 and DAG regulate intracellular Ca\n 2+<\/jats:sup>\n levels and protein phosphorylation by protein kinase C, respectively.\n PLC\u03b2<\/jats:italic>\n enzymes are under the control of GPCR signaling through direct interactions with G proteins\n G\u03b2\u03b3<\/jats:italic>\n and\n G\u03b1<\/jats:italic>\n \n q<\/jats:italic>\n <\/jats:sub>\n and have been shown to be coincidence detectors for dual stimulation of\n G\u03b1<\/jats:italic>\n \n q<\/jats:italic>\n <\/jats:sub>\n and G\n \u03b1<\/jats:italic>\n i<\/jats:sub>\n coupled receptors.\n PLC\u03b2s<\/jats:italic>\n are aqueous-soluble cytoplasmic enzymes, but partition onto the membrane surface to access their lipid substrate, complicating their functional and structural characterization. Using newly developed methods, we recently showed that\n G\u03b2\u03b3<\/jats:italic>\n activates\n PLC\u03b23<\/jats:italic>\n by recruiting it to the membrane. Using these same methods, here we show that\n G\u03b1<\/jats:italic>\n \n q<\/jats:italic>\n <\/jats:sub>\n increases the catalytic rate constant,\n k<\/jats:italic>\n \n cat<\/jats:italic>\n <\/jats:sub>\n , of\n PLC\u03b23<\/jats:italic>\n . Since stimulation of\n PLC\u03b23<\/jats:italic>\n by\n G\u03b1<\/jats:italic>\n \n q<\/jats:italic>\n <\/jats:sub>\n depends on an autoinhibitory element (the X-Y linker), we propose that\n G\u03b1<\/jats:italic>\n \n q<\/jats:italic>\n <\/jats:sub>\n produces partial relief of the X-Y linker autoinhibition through an allosteric mechanism. We also determined membrane-bound structures of the\n PLC\u03b23-G\u03b1<\/jats:italic>\n \n q<\/jats:italic>\n ,\n <\/jats:sub>\n and\n PLC\u03b23-G\u03b2\u03b3(2)-G\u03b1<\/jats:italic>\n \n q<\/jats:italic>\n <\/jats:sub>\n complexes, which show that these G proteins can bind simultaneously and independently of each other to regulate\n PLC\u03b23<\/jats:italic>\n activity. The structures rationalize a finding in the enzyme assay, that co-stimulation by both G proteins follows a product rule of each independent stimulus. We conclude that baseline activity of\n PLC\u03b23<\/jats:italic>\n is strongly suppressed, but the effect of G proteins, especially acting together, provides a robust stimulus upon G protein stimulation.\n <\/jats:p>\n \n Significance Statement<\/jats:title>\n \n For certain cellular signaling processes, the background activity of signaling enzymes must be minimal and stimulus-dependent activation robust. Nowhere is this truer than in signaling by\n PLC\u03b23<\/jats:italic>\n , whose activity regulates intracellular Ca\n 2+<\/jats:sup>\n , phosphorylation by Protein Kinase C, and the activity of numerous ion channels and membrane receptors. In this study we show how\n PLC\u03b23<\/jats:italic>\n enzymes are regulated by two kinds of G proteins,\n G\u03b2\u03b3<\/jats:italic>\n and\n G\u03b1<\/jats:italic>\n \n q<\/jats:italic>\n <\/jats:sub>\n . Enzyme activity studies and structures on membranes show how these G proteins act by separate, independent mechanisms, leading to a product rule of co-stimulation when they act together. The findings explain how cells achieve robust stimulation of\n PLC\u03b23<\/jats:italic>\n in the setting of very low background activity, properties essential to cell health and survival.\n <\/jats:p>\n <\/jats:sec>","DOI":"10.1101\/2023.08.29.555394","type":"posted-content","created":{"date-parts":[[2023,8,30]],"date-time":"2023-08-30T18:15:12Z","timestamp":1693419312000},"source":"Crossref","is-referenced-by-count":0,"title":["The mechanism of\n \n G\u03b1\n q<\/sub>\n <\/i>\n regulation of\n PLC\u03b23<\/i>\n -catalyzed\n PIP2<\/i>\n hydrolysis"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6738-7017","authenticated-orcid":false,"given":"Maria E.","family":"Falzone","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Roderick","family":"MacKinnon","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2023090207550624000_2023.08.29.555394v1.1","doi-asserted-by":"publisher","DOI":"10.1042\/bj0630223"},{"key":"2023090207550624000_2023.08.29.555394v1.2","doi-asserted-by":"publisher","DOI":"10.1042\/bj0790193"},{"key":"2023090207550624000_2023.08.29.555394v1.3","doi-asserted-by":"crossref","first-page":"795","DOI":"10.1016\/j.tibs.2019.04.001","article-title":"Tools