Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2005;1(2):51-66.
doi: 10.7150/ijbs.1.51. Epub 2005 Apr 1.

The GAPs, GEFs, and GDIs of heterotrimeric G-protein alpha subunits

David P Siderovski  1 , Francis S Willard
Affiliations
Review

The GAPs, GEFs, and GDIs of heterotrimeric G-protein alpha subunits

David P Siderovski et al. Int J Biol Sci. 2005.

Abstract

The heterotrimeric G-protein alpha subunit has long been considered a bimodal, GTP-hydrolyzing switch controlling the duration of signal transduction by seven-transmembrane domain (7TM) cell-surface receptors. In 1996, we and others identified a superfamily of "regulator of G-protein signaling" (RGS) proteins that accelerate the rate of GTP hydrolysis by Galpha subunits (dubbed GTPase-accelerating protein or "GAP" activity). This discovery resolved the paradox between the rapid physiological timing seen for 7TM receptor signal transduction in vivo and the slow rates of GTP hydrolysis exhibited by purified Galpha subunits in vitro. Here, we review more recent discoveries that have highlighted newly-appreciated roles for RGS proteins beyond mere negative regulators of 7TM signaling. These new roles include the RGS-box-containing, RhoA-specific guanine nucleotide exchange factors (RGS-RhoGEFs) that serve as Galpha effectors to couple 7TM and semaphorin receptor signaling to RhoA activation, the potential for RGS12 to serve as a nexus for signaling from tyrosine kinases and G-proteins of both the Galpha and Ras-superfamilies, the potential for R7-subfamily RGS proteins to couple Galpha subunits to 7TM receptors in the absence of conventional Gbetagamma dimers, and the potential for the conjoint 7TM/RGS-box Arabidopsis protein AtRGS1 to serve as a ligand-operated GAP for the plant Galpha AtGPA1. Moreover, we review the discovery of novel biochemical activities that also impinge on the guanine nucleotide binding and hydrolysis cycle of Galpha subunits: namely, the guanine nucleotide dissociation inhibitor (GDI) activity of the GoLoco motif-containing proteins and the 7TM receptor-independent guanine nucleotide exchange factor (GEF) activity of Ric8/synembryn. Discovery of these novel GAP, GDI, and GEF activities have helped to illuminate a new role for Galpha subunit GDP/GTP cycling required for microtubule force generation and mitotic spindle function in chromosomal segregation.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest: The authors declare that no conflict of interest exists.

