RGS14 is a complex signaling protein that contains an RGS domain, tandem Ras/Rap binding domains, and a GL domain. Previous studies have focused largely on the presumed function of RGS14 as a regulator of GPCR-G protein signaling (6
). However, findings here and elsewhere (8
) strongly suggest that RGS14 serves as a scaffold that integrates unconventional G protein signaling events rather than as a conventional RGS protein. In support of this idea, we show that RGS14 functionally interacts with Ric-8A, a defined regulator of unconventional G protein signaling pathways (22
). Our key findings indicate the following: 1) RGS14 and Ric-8A co-localize at the plasma membrane with wild-type Gαi1; 2) RGS14 and Ric-8A interact with each other in cells; 3) Ric-8A stimulates dissociation of the RGS14:Gαi1-GDP complex in cells and in vitro
; 4) Ric-8A serves as a GEF to facilitate nucleotide exchange (e.g.
GTPγS binding) on the Gαi1 that it liberates from RGS14; 5) the capacity of Ric-8A to overcome the inhibitory effects of RGS14 on Gαi1 nucleotide exchange and GTPase activity depends on the molar ratio of RGS14 relative to Ric-8A; 6) RGS14 and Ric-8A bind to both distinct and overlapping regions of Gαi1; and 7) native RGS14 and Ric-8A co-exist within the same hippocampal neurons.
Our findings indicate that Ric-8A can functionally regulate the activation state of the RGS14:Gαi1-GDP signaling complex, which may potentially play a role in hippocampal signaling functions since RGS14 expression is highly restricted to this brain region. In this regard, RGS14 shows structural and mechanistic parallels with two other brain proteins, LGN (mPins) and AGS3. Like RGS14, these proteins contain GL domains that form stable complexes with Gαi1-GDP, and LGN has been shown to be recruited to the plasma membrane in cells to form an LGN:Gαi1-GDP complex (22
). Similar to its effects on RGS14, Ric-8A also recognizes and induces dissociation of both the AGS3:Gαi1-GDP and LGN:Gαi1-GDP complexes, subsequently facilitating GTP binding to free Gαi1 (22
). As is the case with RGS14, excess amounts of both LGN and AGS3 have been shown to inhibit Ric-8A effects on Gαi1, suggesting competition between these GL proteins and Ric-8A for Gαi1 binding (22
). Taken together, our findings strongly suggest that RGS14 acts as a GL protein as well as an RGS protein.
RGS14 and Ric-8A co-localize with Gαi1-GDP at the plasma membrane in cells
Our cellular localization findings () suggest that Ric-8A, RGS14, and Gαi1 may functionally interact at the plasma membrane in cells. Since both Ric-8A and RGS14 directly bind to inactive Gαi1 in cells (6
), we examined the subcellular localization of both Ric-8A and RGS14 in the presence of wild-type Gαi1. While a majority of Ric-8A is recruited to the plasma membrane in the presence of wild-type Gαi1, almost all Ric-8A is recruited to the plasma membrane when expressed with both wild-type Gαi1 and RGS14 (). The fact that Ric-8A and RGS14 co-localize at the same time with Gαi1 at the plasma membrane supports the possibility that these proteins functionally interact together through sequential formations/dissociations of RGS14:Gαi1 and Ric-8A:Gαi1 complexes, and perhaps through formation of a transient ternary RGS14:Gαi1-GDP:Ric-8A complex. Our data throughout support both the idea of the formation of RGS14:Gαi1 and Ric-8A:Gαi1 complexes and the concept that Gαi1 is exchanged between RGS14 and Ric-8A before dissociation as free Gαi1-GTP.
Ric-8A induces dissociation of the RGS14:Gαi1-GDP complex and subsequently facilitates nucleotide exchange on Gαi1
Mechanistically, our results show that Ric-8A interacts with the RGS14:Gαi1 complex to regulate its activation state. In the absence of nucleotide, Ric-8A forces Gαi1 dissociation from RGS14 to form a stable (and presumably nucleotide free (23
)) Ric-8A:Gαi1 complex. In the presence of GTPγS, Ric-8A-induced dissociation of RGS14:Gαi1 allows Ric-8A to act as a GEF towards free Gαi1, which results in a rapid uncoupling of the Ric-8A:Gαi1 complex and formation of free Gαi1-GTPγS. Our findings are consistent with previous reports describing Ric-8A regulation of other GL:Gαi1-GDP complexes both in the presence and absence of exogenous GTP (22
). While these intermediate ternary biochemical complexes can be isolated under controlled experimental conditions, the lifetime of an RGS14:Gαi1-GDP:Ric-8A complex in cells is likely very transient (24
). This is reflected by our failure to observe a stable heterotrimeric RGS14:Gαi1-GDP:Ric-8A complex in cells or as purified proteins; in both cases, Ric-8A seems to displace Gαi1 from RGS14 (Figs. and ). However, such a transition complex must exist since Gαi1 transfer occurs from RGS14 to Ric-8A (). We observed Ric-8A/RGS14 complex formation in cells (), but failed to observe this with purified proteins (, and data not shown). Reasons for the discrepancy between these two findings are unclear. We do not observe a stable Ric-8A/RGS14 complex when native RGS14 is co-immunoprecipitated from mouse brain (data not shown), though this does not definitively rule out such a complex. One possibility is that our observed cellular interactions are due to post-translation modifications (e.g.
