RGS14 is a highly unusual signalling protein that contains two distinct Gα interaction sites (RGS domain and GL domain) and tandem (R1 and R2) RBD domains that are known in other proteins to bind Ras and Rap GTPases. Indeed, RGS14 was first identified as a novel Rap1/2 binding partner [7
]. We and others have shown previously that the RGS domain of RGS14 is a promiscuous and nonselective GAP for active forms of Giα and Goα, and that the GL domain is a selective GDI for Giα, but not Goα [7
]. We subsequently reported that the GL motif of RGS14 selectively binds inactive Giα1-GDP and Giα3-GDP, but not Giα2, and that both of these Giα isoforms strongly recruit RGS14 to the plasma membrane to form a tight complex with the GL domain to influence the dynamic subcellular localization of RGS14 [14
]. Here we expand those findings to show that RGS14 binds activated H-Ras and its Raf kinase effectors (Raf-1, B-Raf and A-Raf) to modulate their signalling. H-Ras and Raf each bind RGS14 independently, but the two proteins appear to facilitate one another’s binding to RGS14. Alternatively RGS14 may facilitate formation of the H-Ras/Raf complex which, in turn, binds more efficiently to RGS14. Active H-Ras selectively binds the R1 RBD domain of RGS14 and the Raf kinases also bind within the RBD region. We find that expression of RGS14 markedly inhibits PDGF-stimulated activation of Erk1/2 and that this inhibition is dependent on H-Ras binding but is reversed by co-expression of Giα1. In follow-up studies to understand this better, we found that Giα1 binding inhibited Raf binding to RGS14, suggesting that the binding of Giα1 and Raf are mutually exclusive. These findings demonstrate that RGS14 regulates the activity of multiple GTPases with distinct functions, and are consistent with the idea that RGS14 is a newly appreciated integrator of G protein (Giα) and Ras/Raf signalling.
RGS14 is one of only two known proteins (RGS12 the other) that contains three different binding domains for distinct GTPases, and it sits at the intersection of three families of signalling proteins that contain either an RGS domain, a GL domain(s) or an RBD domain(s). Recent studies indicate that proteins containing GL domains (GL proteins) modulate unconventional G protein signalling pathways and events that do not involve cell surface GPCRs [15
]. Much of the previous work on RGS14 focuses on its presumed role as an RGS protein that modulates GPCR/G protein signalling. However, based on our findings here and our understanding of other proteins that contain GL domains, we propose that RGS14 is a newly appreciated integrator of unconventional G protein signalling and MAPkinase signalling. Our reported findings provide the framework of a model to describe how these functionally opposing domains work together to bind and modulate the functions of inactive and active Gα, and Ras/Raf/MAP kinase signalling.
Our proposed model highlights the GL domain as the first point of contact between Gα and RGS14, which differs from conventional models that highlight the RGS domain in this role [2
]. We envision RGS14 to function primarily as a GL protein that also contains an RGS domain. We have proposed that RGS14 is complexed with subsets of Giα1-GDP or Giα3-GDP via its GL domain and these complexes are devoid of Gβγ [14
]. Our findings presented here are consistent with additional features of this model. We postulate that a non-receptor GEF such as Ric8A and/or possibly others [17
] stimulates nucleotide exchange and GTP binding to Giα which, in turn, promotes dissociation of RGS14 because the GL domain does not bind Gα-GTP. This is consistent with our recent observation that Ric8A interacts with RGS14 to promote Giα1 dissociation (C. Vellano et al, unpublished observation). Once free from Giα1, RGS14 is available to bind active H-Ras and Raf kinase to inhibit MAPKinase signalling, as we show here. In this model, the lifetime of the RGS14-Ras/Raf complex is limited by the RGS domain, which could restore Giα-GDP and promote reformation of Giα-GDP/GL-RGS14 complex, and consequential dissociation of Raf and H-Ras. Consistent with this idea, we find that Giα1 and Raf-1 binding to RGS14 is mutually exclusive. An arrangement that incorporates the RGS domain into the same protein restricts the RGS domain pairing to the Gα pre-selected by the GL domain (Giα1 or Giα3). This configuration imparts spatially restricted RGS/Gα selectivity and eliminates the necessity for intrinsic RGS/Gα selectivity. This is consistent with our earlier observation that the RGS domain is a non-selective GAP for Gi/oα [10
]. We speculate that the RGS domain activity is regulated and unavailable as a GAP until the correct conformation is achieved. Consistent with this idea, we observe that both the GL domain [24
] and the N-terminal region containing the RGS domain of RGS14 are phosphorylated (F.J. Shu, D.P. Cowan et al, unpublished observation), which could be important mechanisms for regulating these protein interactions.
Such a model exhibits many mechanistic parallels with established models of conventional GPCR/Gα signalling (1) except that a non-receptor GEF substitutes for a GPCR and the GL motif of RGS14 substitutes for Gβγ [23
]. Of note, similar models have been proposed for proteins known to regulate cell division (but unrelated to MAPkinase signalling) in lower eukaryotes including worms and flies [32
]. However, distinct GL and RGS proteins are thought to act in these systems whereas, in the case of RGS14 shown here, the RGS activity is already built into the GL protein and pre-positioned nearby to terminate RGS14 inhibition of Ras/Raf signalling. While some components of the working model are speculative, most are consistent with our observed findings and provide a framework for further experiments. Our previous work shows that RGS14 can modulate conventional GPCR/Gα (e.g.