Figures

Figure 1
Figure 1
Standard model of the guanine nucleotide cycle governing 7TM receptor-mediated activation of heterotrimeric G protein-coupled signaling. The Gβγ heterodimer serves to couple Gα to the receptor and also to inhibit its spontaneous release of GDP (i.e., acting as a guanine nucleotide dissociation inhibitor or “GDI” for Gα 17, 18). Ligand-occupied, 7TM cell-surface receptors stimulate signal onset by acting as guanine nucleotide exchange factors (GEFs) for Gα subunits, facilitating GDP release, subsequent binding of GTP, and release of the Gβγ dimer. Both the GTP-bound Gα and liberated Gβγ moieties are then able to modulate the activity of various enzymes, ion channels, and other effectors. Regulator of G-protein signaling (RGS) proteins stimulate signal termination by acting as GTPase-accelerating proteins (GAPs) for Gα, dramatically enhancing their intrinsic rate of GTP hydrolysis.
Figure 2
Figure 2
Multidomain architecture of representative members from all subfamilies of the mammalian RGS protein superfamily. Two alternative nomenclatures have been proposed for several RGS protein subfamilies , . RZ- or A-subfamily members such as RGS17 are characterized by an N-terminal poly-cysteine region (“Cys”) thought to be reversibly palmitoylated . R4- or B-subfamily members include RGS2 and RGS21 that are described in the text. RGS11 was the first member of the R7- or C-subfamily to be shown to bind Gβ5 via its Gγ-like or “GGL” domain . Of the three members of the R12- or D-subfamily (RGS10, RGS12, RGS14), RGS10 is the smallest and comprises little more than an RGS-box , whereas both RGS12 and RGS14 have tandem Ras-binding domains (RBDs) and a C-terminal Gαi/o-Loco interaction (GoLoco) motif , and RGS12 additionally has N-terminal PDZ (PSD95/Dlg/ZO-1 homology) and PTB (phosphotyrosine-binding) domains . Axin and Axin2 (a.k.a. Axil) are negative regulators of the Wnt signaling pathway and comprise the RA- or E-subfamily; neither protein has been shown to interact with Gα subunits, but rather interact with the tumor suppressor protein adenomatous polyposis coli (APC) using the RGS-box . Axin and Axil also contain other domains that interact with β-catenin, the kinase GSK3β, the phosphatase PP2A, and the protein Dishevelled (“DIX” domain) . The GEF- or F-subfamily includes three RhoA-specific guanine nucleotide exchange factors (“GEFs”) with canonical Dbl-homology (DH) and pleckstrin-homology (PH) domains: p115-RhoGEF, PDZ-RhoGEF, and leukemia-associated RhoGEF (LARG); the latter two RhoGEFs each possess an N-terminal PDZ domain, as described in the text. In 1996, we were the first group to identify an N-terminal RGS-box within each member of the G protein-coupled receptor kinase family (known as the GRK- or G-subfamily in the context of the RGS protein superfamily). At least three sorting nexins (SNX13, SNX14, SNX25) have RGS-boxes between phosphatidylinositol-binding (PX) and PX-associated (PXA) domains and thus comprise the SNX- or H-subfamily of RGS proteins. Zheng and colleagues reported that SNX13 (a.k.a. “RGS-PX1”) could act as a GAP for the adenylyl-cyclase-stimulatory isoform of Gα (Gαs) ; however, this report has yet to be confirmed in the literature. TM, putative transmembrane regions. The multiple RGS-box family members D-AKAP2 and RGS22 fall outside the eight established subfamilies; the superscript designations of their RGS-boxes match that used in Figure 3.
Figure 3
Figure 3
Relationship between RGS-box sequences of all 37 human RGS proteins identified to date. Unrooted dendrogram was generated by Clustal-W and TreeView using sequences identified by the SMART profile for RGS-boxes as well as those identified by protein-fold recognition algorithms . Subfamily designations and identification of isolated RGS-box sequences from multi-RGS-containing proteins D-AKAP2 and RGS22 are as described for Figure 2. Note that there is no RGS15, contrary to an early report .
Figure 4
Figure 4
