Abstract
The enzyme phospholipase C-β (PLCβ) is a crucial regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to members of the Gq family of heterotrimeric G proteins. We have determined atomic structures of two invertebrate homologs of PLCβ (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLCβ3 considerably increase basal activity and diminish stimulation by Gαq. Gαq binding requires displacement of the autoinhibitory helix from the catalytic core, thus providing an allosteric mechanism for activation of PLCβ.
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Acknowledgements
We thank E. Ross (University of Texas Southwestern Medical Center at Dallas) for the vector encoding human PLCβ3, and G. Tall (University of Rochester) for the baculovirus vector expressing glutathione S-transferase (GST)-tagged Ric8A and for insight into how to increase yields of Gαq, before publication of his work. We also thank P. Backlund (Section on Mass Spectrometry and Metabolism, National Institute of Child Health & Human Development) for mass spectrometry of PLC21 samples. This work was supported by US National Institutes of Health grants HL071818 and HL086865 (J.J.G.T.) and by the Intramural Research program of the National Institute on Deafness and Other Communication Disorders, US National Institutes of Health (J.K.N.). Our research used the Cell and Molecular Biology Core of the Michigan Diabetes Research and Training Center, supported by DK20572. Use of the Advanced Photon Source at Argonne National Laboratory was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and Michigan's Technology Tri-Corridor (grant 085P1000817).
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A.M.L., V.M.T., J.K.N. and J.J.G.T. designed the overall experimental approach. J.G., S.C. and J.K.N. purified LPLC21 and SPLC21, and cloned and sequenced cDNA encoding SPLC21. K.C.S. crystallized LPLC21. A.M.L. crystallized SPLC21 and determined the crystal structures of LPLC21 and SPLC21. A.M.L. and V.M.T. cloned, expressed and purified human PLCβ3 variants. V.M.T. cloned, expressed and purified Gαq. A.M.L. did all activity-based assays. D.M.T. helped design and, together with V.D.D., conducted Thermo Fluor and FCPIA assays. A.M.L., V.M.T. and J.J.G.T. wrote the manuscript.
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Lyon, A., Tesmer, V., Dhamsania, V. et al. An autoinhibitory helix in the C-terminal region of phospholipase C-β mediates Gαq activation. Nat Struct Mol Biol 18, 999–1005 (2011). https://doi.org/10.1038/nsmb.2095
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DOI: https://doi.org/10.1038/nsmb.2095
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