M2ChoR/Giα) signalling in membranes, and the model presented here does not rule out a parallel role for RGS14 in conventional GPCR/Gα signalling, as we have proposed [10
]. RGS14 may serve a (poorly understood) role in unconventional G protein signalling pathways involving non-receptor (GPCR) GEFs alone, or these in concerted action with GPCRs.
RGS14’s closest relative, RGS12, also was shown to bind H-Ras and B-Raf to inhibit PDGF Erk phosphorylation, though no Giα regulation of this activity was reported. RGS12 inhibition of PDGF signalling may be mediated by direct binding to the PDGF receptor [36
] suggesting that RGS14 and RGS12 serve distinct functions. Consistent with this idea, RGS14 and RGS12 share both overlapping and distinct sequence and predicted domains structures (63% identity; 70% similarity), and have very different patterns of subcellular distribution. RGS12 localizes to punctate structures in the nucleus and cytosol [37
], while RGS14 is distributed diffusely throughout the cytosol where it shuttles in/out of the nucleus and also accumulates at centrosomes and the plasma membrane with Giα1/3 [11
The exact mechanism by which RGS14 inhibits PDGF-stimulated MAPkinase signalling is unclear. We propose that RGS14 could serve as a molecular scaffold that sequesters active H-Ras and Raf from their signalling pathways to passively inhibit signalling. We favor this idea which is consistent with our proposed model. Alternatively, RGS14 could directly alter H-Ras or Raf-1 activity, perhaps by stimulating Ras GTPase activity and/or inhibiting Raf kinase activity. H-Ras binds specifically to the R1 RBD, and previous work has shown that the RBD domains of other proteins simply serve as binding sites for active Ras-GTP or Rap-GTP, but do not directly alter GTP binding to or GTP hydrolysis activity of these GTPases [5
]. Consistent with these reports, we previously showed the RGS14 failed to alter Rap GTP binding/GTPase activity [10
]. This would suggest that RGS14’s effect on MAPkinase signalling is likely that of a passive scaffold that sequesters H-Ras and Raf. Alternatively RGS14 may serve to redirect Ras/Raf kinase activity away from MEK/Erk towards a different substrate and pathway. In this regard, RGS14 may act in a fashion similar to the well-described scaffold 14:3:3, which also binds Raf kinases to passively modulate their signalling activity. Further studies are required to determine if RGS14 redirects Raf kinase activity or if it competes with 14:3:3 for Raf binding.
Our previous work has shown that RGS14 is most highly expressed in neurons of adult brain [10
], most notably within the hippocampus (Sarah E. Lee, et al, unpublished observation, and also http://www.brain-map.org
). GL proteins studied thus far have been shown to be important for cell division, but also for neuronal development and synaptic plasticity in both lower and higher eukaryotes [16
]. Several studies have implicated a possible role for RGS14 in cell division [28
]. However, since RGS14 is highly enriched in mitotic incompetent neurons of adult brain, it must serve functions distinct from (or in addition to) those relating to cell division. Within hippocampal neurons, MAPkinase signalling pathways play a crucial role in regulating various aspects of neuronal differentiation and synaptic plasticity [22
]. It is possible that RGS14 serves an important, though as yet poorly defined role in regulating these processes. Other signalling proteins that share similar properties with RGS14 as scaffolds, GL proteins, and/or as regulators of MAPkinase signalling have been shown to play important roles in synaptic plasticity. For example, the mammalian partner of in
sceutable (mPins, aka
LGN or AGS5) protein and RGS14 both contain GL/GPR motifs, are modulated by Ric8A [31
], and are stabilized by Giα-GDP binding at the plasma membrane and centrosomes. In addition, mPins/LGN/AGS5 is reported to regulate opposing pulling forces during cell division of mitotic competent non-neuronal cells [41
]. However, mPins/LGN/AGS5 also is expressed in mitotic incompetent adult hippocampal neurons (as is RGS14) and is enriched in synaptic membranes where it associates with PSD-95 and MAGUK scaffolding proteins in a Giα1-dependent manner to influence their trafficking and NMDA receptor surface expression [42
]. Of note, we observe here () that Giα1 does not bind Raf-1, yet is capable of recruiting this kinase to the plasma membrane along with RGS14, suggesting the involvement of a larger multi-protein signalling complex like PSD-95 or MAGUK. This also is consistent with a recent report showing that the FRMPD1 protein serves as a scaffold to regulate the subcellular localization and interactions of the GPR/GL-protein AGS3 and Giα3 [43
]. Other scaffolding proteins play a key role in synaptic plasticity. Loss of Shank, a key scaffolding component of the postsynaptic density (PSD) of hippocampal neurons, results in mice that exhibit smaller dendritic spines, decreased synaptic transmission, but enhanced learning and memory [44
]. Also, loss of the adaptor protein N-Shc/Shc-C, which directly links NMDA and BDNF-receptors to MAPkinase signalling in hippocampal neurons, yields mice that exhibit enhanced spatial learning and memory [45
]. These reports support the idea that RGS14 may serve functions complimentary to those of other scaffolding proteins in neuronal synaptic plasticity.