Membrane targeting strategies employed by multi-domain RGS proteins. (A) The R7 RGS proteins form obligate heterodimers with Gβ5 via a Gγ-like sequence (the “GGL” domain) N-terminal to the RGS-box . This GGL/Gβ5 interaction could allow R7 RGS proteins to act as conventional Gβγ subunits in coupling Gα subunits to 7TM receptors, thereby localizing RGS-box-mediated GAP activity to particular receptors . The DEP domain of RGS9-1 interacts with a membrane-anchoring protein (R9AP) ; analogous interactors may exist for the DEP domains of other R7 subfamily members . (B) The PDZ domain of RGS12 is able to bind the C-terminus of the IL-8 receptor CXCR2 (at least in vitro) . The RGS12 PTB domain binds the synprint (“synaptic protein interaction”) region of the N-type calcium channel (Cav2.2); this interaction is dependent on neurotransmitter-mediated phosphorylation of the channel by Src . (C) The AtRGS1 protein of Arabidopsis thaliana (thale cress) has a unique structure for an RGS protein: an N-terminus resembling a 7TM receptor and a C-terminal RGS-box . Although a ligand is not known for the 7TM portion of AtRGS1, a simple sugar is most likely . (D) The transmembrane receptor Plexin-B1 couples binding of the membrane-bound semaphorin Sema4D to RhoA activation via an interaction with the PDZ domain of PDZ-RhoGEF (and of the related RGS-RhoGEF LARG) . Domain abbreviations : IPT, immunoglobulin-like fold found in plexins, Met and Ron tyrosine kinase receptors, and intracellular transcription factors; PSI, domain found in plexins, semaphorins, and integrins; Sema, semaphorin domain.
Figure 5
Figure 5
The GoLoco motif, a Gαi/o-interacting polypeptide found singly or in arrays in various proteins, binds across the face of Gα. (A) Alignment of the highly-conserved, 19 amino-acid GoLoco motif signatures from human RGS12 and RGS14 is colored according to side-chain chemistry using Clustal-X defaults . Consensus symbols for amino-acid character are hyphen (-), acidic; Φ, hydrophobic; Ψ, large aliphatic; (+), basic; and π, small side chain. Domain abbreviations: RapGAP, Rap-specific GTPase-activating protein domain. (B) Our structure of Gαi1 (green with translucent space-filling shell; switch regions in blue) bound to a 36-amino acid peptide derived from the GoLoco motif-region of RGS14 . The GoLoco peptide (yellow) binds across both Ras-like and all α-helical lobes within Gαi1, trapping GDP (brown) within its binding site. Note the relative position of the GoLoco triad's arginine “finger” (yellow in ball-and-stick representation) reaching into the GDP binding site. Gαi1-specific contacts from the α-helical lobe to the C-terminal portion of the GoLoco peptide are denoted in magenta.
Figure 6
Figure 6
Relationship between GoLoco motif sequences from all GoLoco proteins identified to date. Unrooted dendrogram was generated by Clustal-W and TreeView using GoLoco motif sequences originally published in our comprehensive review of the GoLoco motif . The individual GoLoco motifs from tandem arrays (Fig. 5A) are numbered GL1 to GL4 starting from the first N-terminal motif. Species abbreviations: a, Anopheles gambiae (mosquito); Cb, Caenorhabditis briggsae; Ce, Caenorhabditis elegans; d, Drosophila; f, Fugu rubripes; h, human; m, mouse; r, rat; z, Danio rerio (zebrafish). Note that there are two LGN-like proteins encoded by the genome of Fugu rubripes; we have denoted the one more closely-related to mammalian LGN/GPSM2 as fGPSM2 (Ensembl translation ID SINFRUP00000160450) and the more distantly-related protein as fLGN2 (Ensembl SINFRUP00000146023). Two conclusions can immediately be drawn from this analysis: (i) Drosophila Pins, which has only three GoLoco motifs (Fig. 8) vs the four motifs of GPSM1 and GPSM2 proteins, has lost the second motif, since dPins GL2 is most similar to GL3 of GPSM1/2 proteins, and dPins GL3 is most similar to GL4 of GPSM1/2 proteins; (ii) the triple GoLoco motif protein GPSM3 (a.k.a. G18) is not directly related to the tandem GoLoco arrays of GPSM1/2 proteins (i.e., GPSM3 is not merely missing the N-terminal TPR region of GPSM1/2).
Figure 7
Figure 7
Experimental paradigm used to demonstrate that GoLoco motif-derived peptides can uncouple heterotrimeric G-protein signaling, but have no intrinsic ability to directly activate Gβγ-dependent signaling. (A) The dopamine agonist quinpirole, when applied to AtT-20 cells stably or transiently expressing the D2-dopamine receptor , activates Gi-heterotrimers and elicits potassium current via the Kir3.1/3.2 channel (as activated by free Gβγ). Upon GTP hydrolysis by Gα, the heterotrimer of Gα·GDP and Gβγ subunits can reform, restoring the “coupled” receptor/G-protein complex. However, in the presence of GoLoco motif-derived peptides, Gα can become trapped in a Gα·GDP/GoLoco complex, preventing Gβγ reassociation and re-coupling to the 7TM receptor. (B) Sample electrophysiological data in support of the model presented in part (A). AtT-20 cells under whole-cell patch-clamp configuration to measure potassium currents were treated with repeated quinpirole pulses (orange circles) or buffer (open circles) after loading with 10 μM RGS12 GoLoco peptide (left panel) or control scrambled peptide (right panel).