for Understanding Nanoscale Lipid Regulation of Ion Channels","volume":"44","year":"2019","journal-title":"Trends in Biochemical Sciences"},{"key":"2023090207550624000_2023.08.29.555394v1.4","doi-asserted-by":"crossref","first-page":"1560","DOI":"10.1016\/j.bbamem.2013.10.023","article-title":"Lipid modulation of ion channels through specific binding sites","volume":"1838","year":"2014","journal-title":"Biochimica et Biophysica Acta - Biomembranes"},{"key":"2023090207550624000_2023.08.29.555394v1.5","doi-asserted-by":"crossref","unstructured":"S. B. Hansen (2015) Lipid agonism: The PIP2 paradigm of ligand-gated ion channels. in Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids (Elsevier B.V.), pp 620\u2013628.","DOI":"10.1016\/j.bbalip.2015.01.011"},{"key":"2023090207550624000_2023.08.29.555394v1.6","doi-asserted-by":"crossref","unstructured":"G. Kadamur , E. M. Ross (2013) Mammalian Phospholipase C. in Annual Review of Physiology, pp 127\u2013154.","DOI":"10.1146\/annurev-physiol-030212-183750"},{"key":"2023090207550624000_2023.08.29.555394v1.7","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.bi.56.070187.001111"},{"key":"2023090207550624000_2023.08.29.555394v1.8","doi-asserted-by":"crossref","unstructured":"A. M. Lyon , J. J. G. Tesmer (2013) Structural insights into phospholipase C-\u03b2 function. in Molecular Pharmacology, pp 488-500.","DOI":"10.1124\/mol.113.087403"},{"key":"2023090207550624000_2023.08.29.555394v1.9","doi-asserted-by":"crossref","unstructured":"A. M. Lyon , J. A. Begley , T. D. Manett , J. J. G. Tesmer (2014) Molecular mechanisms of phospholipase C \u03b23 autoinhibition. in Structure, pp 1844\u20131854.","DOI":"10.1016\/j.str.2014.10.008"},{"key":"2023090207550624000_2023.08.29.555394v1.10","doi-asserted-by":"crossref","unstructured":"A. M. Lyon , S. Dutta , C. A. Boguth , G. Skiniotis , J. J. G. Tesmer (2013) Fulllength G\u03b1q-phospholipase C-\u03b23 structure reveals interfaces of the C-terminal coiled-coil domain. in Nature Structural and Molecular Biology (Nature Publishing Group), pp 355\u2013362.","DOI":"10.1038\/nsmb.2497"},{"key":"2023090207550624000_2023.08.29.555394v1.11","doi-asserted-by":"crossref","unstructured":"A. M. Lyon et al. (2011) An autoinhibitory helix in the C-terminal region of phospholipase C-\u03b2 mediates G\u03b1q activation. in Nature Structural and Molecular Biology (Nature Publishing Group), pp 999\u20131005.","DOI":"10.1038\/nsmb.2095"},{"key":"2023090207550624000_2023.08.29.555394v1.12","doi-asserted-by":"crossref","unstructured":"S. N. Hicks et al. (2008) General and Versatile Autoinhibition of PLC Isozymes. in Molecular Cell, pp 383\u2013394.","DOI":"10.1016\/j.molcel.2008.06.018"},{"key":"2023090207550624000_2023.08.29.555394v1.13","doi-asserted-by":"crossref","unstructured":"T. H. Charpentier et al. (2014) Membrane-induced allosteric control of phospholipase C-\u03b2 isozymes. in Journal of Biological Chemistry, pp 29545\u201329557.","DOI":"10.1074\/jbc.M114.586784"},{"key":"2023090207550624000_2023.08.29.555394v1.14","doi-asserted-by":"crossref","first-page":"e2301121120","DOI":"10.1073\/pnas.2301121120","article-title":"G\u03b2\u03b3<\/i> activates PIP2<\/i> hydrolysis by recruiting and orienting PLC\u03b2<\/i> on the membrane surface","volume":"120","year":"2023","journal-title":"Proceedings of the National Academy of Sciences"},{"key":"2023090207550624000_2023.08.29.555394v1.15","doi-asserted-by":"crossref","unstructured":"J. R. Hepler et al. (1996) Functional importance of the amino terminus of Gq\u03b1. In Journal of Biological Chemistry, pp 496\u2013504.","DOI":"10.1074\/jbc.271.1.496"},{"key":"2023090207550624000_2023.08.29.555394v1.16","doi-asserted-by":"crossref","unstructured":"G. L. Waldo et al. (2010) Kinetic Scaffolding Mediated by a Phospholipase C\u2013b and Gq Signaling Complex. in Science.","DOI":"10.1126\/science.1193438"},{"key":"2023090207550624000_2023.08.29.555394v1.17","doi-asserted-by":"crossref","unstructured":"R. A. Rebres et al. (2011) Synergistic Ca2+ responses by G\u03b1i- and G\u03b1q-coupled G-protein-coupled receptors require a single PLC\u03b2 isoform that is sensitive to both G\u03b2\u03b3 and