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A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators
Published on: February 20, 2018
Activation of Phospholipase C β by Gβγ and Gαq Involves C-Terminal Rearrangement to Release Autoinhibition
Isaac J Fisher1, Meredith L Jenkins2, Gregory G Tall3
1Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109, USA; Department of Pharmacology and Physiology, University of Rochester School of Medicine, Rochester, NY 14629, USA.
Abstract:
Phospholipase C (PLC) enzymes hydrolyze phosphoinositide lipids to inositol phosphates and diacylglycerol. Direct activation of PLCβ by Gαq and/or Gβγ subunits mediates signaling by Gq and some Gi coupled G-protein-coupled receptors (GPCRs), respectively. PLCβ isoforms contain a unique C-terminal extension, consisting of proximal and distal C-terminal domains (CTDs) separated by a flexible linker. The structure of PLCβ3 bound to Gαq is known, however, for both Gαq and Gβγ; the mechanism for PLCβ activation on membranes is unknown. We examined PLCβ2 dynamics on membranes using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Gβγ caused a robust increase in dynamics of the distal C-terminal domain (CTD). Gαq showed decreased deuterium incorporation at the Gαq binding site on PLCβ. In vitro Gβγ-dependent activation of PLC is inhibited by the distal CTD. The results suggest that disruption of autoinhibitory interactions with the CTD leads to increased PLCβ hydrolase activity.
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