Figure 8
Figure 8
Known interactions between apical polarity proteins PAR3, PAR6, atypical PKC (aPKC), and Drosophila Inscuteable (dInsc), Partner of Inscuteable (Pins), and Gαi in Drosophila neuroblast asymmetric cell division. ARM, homology to Armadillo repeats. Red circles, TPR motifs; yellow rectangles, GoLoco motifs.
Figure 9
Figure 9
Alignment of C. elegans GPR-1 and GPR-2 protein sequences. Although individual tetratricopeptide repeats (TPRs) are not easily identified within the N-termini of GPR-1 and GPR-2 (as they are within mammalian and Drosophila homologs; Figs. 5A & 8), amino-acids 14 to 341 (boxed in grey) are predicted by protein-fold recognition algorithms (e.g., ref. 36) to fold into a right-handed alpha-alpha superhelix (SCOP identifier d1ld8a_; expect value 5 x 10-5) similar to folds exhibited by TPR-containing proteins . Also boxed is the conserved GoLoco motif signature (amino-acids 425-442) and its consensus as defined in . Consensus symbols for amino-acid character are hyphen (-), acidic; Φ, hydrophobic; Ψ, large aliphatic; (+), basic; and π, small side chain.
Figure 10
Figure 10
A. Summary of data implicating Gα and its regulators in generation of asymmetric anterior/posterior forces acting on the mitotic spindle of one-cell C. elegans zygotes during ACD (reviewed in 91). B. Model of a novel Gα nucleotide cycle incorporating the data of part A. RGS-7 contains N-terminal homology to conserved region 2 (“C2”) of protein kinase C and an RGS-box presumed in the model to exert GAP activity. Force arrow denotes speculation that, at least in the nematode, the GTP-bound form of GOA-1/GPA-16 interacts, directly or indirectly, with astral MTs to generate force on the mitotic spindle.
Figure 11
Figure 11
Predicted effects of RIC-8 and RGS-7 on the Gα nucleotide cycle underlying MT force generation in ACD. A. Steady-state GTP hydrolysis of Gα subunits is known to be limited by nucleotide exchange (kex), not by the intrinsic GTPase rate (kcat) , . B. RGS‑7 GAP activity, while by definition accelerating the intrinsic GTPase rate of Gα (kcat' > kcat), will have no effect on steady-state GTP hydrolysis, as the limiting rate for isolated Gα subunits remains GDP release, a key component of kex , . C. RIC-8 GEF activity should accelerate nucleotide exchange (kex' > kex) and increase observed rate of steady-state GTP hydrolysis (up to the new limiting rate of kcatif kex' > kcat). D. Addition of both RIC-8 GEF and RGS-7 GAP activities should dramatically accelerate steady-state GTP hydrolysis vs the basal condition in (A), up to whichever component rate is limiting (kex'or kcat'). E. A graphical representation of predicted steady state GTP hydrolysis by Gα in the presence of these two novel G-protein cycle components. The ordinate represents the amount of inorganic phosphate (Pi) released, and the abscissa represents time.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Gilman AG. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615–49. - PubMed
    1. Hamm HE. The many faces of G protein signaling. J Biol Chem. 1998;273(2):669–72. - PubMed
    1. Offermanns S. G-proteins as transducers in transmembrane signalling. Prog Biophys Mol Biol. 2003;83(2):101–30. - PubMed
    1. De Vries L. et al. GAIP, a protein that specifically interacts with the trimeric G protein G alpha i3, is a member of a protein family with a highly conserved core domain. Proc Natl Acad Sci U S A. 1995;92(25):11916–20. - PMC - PubMed
    1. Dohlman HG. et al. Sst2, a negative regulator of pheromone signaling in the yeast Saccharomyces cerevisiae: expression, localization, and genetic interaction and physical association with Gpa1 (the G-protein alpha subunit) Mol Cell Biol. 1996;16(9):5194–209. - PMC - PubMed

MeSH terms

Substances