G\u03b1q. in Journal of Biological Chemistry, pp 942\u2013951.","DOI":"10.1074\/jbc.M110.198200"},{"key":"2023090207550624000_2023.08.29.555394v1.18","doi-asserted-by":"crossref","unstructured":"T. I. A. Roach et al. (2008) Signaling and cross-talk by C5a and UDP in macrophages selectively use PLC\u03b23 to regulate intracellular free calcium. in Journal of Biological Chemistry, pp 17351\u201317361.","DOI":"10.1074\/jbc.M800907200"},{"key":"2023090207550624000_2023.08.29.555394v1.19","doi-asserted-by":"crossref","unstructured":"F. Philip , G. Kadamur , R. G. Silos , J. Woodson , E. M. Ross (2010) Synergistic activation of phospholipase C-\u03b23 by G\u03b1q and G\u03b2\u03b3 describes a simple two-state coincidence detector. in Current Biology, pp 1327\u20131335.","DOI":"10.1016\/j.cub.2010.06.013"},{"key":"2023090207550624000_2023.08.29.555394v1.20","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1016\/j.bej.2012.01.010","article-title":"On the Lambert W function and its utility in biochemical kinetics","volume":"63","year":"2012","journal-title":"Biochemical Engineering Journal"},{"key":"2023090207550624000_2023.08.29.555394v1.21","doi-asserted-by":"crossref","unstructured":"M. J. W. Adjobo-Hermans et al. (2013) PLC\u03b2 isoforms differ in their subcellular location and their CT-domain dependent interaction with G\u03b1q. in Cellular Signalling (Elsevier Inc.), pp 255\u2013263.","DOI":"10.1016\/j.cellsig.2012.09.022"},{"key":"2023090207550624000_2023.08.29.555394v1.22","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1002553"},{"key":"2023090207550624000_2023.08.29.555394v1.23","doi-asserted-by":"crossref","unstructured":"W. Huang et al. (2018) A membrane-associated, fluorogenic reporter for mammalian phospholipase C isozymes. in Journal of Biological Chemistry, pp 1728\u20131735.","DOI":"10.1074\/jbc.RA117.000926"},{"key":"2023090207550624000_2023.08.29.555394v1.24","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA118.005291"},{"key":"2023090207550624000_2023.08.29.555394v1.25","first-page":"62","volume-title":"in Methods in Enzymology","volume":"295","year":"1998"},{"key":"2023090207550624000_2023.08.29.555394v1.26","doi-asserted-by":"crossref","unstructured":"A. M. Lyon , V. G. Taylor , J. J. G. Tesmer (2014) Strike a pose: G\u03b1q complexes at the membrane. in Trends in Pharmacological Sciences (Elsevier Ltd), pp 23\u201330.","DOI":"10.1016\/j.tips.2013.10.008"},{"key":"2023090207550624000_2023.08.29.555394v1.27","doi-asserted-by":"crossref","first-page":"2550","DOI":"10.1038\/s41467-023-38281-3","article-title":"Molecular architecture of the G\u03b1i-bound TRPC5 ion channel | Nature Communications","volume":"14","year":"2023","journal-title":"Nature Communications"},{"key":"2023090207550624000_2023.08.29.555394v1.28","doi-asserted-by":"crossref","first-page":"1045","DOI":"10.1038\/s41467-022-28685-y","article-title":"Structural basis of adenylyl cyclase 9 activation","volume":"13","year":"2022","journal-title":"Nature Communications"},{"key":"2023090207550624000_2023.08.29.555394v1.29","doi-asserted-by":"crossref","unstructured":"B. Khanppnavar et al. (2023) Structural basis of activation and inhibition of the Ca2+\/calmodulin-sensitive adenylyl cyclase 8. (bioRxiv).","DOI":"10.1101\/2023.03.03.531047"},{"key":"2023090207550624000_2023.08.29.555394v1.30","doi-asserted-by":"publisher","DOI":"10.1126\/science.aav0778"},{"key":"2023090207550624000_2023.08.29.555394v1.31","doi-asserted-by":"publisher","DOI":"10.1038\/s41388-020-01569-5"},{"key":"2023090207550624000_2023.08.29.555394v1.32","doi-asserted-by":"crossref","first-page":"eabj4243","DOI":"10.1126\/scisignal.abj4243","article-title":"Uveal melanoma\u2013 associated mutations in PLC\u03b24 are constitutively activating and promote melanocyte proliferation and tumorigenesis","volume":"14","year":"2021","journal-title":"Science Signaling"},{"key":"2023090207550624000_2023.08.29.555394v1.33","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1111\/pcmr.12708","article-title":"Oncogenic signaling in uveal melanoma","volume":"31","year":"2018","journal-title":"Pigment Cell & Melanoma Research"},{"key":"2023090207550624000_2023.08.29.555394v1.34","doi-asserted-by":"crossref","unstructured":"E. E. Garland-Kuntz