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Hydrogen‐Deuterium exchange reveals distinct activation states of PLCβ2 by G‐proteins
Author(s) -
Fisher Isaac,
Jenkins Meredith,
Tall Greg,
Burke John,
Smrcka Alan
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.686.2
Subject(s) - phospholipase c , gq alpha subunit , chemistry , diacylglycerol kinase , g protein , pleckstrin homology domain , phosphatidylinositol , g protein coupled receptor , hydrogen–deuterium exchange , receptor , stereochemistry , biochemistry , signal transduction , protein kinase c , organic chemistry , hydrogen
G‐protein coupled receptors (GPCRs) are an important class of cell surface receptors that transduce extracellular stimuli to intracellular signaling events via G‐proteins. One major effector of G proteins is phospholipase C (PLC), which hydrolyzes Phosphatidylinositol 4,5 bisphosphate (PIP 2 ) into Diacylglycerol (DAG) and Inositol 1,4,5, trisphosphate (IP 3 ). PLCβ2 and PLCβ3 isoforms are directly activated by binding Gα q , Gβγ or both. The mechanism of activation of PLCβ2 by Gβγ is unknown, and the binding site(s) for Gβγ on PLCβ remain controversial. Here, we use hydrogen‐deuterium exchange mass spectrometry (HDXMS) to understand the conformational dynamics of PLCβ and to answer open questions in the field concerning the mechanism of activation of PLC by Gβγ and Gα q . For these experiments, several complexes were examined with HDXMS: PLCβ2 alone, PLCβ2+ PIP 2 /Phosphoethanolamine (PE)/Phosphatidylserine (PS) vesicles, or PLCβ2+ PIP 2 /PE/PS vesicles plus Gβγ or Gα q ‐AlF 4 . Incubation of PLCβ2 with PIP 2 /PE/PS vesicles strongly protects regions in the C‐terminus, confirming that this region is involved in membrane binding. In the presence of Gα q ‐AlF 4 , PLCβ2 shows protection from deuterium exchange in the helix‐loop‐helix region of the proximal C‐terminal domain (CTD) and the EF‐hand domain on PLCβ2 that exactly correspond to regions shown to interact with Gα q in the Gα q ‐PLCβ3 crystal structure. These data validate that our approach accurately reports bona fide protein‐protein and protein‐lipid interactions. Incubation of Gβγ with PLCβ2 does not reveal any regions of strong protection, but rather causes strong increases in deuterium exchange in the of the CTD of PLCβ2, indicating that the CTD of PLC undergoes a large conformational rearrangement upon Gβγ binding. This increase in deuterium exchange of the CTD of PLC is not seen upon Gα q activation. This indicates that there are distinct conformational states of PLCβ induced by Gα q and Gβγ interactions, suggesting that Gα q and Gβγ use different mechanisms for activation of PLCβ. Finally, these data suggest a mechanism for activation of PLC by Gβγ, where Gβγ binding to PLC breaks an autoinhibitory interaction involving the CTD of PLC and either the membrane or another portion of PLCβ2. Support or Funding Information This work is supported by NIH R01 GM081772 to AVS. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .