Mittal V and Linder ME, J Biol Chem. 2004 Nov 5;279(45):46772-8. Epub 2004 Aug 26.
Regulators of G protein signaling (RGS) modulate G protein activity by functioning as GTPase-activating proteins (GAPs) for alpha-subunits of heterotrimeric G proteins. RGS14 regulates G protein nucleotide exchange and hydrolysis by acting as a GAP through its R
GS domain and as a guanine nucleotide dissociation inhibitor (GDI) through its GoLoco motif. RGS14 exerts GDI activity on Galphai1, but not Galphao. Selective interactions are mediated by contacts between the alphaA and alphaB helices of the Galphai1 helical domain and the GoLoco C terminus (Kimple, R. J., Kimple, M. E., Betts, L., Sondek, J., and Siderovski, D. P. (2002) Nature 416, 878-881). Three isoforms of Galphai exist in mammalian cells. In this study, we tested whether all three isoforms were subject to RGS14 GDI activity. We found that RGS14 inhibits guanine nucleotide exchange on Galphai1 and Galphai3 could, but not Galphai2. Galphai2 be rendered sensitive to RGS14 GDI activity by replacement of residues within the alpha-helical domain. In addition to the contact residues in the alphaA and alphaB helices previously identified, we found that the alphaA/alphaB and alphaB/alphaC loops are important determinants of Galphai selectivity. The striking selectivity observed for RGS14 GDI activity in vitro points to Galphai1 and Galphai3 as the likely targets of RGS14-GoLoco regulation in vivo.
Snow BE, etal., Biochem Biophys Res Commun 1997 Apr 28;233(3):770-7.
We report the cloning of two novel rat regulators of G-protein signaling (RGS) cDNAs using a degenerate PCR strategy. The rRgs12 and rRgs14 cDNAs encode predicted polypeptides of 1387 and 544 amino acids, respectively. We have also identified the human orthologu
e of rRgs12 by alignment of cosmid sequences in the database which map the human RGS12 gene to chromosome 4p16.3. Furthermore, we identified human ESTs with high homology to rRgs14 which map to human chromosome 5qter. Northern blot analysis indicates that rRgs14 is expressed at high levels in brain, lung, and spleen, whereas rRgs12 is expressed at high levels in brain and lung and lower levels in testis, heart, and spleen. Analysis of the predicted rRGS12 and rRGS14 polypeptides indicates that they are closely related and possess regions of homology outside of the conserved RGS domain. We have also identified conserved regions in RGS12 which are similar to protein domains found in mouse rhophilin and coiled-coil proteins suggesting possible interactions with ras-like G-proteins.
BACKGROUND: Regulator of G-protein signaling (RGS) proteins have been well-described as accelerators of Galpha-mediated GTP hydrolysis ("GTPase-accelerating proteins" or GAPs). However, RGS proteins with complex domain architectures are now known to regulate much more than Galpha GTPase activity. ... (more)
pan style='font-weight:700;'>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 FINDINGS: Full-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/SIGNIFICANCE: In 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.
Kimple RJ, etal., J Biol Chem. 2001 Aug 3;276(31):29275-81. Epub 2001 May 31.
The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein alpha subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain
not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single G alpha(i/o)-Loco (GoLoco) motif predicted to represent a second G alpha interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with G alpha(i1), G alpha(i2), and G alpha(i3) in their GDP-bound forms. In GTP gamma S binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by G alpha(i1). Both regions also stabilize G alpha(i1) in its GDP-bound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF(4)(-). Our results indicate that both RGS12 and RGS14 harbor two distinctly different G alpha interaction sites: a previously recognized N-terminal RGS box possessing G alpha(i/o) GAP activity and a C-terminal GoLoco region exhibiting G alpha(i) GDI activity. The presence of two, independent G alpha interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple G alpha GAP activity.
MAPkinase signalling is essential for cell growth, differentiation and cell physiology. G proteins and tyrosine kinase receptors each modulate MAPkinase signalling through distinct pathways. We report here that RGS14 is an integrator of G protein and MAPKinase
signalling pathways. RGS14 contains a GPR/GoLoco (GL) domain that forms a stable complex with inactive Gialpha1/3-GDP, and a tandem (R1, R2) Ras binding domain (RBD). We find that RGS14 binds and regulates the subcellular localization and activities of H-Ras and Raf kinases in cells. Activated H-Ras binds RGS14 at the R1 RBD to form a stable complex at cell membranes. RGS14 also co-localizes with and forms a complex with Raf kinases in cells. The regulatory region of Raf-1 binds the RBD region of RGS14, and H-Ras and Raf each facilitate one another's binding to RGS14. RGS14 selectively inhibits PDGF-, but not EGF- or serum-stimulated Erk phosphorylation. This inhibition is dependent on H-Ras binding to RGS14 and is reversed by co-expression of Gialpha1, which binds and recruits RGS14 to the plasma membrane. Gialpha1 binding to RGS14 inhibits Raf binding, indicating that Gialpha1 and Raf binding to RGS14 are mutually exclusive. Taken together, these findings indicate that RGS14 is a newly appreciated integrator of G protein and Ras/Raf signalling pathways.
RGS14 is a multifunctional protein that contains an RGS domain, which binds active Gi/o alpha-GTP, a GoLoco/GPR domain, which binds inactive Gi alpha-GDP, and a tandem Rap1/2 binding domain (RBD). Studies were initiated to determine the roles of these domains an
d their interactions with Gi alpha on RGS14 subcellular localization. We report that RGS14 dynamic subcellular localization in HeLa cells depends on distinct domains and selective interactions with preferred Gi alpha isoforms. RGS14 shuttles rapidly between the nucleus and cytoplasm, and associates with centrosomes during interphase and mitosis. RGS14 localization to the nucleus depends on the RGS and RBD domains, its translocation out of the nucleus depends on the GoLoco/GPR domain, and its localization to centrosomes depends on the RBD domain. Gi alpha subunits (Gi alpha1, 2 and 3) localize predominantly at the plasma membrane. RGS14 binds directly to inactive and active forms of Gi alpha1 and Gi alpha3, but not Gi alpha2, both as a purified protein and when recovered from cells. RGS14 localizes predominantly at the plasma membrane in cells with inactive Gi alpha1 and Gi alpha3, but not Gi alpha2, whereas less RGS14 associates with active Gi alpha1/3 at the plasma membrane. RGS14 binding to inactive, but not active Gi alpha1/3 also prevents association with centrosomes or nuclear localization. Removal or functional inactivation of the GoLoco/GPR domain causes RGS14 to accumulate at centrosomes and in the nucleus, but renders it insensitive to recruitment to the plasma membrane by Gi alpha1/3. These findings highlight the importance of the GoLoco/GPR domain and its interactions with Gi alpha1/3 in determining RGS14 subcellular localization and linked functions.