et al. (2018) Direct observation of conformational dynamics of the PH domain in phospholipases C\u03f5 and \u03b2 may contribute to subfamily-specific roles in regulation. in Journal of Biological Chemistry, pp 17477\u201317490.","DOI":"10.1074\/jbc.RA118.003656"},{"key":"2023090207550624000_2023.08.29.555394v1.35","doi-asserted-by":"crossref","first-page":"105050","DOI":"10.1016\/j.chemphyslip.2021.105050","article-title":"Structure and regulation of phospholipase C\u03b2 and \u03b5 at the membrane","volume":"235","year":"2021","journal-title":"Chemistry and Physics of Lipids"},{"key":"2023090207550624000_2023.08.29.555394v1.36","first-page":"755","article-title":"Electronic tuning of self-healing fluorophores for live-cell and single-molecule imaging","volume":"8","year":"2016","journal-title":"Chem. Sci"},{"key":"2023090207550624000_2023.08.29.555394v1.37","doi-asserted-by":"crossref","unstructured":"C. Zhao , R. MacKinnon , Structural and functional analyses of a GPCR-inhibited ion channel TRPM3. Neuron 0 (2022).","DOI":"10.1016\/j.neuron.2022.10.002"},{"key":"2023090207550624000_2023.08.29.555394v1.38","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M110.178897"},{"key":"2023090207550624000_2023.08.29.555394v1.39","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M101197200"},{"key":"2023090207550624000_2023.08.29.555394v1.40","doi-asserted-by":"publisher","DOI":"10.1016\/S0006-2952(99)00080-5"},{"key":"2023090207550624000_2023.08.29.555394v1.41","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2005.07.007"},{"key":"2023090207550624000_2023.08.29.555394v1.42","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.42166"},{"key":"2023090207550624000_2023.08.29.555394v1.43","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2015.08.008"},{"key":"2023090207550624000_2023.08.29.555394v1.44","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1038\/nmeth.4193","article-title":"MotionCor2: Anisotropic correction of beam-induced motion for improved cryo-electron microscopy","volume":"14","year":"2017","journal-title":"Nature Methods"},{"key":"2023090207550624000_2023.08.29.555394v1.45","first-page":"1","article-title":"SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM","volume":"2","year":"2019","journal-title":"Communications Biology"},{"key":"2023090207550624000_2023.08.29.555394v1.46","doi-asserted-by":"publisher","DOI":"10.1107\/S205225251801463X"},{"key":"2023090207550624000_2023.08.29.555394v1.47","doi-asserted-by":"crossref","unstructured":"J. Zivanov et al., RELION-3 : new tools for automated high-resolution cryo-EM structure determination. bioRxiv 10.1101\/421123, 1-38 (2018).","DOI":"10.1101\/421123"},{"key":"2023090207550624000_2023.08.29.555394v1.48","first-page":"43","article-title":"PHENIX News","volume":"4","year":"2013","journal-title":"Computational Crystallography Newsletter"},{"key":"2023090207550624000_2023.08.29.555394v1.49","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444909042073"},{"key":"2023090207550624000_2023.08.29.555394v1.50","doi-asserted-by":"publisher","DOI":"10.1002\/jcc.20084"},{"key":"2023090207550624000_2023.08.29.555394v1.51","doi-asserted-by":"publisher","DOI":"10.1002\/PRO.3943"},{"key":"2023090207550624000_2023.08.29.555394v1.52","doi-asserted-by":"crossref","first-page":"793","DOI":"10.1038\/nmeth.4347","article-title":"Addressing preferred specimen orientation in single-particle cryo-EM through tilting","volume":"14","year":"2017","journal-title":"Nature Methods"},{"key":"2023090207550624000_2023.08.29.555394v1.53","doi-asserted-by":"publisher","DOI":"10.3109\/14756366.2012.688039"},{"key":"2023090207550624000_2023.08.29.555394v1.54","doi-asserted-by":"publisher","DOI":"10.1021\/bi201284u"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2023.08.29.555394","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T05:08:53Z","timestamp":1768453733000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2023.08.29.555394"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,8,30]]},"references-count":54,"URL":"https:\/\/doi.org\/10.1101\/2023.08.29.555394","relation":{},"subject":[],"published":{"date-parts":[[2023,8,30]]},"subtype":"preprint"}}