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Characterization of glycerophosphodiesterase 4-interacting molecules Gαq/11and Gβ, which mediate cellular lysophospholipase D activity.

07:00 EST 3rd December 2019 | BioPortfolio

Summary of "Characterization of glycerophosphodiesterase 4-interacting molecules Gαq/11and Gβ, which mediate cellular lysophospholipase D activity."

We previously purified lysophospholipase D (lysoPLD), which hydrolyzes lysophosphatidylcholine (lysoPC) to lysophosphatidic acid (LPA), from rat brain and identified the heterotrimeric G protein subunits Gαq and Gβ1 in the lysoPLD active fractions. Tag-affinity purified Gαq exhibits lysoPLD activity but a mutant that affected cellular localization or interaction with the Gβ subunit reduced lysoPLD activity. Size exclusion chromatography revealed that active lysoPLD is a much higher molecular weight complex than is heterotrimeric G protein, suggesting the presence of other components. Liquid chromatography-tandem mass spectrometry of lysoPLD purified from rat brain identified glycerophosphodiesterase 4 (GDE4), recently reported as lysoPLD, in the same fraction as G proteins. The over-expressed and tag-purified Gαq fractions, which exhibit lysoPLD activity, contained GDE4. Exogenously expressed GDE4 was co-immunoprecipitated with endogenous Gαq and Gβ and exhibited high lysoPLD activity. The results of confocal microscopy and cell fractionation experiments indicated that exogenously expressed GDE4 in cells mainly localized at the ER and partially co-localized with Gαq protein at the plasma membrane. Proteinase K protection assay results suggested that the catalytic domain of GDE4 faces the lumen/extracellular space. Mutations at the conserved amino acids in the C-terminus cytoplasmic regions amongst GDE1, 4 and 7, dramatically suppressed GDE4 enzyme activities. When both the Gαq and Gα11 genes in Neuro2A cells were disrupted using the CRISPR-Cas9 system, endogenous lysoPLD activity was partially reduced but rescued by over-expression of Gαq. These results suggest that GDE4 is a new effector of G protein signaling that produces bioactive phospholipid LPA and/or modulates membrane homeostasis.

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Name: The Biochemical journal
ISSN: 1470-8728
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