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Identifying a Phospholipase C Epsilon Region that Interacts with Gbeta Gamma
Author(s) -
Madukwe Jerry,
Malik Sundeep,
Smrcka Alan
Publication year - 2017
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.31.1_supplement.816.2
Subject(s) - phospholipase c , gtpase , microbiology and biotechnology , heterotrimeric g protein , gq alpha subunit , g protein , phospholipase , g protein coupled receptor , golgi apparatus , guanine nucleotide exchange factor , biology , phosphatidylinositol 4,5 bisphosphate , chemistry , biochemistry , signal transduction , phosphatidylinositol , enzyme , endoplasmic reticulum
Many hormones and neurotransmitters function by activating isozymes of Phospholipase C (PLC) family through G protein coupled receptors (GPCRs). PLCs hydrolyze membrane phospholipids to generate downstream signaling molecules essential for normal function of the cell. Chronic PLC activation has been implicated in diseases that affect the body. The PLCɛ isoform has been shown by our laboratory to be scaffolded at the nuclear envelope yet responds to plasma membrane (PM) endothelin 1 (ET‐1) receptor stimulus. ET‐1 stimulates phosphatidyl inositol 4‐phosphate (PI4P) hydrolysis in the perinuclear Golgi apparatus generating diacyglycerol and activating a downstream nuclear pool of PKD in neonatal cardiac ventricular myocytes (NRVM). This signaling process leads to reactivation of the fetal gene program and pathologic myocyte hypertrophy (Zhang et al. Cell, 2013). Small GTPases and Heterotrimeric G protein activate PLCɛ. We have shown that the PM endothelin‐1 receptor stimulation of Golgi PI4P depletion is dependent on Gβγ dimer (Malik et al. Mol. Biol. Cell, 2015). Other groups have shown that PLCɛ co‐expressed with Gβγ and a select group of constitutively active small GTPases produce marked increases in inositol phosphate accumulation in COS7 cells. Domains of PLCɛ that interacts with small GTPases for its activation have previously been defined. However, it is not known whether PLCɛ interacts with Gβγ directly for its activation, or which region is required. Our laboratory has previously shown that Gβγ directly interacts with PLCβ2 in vitro in a region within its catalytic domain. We hypothesize that a region within the catalytic domain of PLCɛ interacts with Gβγ directly and propose to identify this region. We reexamined PLCβ2 and PLCɛ activation by Gβγ, Gq and small GTPases (hRasG12V) in COS7 cells co‐transfection assay and observed marked increases in inositol phosphate accumulation. We then identified a region of PLCɛ that aligns with the catalytic domain of PLCβ2. We are currently making mutations within the catalytic domain and other regions of PLCɛ which will be tested for Gβγ activation using the COS7 cell co‐transfection assay. We will also determine if purified Gβγ can interact with and activate PLCɛ in vitro .