G protein βγ subunits directly interact with and activate phospholipase CΕ

Jerry C Madukwe,Elisabeth E Garland-Kuntz,Angeline M Lyon,Alan V Smrcka

doi:10.1074/jbc.ra118.002354

Abstract

Phospholipase C (PLC) enzymes hydrolyze membrane phosphatidylinositol 4,5 bisphosphate (PIP2) and regulate Ca2+ and protein kinase signaling in virtually all mammalian cell types. Chronic activation of the PLCϵ isoform downstream of G protein-coupled receptors (GPCRs) contributes to the development of cardiac hypertrophy. We have previously shown that PLCϵ-catalyzed hydrolysis of Golgi-associated phosphatidylinositol 4-phosphate (PI4P) in cardiac myocytes depends on G protein βγ subunits released upon stimulation with endothelin-1. PLCϵ binds and is directly activated by Ras family small GTPases, but whether they directly interact with Gβγ has not been demonstrated. To identify PLCϵ domains that interact with Gβγ, here we designed various single substitutions and truncations of WT PLCϵ and tested them for activation by Gβγ in transfected COS-7 cells. Deletion of only a single domain in PLCϵ was not sufficient to completely block its activation by Gβγ, but blocked activation by Ras. Simultaneous deletion of the C-terminal RA2 domain and the N-terminal CDC25 and cysteine-rich domains completely abrogated PLCϵ activation by Gβγ, but activation by the GTPase Rho was retained. In vitro reconstitution experiments further revealed that purified Gβγ directly interacts with a purified fragment of PLCϵ (PLCϵ-PH-RA2) and increases PIP2 hydrolysis. Deletion of the RA2 domain decreased Gβγ binding and eliminated Gβγ stimulation of PIP2 hydrolysis. These results provide first evidence that Gβγ directly interacts with PLCϵ and yield insights into the mechanism by which βγ subunits activate PLCϵ.

Highlights

Phospholipase C (PLC) enzymes hydrolyze membrane phosphatidylinositol 4,5 bisphosphate (PIP2) and regulate Ca2؉ and protein kinase signaling in virtually all mammalian cell types
We have previously shown that PLC⑀-catalyzed hydrolysis of Golgi-associated phosphatidylinositol 4-phosphate (PI4P) in cardiac myocytes depends on G protein ␤␥ subunits released upon stimulation with endothelin-1
We recently found that G␤␥-dependent regulation of PLC⑀ at the Golgi apparatus in cardiac myocytes regulates local PI4P hydrolysis, protein kinase D (PKD) activation, gene expression, and hypertrophy [18]

Summary

Results

G protein– dependent activation of PLC⑀ is difficult to reconstitute with purified G proteins [12, 17], so to understand the mechanism by which G␤␥ regulates PLC⑀, we confirmed previous work showing that G␤␥ co-transfected with PLC⑀ increases accumulation of inositol phosphates (IP) in COS-7 cells labeled with [3H]inositol [16]. Co-transfection of this PLC⑀ mutant with RhoG14V resulted in some Rho-stimulated accumulation of IP, confirming that this construct can be activated to some level (Fig. 4D, right panel). In this case co-transfection with RhoG14V did not result in accumulation of IP above levels of cells transfected with G␤␥ or Rho alone (Fig. 4E) These data together suggest that PLC⑀ activation by G␤␥ requires both the N (cysteine-rich and CDC25 domains) and C termini (RA2 domain) for full activation of PLC⑀. 7B) and the lack of activation of this construct in COS-7 cells (Fig. 4D) Overall, these results suggest that the activation of PLC⑀ involves a direct interaction with G␤␥ that requires the C terminus RA2 domain and the N-terminal cysteine-rich and CDC25 domains

Discussion

Materials and plasmids

Phospholipase C activity assay

Statistical analysis