fatty acylation, phosphorylation) on either protein that promote a favorable conformation for binding. Alternatively, an intermediary protein may facilitate an interaction which may be independent of any Ric-8A effects on the RGS14:Gαi1-GDP complex (as is the case with Frmpd1 and AGS3 (37
)). Recovered Ric-8A bound to RGS14 () may also be the result of native Gαi1 bridging the two proteins together, however our dissociation data () does not support this idea. Such an intermediary protein bringing Ric-8A and RGS14 together may facilitate RGS14 to “switch” from regulating G protein signaling to regulating H-Ras/Raf-1-madiated MAP kinase signaling (8
) (or other unknown signaling pathways). The role of Ric-8A in this context remains to be studied.
Ric-8A accelerates nucleotide exchange and GTPase activity of Gαi1 following RGS14:Gαi1-GDP dissociation
We observe that Ric-8A accelerates both GTPγS binding to and the steady-state GTPase activity of Gαi1 in the presence of RGS14, however these Ric-8A effects can be reversed by increasing concentrations of RGS14 (Figs. -); this was the case for both full-length RGS14 and truncated RGS14 missing the RGS domain (ΔRGS14). Even with a dominant GDI function, Ric-8A is able to overcome ΔRGS14 inhibition of GTPγS binding to Gαi1, stimulating over a 20-fold increase in Gαi1 nucleotide binding when introduced to the ΔRGS14:Gαi1-GDP complex (). A five-fold excess of ΔRGS14 to Ric-8A completely inhibits this Ric-8A-induced GTPγS binding, indicating that ΔRGS14 maintains Gαi1 in an inactive state. Full-length RGS14 appears to be as effective as ΔRGS14 at inhibiting Gαi1-directed steady-state GTP hydrolysis, both alone and in the presence of Ric-8A (). The presence of the RGS domain and its GAP activity might be expected to enhance GTP hydrolysis. However, it is likely that nucleotide exchange, and not GTP hydrolysis, is rate-limiting under the experimental conditions used. In this case, the GAP activity of the RGS domain would not be apparent in this in vitro assay, but is necessarily important in the context of cellular signaling.
Like we observe with the GTPγS binding assay, Ric-8A is able to overcome RGS14 inhibition of steady-state Gαi1 GTPase activity, catalyzing a 2.4-fold increase in Gαi1 steady-state GTPase activity when introduced to the RGS14:Gαi1-GDP complex (). Again, increasing concentrations of RGS14 inhibit Ric-8A effects on Gαi1 GTP hydrolysis (). Since the GEF activity of Ric-8A serves to enhance GDP release and increase the velocity of and/or eliminate the rate-limiting step in nucleotide exchange and hydrolysis, enhanced RGS14 binding to Gαi1-GDP would result in increased GDI activity reflected as an inhibition of GTPγS binding and steady-state GTPase activity that is more difficult for Ric-8A to overcome (as we observe). Therefore, RGS14 may bind Gαi1-GDP and hinder Ric-8A (by competitive or non-competitive inhibition) from binding and catalyzing Gαi1-directed GTP binding and hydrolysis.
Ric-8A and RGS14 bind Gαi1 at both distinct and overlapping sites
In studies designed to identify sites(s) of RGS14 and Ric-8A interactions on Gαi1 (), we found that RGS14 and Ric-8A compete for an overlapping binding site on the extreme C-terminus of Gαi1. Whereas residue N149 of Gαi1 has been shown to interact with the GL domain of RGS14 (30
), identified binding sites on Gαi1 for Ric-8A were previously unknown. A recent study suggests that Ric-8A binds to the extreme C-terminus of Gαi1 since pertussis toxin-stimulated modification of C351 within this region inhibits Ric-8A activation of Gαi1 in cells (29
). By comparing the binding properties of Gαi1 (N149I) (which does not bind RGS14 (31
)) and Gαi1-ΔCT (missing the last 11 amino acids including C351), we determined that Ric-8A and RGS14 share distinct and overlapping binding regions on Gαi1 (). The presence of an overlapping binding region correlates with our other data (Figs. and ) that shows increasing concentrations of RGS14 block Ric-8A GEF activity towards Gαi1. Taken together, these findings are consistent with the idea that RGS14 and Ric-8A compete for the exact same or very proximal residues within the extreme C-terminal 11 amino acids of Gαi1. Since RGS14 binds N149 of Gαi1 and Ric-8A does not, it is also possible that RGS14 and Ric-8A are acting on distinct and overlapping regions of Gαi1 at the same time. RGS14 may interact with Gαi1 at residue N149 to carry out additional functions and/or to affect Ric-8A:Gαi1 interactions by allosteric modulation. These findings are the first to show any binding site for Ric-8A on Gαi1, and also the first to show a second binding region on Gαi1 for RGS14. Solved co-crystal structures of the RGS14:Gαi1 and the Ric-8A:Gαi1 complexes will be necessary to precisely define the binding interfaces between these proteins.
