Regulator of G-Protein Signaling 14 (RGS14) Is a Selective H-Ras Effector

Francis S Willard,Adam J Kimple,Randall J Kimple,Emily A Oestreich,Nathaniel A Sowa,Declan A Doyle,Mark J Zylka,Xiaoyan Li,Melinda D Willard,Meera Soundararajan,Channing J Der,David P Siderovski,William D Snider

doi:10.1371/journal.pone.0004884

Francis S Willard, Adam J Kimple + Show 11 more

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BackgroundRegulator of G-protein signaling (RGS) proteins have been well-described as accelerators of Gα-mediated GTP hydrolysis (“GTPase-accelerating proteins” or GAPs). However, RGS proteins with complex domain architectures are now known to regulate much more than Gα GTPase activity. RGS14 contains tandem Ras-binding domains that have been reported to bind to Rap- but not Ras GTPases in vitro, leading to the suggestion that RGS14 is a Rap-specific effector. However, more recent data from mammals and Drosophila imply that, in vivo, RGS14 may instead be an effector of Ras.Methodology/Principal FindingsFull-length and truncated forms of purified RGS14 protein were found to bind indiscriminately in vitro to both Rap- and Ras-family GTPases, consistent with prior literature reports. In stark contrast, however, we found that in a cellular context RGS14 selectively binds to activated H-Ras and not to Rap isoforms. Co-transfection / co-immunoprecipitation experiments demonstrated the ability of full-length RGS14 to assemble a multiprotein complex with components of the ERK MAPK pathway in a manner dependent on activated H-Ras. Small interfering RNA-mediated knockdown of RGS14 inhibited both nerve growth factor- and basic fibrobast growth factor-mediated neuronal differentiation of PC12 cells, a process which is known to be dependent on Ras-ERK signaling.Conclusions/SignificanceIn cells, RGS14 facilitates the formation of a selective Ras·GTP-Raf-MEK-ERK multiprotein complex to promote sustained ERK activation and regulate H-Ras-dependent neuritogenesis. This cellular function for RGS14 is similar but distinct from that recently described for its closely-related paralogue, RGS12, which shares the tandem Ras-binding domain architecture with RGS14.

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Many extracellular signaling molecules exert their cellular effects through activation of G protein-coupled receptors (GPCRs) [1,2,3]
We examined the ability of GSTRGS14 fusion proteins to interact with wild-type and activated Rap2A and Rap2B
Interactions were observed with both Rap2A and Rap2B, and this binding appeared to be mediated by the first RBD in the tandem array; in contrast to the interaction with H-Ras (Figure 1A), the interaction was independent of the nucleotide state of Rap2A/2B (Figure 1B and 1C). (Note that endogenous RapGEF activity in HEK 293T cells could result in a significant amount of wild type Rap protein being GTP-bound.)

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Introduction

Many extracellular signaling molecules exert their cellular effects through activation of G protein-coupled receptors (GPCRs) [1,2,3]. GPCRs are seven transmembrane spanning proteins coupled to a membrane-associated heterotrimeric complex that is comprised of a GTP-hydrolyzing Ga subunit and a Gbc dimeric partner [1,2]. The duration of G-protein signaling through effectors is thought to be controlled by the lifetime of the Ga subunit in its GTP-bound form [2,4]. The lifetime of Ga?GTP is modulated by RGS (regulators of G-protein signaling) domain-containing proteins [4]. Many RGS proteins catalyze rapid GTP hydrolysis by isolated Ga subunits in vitro and attenuate or modulate GPCR-initiated signaling in vivo [4,5,8]; RGS proteins are considered key desensitizers of heterotrimeric Gprotein signaling pathways [4,8]. More recent data from mammals and Drosophila imply that, in vivo, RGS14 may instead be an effector of Ras

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