Mechanisms for Regulation of RhoA by Hormones
Carter, Angela Michelle
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The family of RGS-RhoGEFs, which consists of p115RhoGEF, LARG, and PDZ-RhoGEF (PRG), are specific guanine nucleotide exchange factors (GEF) for the monomeric GTPase, RhoA. Like most RhoGEFs, these proteins contain tandem DH•PH domains that mediate their nucleotide exchange activity. Members of this family also contain a regulator of G-protein signaling (RGS) domain that interacts directly with the α subunit of G₁₂ heterotrimeric GTPases and enhances the rate of intrinsic GTP hydrolysis by these proteins. While activated Gα₁₃ modestly stimulates the exchange activity of p115RhoGEF and LARG, it does not stimulate the intrinsic activity of PRG. All three RGS-RhoGEFs localize to the plasma membrane upon expression of activated Gα₁₃ subunits indicating that regulation of the cellular localization of these RhoGEFs may be a fundamental mechanism for controlling their activity. These studies examine translocation and formation of signaling complexes as mechanisms for regulating the RGS-RhoGEFs by hormones. A small molecule regulated heterodimerization system was used to rapidly and directly control the localization of the RGS-RhoGEFs. Acute localization of the RGS-RhoGEFs to the plasma membrane activates RhoA within minutes and to levels that are comparable to activation of RhoA by stimulation of EDG receptors with hormone. The activity of membrane localized RhoGEFs is not dependent on activation of Gα₁₃. These data demonstrate that simple translocation of the RhoGEFs can drive activation of the GTPases via the intrinsic exchange activity of the GEFs. The conserved RGS domain was identified as a module capable of localizing the RhoGEFs by association with activated Gα₁₃ in response to hormone stimulation. Evidence is also provided that PDZ domains of PRG interact with selected EDG receptors and PH domains of various Lbc-RhoGEFs interact with activated RhoA. These interactions provide multiple anchoring points that may act cooperatively to secure the RhoGEFs to the plasma membrane in proximity to their substrate RhoA. Formation of such signaling complexes between receptors, G proteins, RhoGEFs, and RhoA may be further aided by constitutive oligomerization of EDG receptors. Formation of high order stable signaling complexes may be required to hold RhoGEFs at the plasma membrane for the timeframe necessary to modulate reorganization of actin cytoskeletal structures.