Working model for how Ric-8A regulates the RGS14:Gai1-GDP signaling complex
Since RGS14 was first identified as a Rap binding protein that contains an RGS domain (7
), much of the previous work on this protein has focused on its presumed role as an RGS protein that modulates GPCR/G protein signaling (6
). However, our findings here combined with findings elsewhere (8
) suggest that RGS14 may serve as a GL protein that integrates unconventional Ric-8A/G protein signaling with Ras/Raf/MAP kinase signaling (7
). These findings provide a framework for a working model (Fig. S2
) to describe how these proteins and the functionally opposed RGS and GL domains work together to bind and modulate the functions of Ric-8A, inactive Gαi-GDP, and active Gαi-GTP. Our proposed model highlights the GL domain as the first point of contact between Gαi and RGS14 rather than the RGS domain. In its basal resting state, RGS14 exists in a stable complex with Gαi1-GDP at the cell membrane. We postulate that following a signaling event (as yet undefined), Ric-8A recognizes the RGS14:Gαi1-GDP complex to stimulate nucleotide exchange and GTP binding to Gαi1, which then promotes dissociation of RGS14 (because the GL domain does not bind Gα-GTP). Of note, a role for a GPCR in this activation step cannot be ruled out. Once free from Gαi1, RGS14 would be available to act on other downstream binding partners (e.g
. active H-Ras and Raf kinases to modulate MAP kinase signaling) (7
). In this model, we envision that the lifetime of this newly-formed RGS14 signaling complex is limited by the RGS domain, which acts on nearby Gαi1-GTP to restore Gαi1-GDP and to promote reformation of the Gαi1-GDP:GL-RGS14 complex. This event is coupled with dissociation of RGS14 from its binding partners and a return to the basal resting state. An attractive feature of this model is that the structural configuration of RGS14 that incorporates both the RGS domain and GL domain into the same protein could serve to spatially restrict the function of the RGS domain towards the pre-bound Gα, thus eliminating the need for strict intrinsic RGS/Gα selectivity (i.e. even though the RGS domain is capable of acting on other Gα, it will only act on the one that is nearby). This idea is consistent with earlier observations that the RGS domain is a non-selective GAP for Gαi/o (6
)), while the GL domain is specific for Gαi1 and Gαi3 (11
). This proposed activation/deactivation cycle (Fig. S2
) is entirely consistent with our findings here and with previous findings (8
), and future studies will examine untested steps in this model.
RGS14 and Ric-8A are brain proteins important for hippocampal functions
We find that native RGS14 and Ric-8A co-exist and co-localize within the same neurons of the CA2 and CA1 sub-regions of the hippocampus (). These findings highlight the likelihood for functional interplay between Ric-8A and RGS14 in hippocampal signaling pathways. Our findings here and those in previous reports (33
) indicate that Ric-8A is widely expressed in brain, including but not limited to those hippocampal neurons that contain RGS14. Thus, Ric-8A must also serve roles in addition to regulation of the RGS14:Gαi1-GDP signaling complex. In this regard, LGN/mPins, AGS3, and other proteins that contain GL domains are also highly enriched in various brain regions (39
). Furthermore, we observe via size-exclusion chromatography that most of the Ric-8A in soluble brain lysates exists as an uncomplexed monomer (data not shown). Therefore, it is possible that Ric-8A acts as a master regulator of multiple GL:Gαi-GDP signaling complexes involved with brain signaling. Consistent with this idea, both LGN/mPins and AGS3 have each been reported to serve important roles in synaptic plasticity in brain (15
). Genetic deletion of Ric-8A is reported to alter hippocampal learning behavior (32
). Of particular relevance to these reports and our findings here, we observe that RGS14 is expressed almost exclusively in CA2 neurons of mouse hippocampus and that genetic deletion of RGS14 in mouse brain results in animals with a targeted enhancement of hippocampal-based learning and memory and synaptic plasticity in CA2 neurons (34
). These studies, combined with our results here and other reports showing that the RGS14 binding partners H-Ras, Rap2 and Raf-1 are also important for hippocampal learning and memory (43
) strongly suggest that RGS14 is a newly appreciated multifunctional GL and RGS protein that integrates unconventional Ric-8A/Gαi and MAP kinase signaling pathways important for hippocampal cognitive processing.