Regulated Localization Is Sufficient for Hormonal Control of Regulator of G Protein Signaling Homology Rho Guanine Nucleotide Exchange Factors (RH-RhoGEFs)

Angela M Carter,Stephen Gutowski,Paul C Sternweis

doi:10.1074/jbc.m114.564930

Abstract

The regulator of G protein signaling homology (RH) Rho guanine nucleotide exchange factors (RhoGEFs) (p115RhoGEF, leukemia-associated RhoGEF, and PDZ-RhoGEF) contain an RH domain and are specific GEFs for the monomeric GTPase RhoA. The RH domains interact specifically with the α subunits of G12 heterotrimeric GTPases. Activated Gα13 modestly stimulates the exchange activity of both p115RhoGEF and leukemia-associated RhoGEF but not PDZ-RhoGEF. Because all three RH-RhoGEFs can localize to the plasma membrane upon expression of activated Gα13, cellular localization of these RhoGEFs has been proposed as a mechanism for controlling their activity. We use a small molecule-regulated heterodimerization system to rapidly control the localization of RH-RhoGEFs. Acute localization of the proteins to the plasma membrane activates RhoA within minutes and to levels that are comparable with activation of RhoA by hormonal stimulation of G protein-coupled receptors. The catalytic activity of membrane-localized RhoGEFs is not dependent on activated Gα13. We further show that the conserved RH domains can rewire two different RacGEFs to activate Rac1 in response to a traditional activator of RhoA. Thus, RH domains act as independent detectors for activated Gα13 and are sufficient to modulate the activity of RhoGEFs by hormones via mediating their localization to substrate, membrane-associated RhoA.

Highlights

Constitutive localization of RH-Rho guanine nucleotide exchange factors (RhoGEFs) to the plasma membrane elevates levels of active RhoA
The system consists of two binding partners, an FKBP that is constitutively localized to the plasma membrane with an N-terminal myristoylation sequence, and a modified FRB that localizes to the cytosol
We constructed chimeras of FRB and various RH-RhoGEFs to allow the regulated localization of exogenously expressed RH-RhoGEFs to the plasma membrane in the presence of rapamycin analog AP21967 (Rapalog)

Summary

Background

Constitutive localization of RH-RhoGEFs to the plasma membrane elevates levels of active RhoA. The regulator of G protein signaling homology (RH) Rho guanine nucleotide exchange factors (RhoGEFs) (p115RhoGEF, leukemia-associated RhoGEF, and PDZ-RhoGEF) contain an RH domain and are specific GEFs for the monomeric GTPase RhoA. Its RH domain binds to the activated ␣ subunits of G␣12 and G␣13, but it does not act as a GTPase-activating protein [11], and the exchange activity of PRG is not modulated in vitro by the presence of activated G␣13 [11] The latter observation, combined with the modest 2- to 4-fold activation of p115RhoGEF and LARG by G␣13, indicates that an additional mechanism likely exists to regulate the activity of the RH-RhoGEFs in vivo. Using chimeric proteins containing RH domains and RacGEF domains, we define the RH domain as a hormone-responsive module that can rewire the Rac pathway to respond to traditional activators of RhoA

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION