Differential Sensitivity of P-Rex1 to Isoforms of G Protein βγ Dimers

Linnia H Mayeenuddin,William E Mcintire,James C Garrison

doi:10.1074/jbc.m506034200

Linnia H Mayeenuddin, William E Mcintire + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m506034200

Copy DOI

Abstract

P-Rex1 is a specific guanine nucleotide exchange factor (GEF) for Rac, which is present in high abundance in brain and hematopoietic cells. P-Rex1 is dually regulated by phosphatidylinositol (3,4,5)-trisphosphate and the Gbetagamma subunits of heterotrimeric G proteins. We examined which of the multiple G protein alpha and betagamma subunits activate P-Rex1-mediated Rac guanine nucleotide exchange using pure, recombinant proteins reconstituted into synthetic lipid vesicles. AlF(-)(4) activated G(s),G(i),G(q),G(12), or G(13) alpha subunits were unable to activate P-Rex1. Gbetagamma dimers containing Gbeta(1-4) complexed with gamma(2) stimulated P-Rex1 activity with EC(50) values ranging from 10 to 20 nm. Gbeta(5)gamma(2) was not able to stimulate P-Rex1 GEF activity. Dimers containing the beta(1) subunit complexed with a panel of different Ggamma subunits varied in their ability to stimulate P-Rex1. The beta(1)gamma(3), beta(1)gamma(7), beta(1)gamma(10), and beta(1)gamma(13HA) dimers all activated P-Rex1 with EC(50) values ranging from 20 to 38 nm. Dimers composed of beta(1)gamma(12) had lower EC(50) values (approximately 112 nm). The farnesylated gamma(11) subunit is highly expressed in hematopoietic cells; surprisingly, dimers containing this subunit (beta(1)gamma(11)) were also less effective at activating P-Rex1. These findings suggest that the composition of the Gbetagamma dimer released by receptor activation may differentially activate P-Rex1.

Highlights

Dissociation inhibitors and/or GTPase activating proteins, the stimulation of Rac-guanine nucleotide exchange factor (GEF) is thought to be the most biologically important mode of Rac activation [1]
P-Rex proteins apparently serve as a link between phospholipid and G protein-coupled receptor (GPCR) signaling and act as coincidence detectors for signals arising from these two pathways [10]
The activation of GPCRs leads to the release of G␤␥ subunits, which have been shown to activate the p110-␤ and p110-␥ isoforms of PtdIns 3-kinase leading to the production of PIP3 in hematopoietic cells such as neutrophils [11,12,13]

Summary

Introduction

Dissociation inhibitors and/or GTPase activating proteins, the stimulation of Rac-GEFs is thought to be the most biologically important mode of Rac activation [1]. The specific GPCR(s) responsible for P-Rex activation in hematopoietic cells has not been identified, the stimulation of Gi-coupled receptors expressed in neutrophils, such as the fMet-Leu-Phe receptor, is likely to lead to the activation of P-Rex via the liberation of G␤␥ subunits. We have demonstrated that the p110␥ isoform of PtdIns 3-kinase is differentially regulated by certain isozymes of G␤␥ [20] Given that both PtdIns 3-kinase and P-Rex are found in cells of hematopoietic origin [7, 11] and have been shown to be regulated by G protein ␤1␥2 subunits, we hypothesized that P-Rex might have a ␤␥ sensitivity profile similar to that of PtdIns 3-kinase. We found that P-Rex is not modulated by activated G␣ subunits but that P-Rex is selectively regulated by G␤␥ subunits

Objectives

Results

Conclusion