In vitro selection of state-specific peptide modulators of G protein signaling using mRNA display

The G protein regulatory (GPR) motif is a approximately 20-residue conserved domain that acts as a guanine dissociation inhibitor (GDI) for G(i/o)(alpha) subunits. Here, we describe the isolation of peptides derived from a GPR consensus sequence using mRNA display selection libraries. Biotinylated G(i)(alpha)(1), modified at either the N or C terminus, serves as a high-affinity binding target for mRNA-displayed GPR peptides. In vitro selection using mRNA display libraries based on the C terminus of the GPR motif revealed novel peptide sequences with conserved residues. Surprisingly, selected peptides contain mutations to a highly conserved Arg in the GPR motif, previously shown to be crucial for binding and inhibition activities. The dominant peptide from the selection, R6A, and a minimal 9-mer peptide, R6A-1, do not contain Arg residues yet retain high affinity (K(D) = 60 and 200 nM, respectively) and specificity for the GDP-bound state of G(i)(alpha)(1), as measured by surface plasmon resonance. The selected peptides also maintain GDI activity for G(i)(alpha)(1), inhibiting both the exchange of GDP in GTPgammaS binding assays and the AlF(4)(-)-stimulated enhancement of intrinsic tryptophan fluorescence. The kinetics of GDI activity, however, are different for the selected peptides and demonstrate biphasic kinetics, suggesting a complex mechanism for inhibition. Like the GPR motif, the R6A and R6A-1 peptides compete with G(betagamma) subunits for binding to G(i)(alpha)(1), suggesting their use as activators of G(betagamma) signaling.     

The R6A-1 peptide binds to switch II of Galphai1 but is not a GDP-dissociation inhibitor

Heterotrimeric G-proteins are molecular switches that convert signals from membrane receptors into changes in intracellular physiology. Recently, several peptides that bind heterotrimeric G-protein alpha subunits have been isolated including the novel Galpha(i1).GDP binding peptides R6A and KB-752. The R6A peptide and its minimized derivative R6A-1 interact with Galpha(i1).GDP. Based on spectroscopic analysis of BODIPYFL-GTPgammaS binding to Galpha(i1), it has been reported that R6A-1 has guanine nucleotide dissociation inhibitor (GDI) activity against Galpha(i1) [W.W. Ja, R.W. Roberts, Biochemistry 43 (28) (2004) 9265-9275]. Using radioligand binding, we show that R6A-1 is not a GDI for Galpha(i1) subunits. Furthermore, we demonstrate that R6A-1 reduces the fluorescence quantum yield of the Galpha(i1)-BODIPYFL-GTPgammaS complex, thus explaining the previously reported GDI activity as a fluorescence artifact. We further show that R6A-1 has significant sequence similarity to the guanine nucleotide exchange factor peptide KB-752 that binds to switch II of Galpha(i1). We use competitive binding analysis to show that R6A-1 also binds to switch II of Galpha subunits.     

Identification of structural features in the G-protein regulatory motif required for regulation of heterotrimeric G-proteins.

The G-protein regulatory (GPR) motif, a conserved 25-30 amino acid domain found in multiple mammalian proteins, stabilizes the GDP-bound conformation of Galpha(i), inhibits guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding to Galpha(i) and competes for Gbetagamma binding to Galpha. To define the core GPR motif and key amino acid residues within a GPR peptide (TMGEEDFFDLLAKSQSKRMDDQRVDLAG), we determined the effect of truncation, insertion, and alanine substitutions on peptide-mediated inhibition of GTPgammaS binding to purified Galpha(i1). The bioactive core GPR peptide consists of 17 amino acids ((7)F-R(23)). Within this core motif, two hydrophobic sectors ((7)FF(8) and (10)LL(11)) and Q(22) are required for bioactivity, whereas M19A and R23A increased IC(50) values by 70-fold. Disruption of spatial relationships between the required sectors in the amino and carboxyl regions of the peptide also resulted in a loss of biological activity. Mutation of three charged sectors ((4)EED(6), R(18), (20)DD(21)) within the 28-amino acid GPR decreased peptide affinity by approximately 10-fold. Alanine substitutions of selected residues within the core GPR peptide differently influenced peptide inhibition of GTPgammaS binding to Galpha(i) versus Galpha(o). These data provide a platform for the development of novel, G-protein-selective therapeutics that inhibit Galpha(i)- mediated signaling, selectively activate Gbetagamma-sensitive effectors, and/or disrupt specific regulatory input to G-proteins mediated by GPR-containing proteins.     

Interaction of activator of G-protein signaling 3 (AGS3) with LKB1, a serine/threonine kinase involved in cell polarity and cell cycle progression: phosphorylation of the G-protein regulatory (GPR) motif as a regulatory mechanism for the interaction of GPR motifs with Gi alpha

Activator of G-protein signaling 3 (AGS3) has a modular domain structure consisting of seven tetratricopeptide repeats (TPRs) and four G-protein regulatory (GPR) motifs. Each GPR motif binds to the alpha subunit of Gi/Go (Gialpha > Goalpha) stabilizing the GDP-bound conformation of Galpha and apparently competing with Gbetagamma for GalphaGDP binding. As an initial approach to identify regulatory mechanisms for AGS3-G-protein interactions, a yeast two-hybrid screen was initiated using the TPR and linker region of AGS3 as bait. This screen identified the serine/threonine kinase LKB1, which is involved in the regulation of cell cycle progression and polarity. Protein interaction assays in mammalian systems using transfected cells or brain lysate indicated the regulated formation of a protein complex consisting of LKB1, AGS3, and G-proteins. The interaction between AGS3 and LKB1 was also observed with orthologous proteins in Drosophila where both proteins are involved in cell polarity. LKB1 immunoprecipitates from COS7 cells transfected with LKB1 phosphorylated the GPR domains of AGS3 and the related protein LGN but not the AGS3-TPR domain. GPR domain phosphorylation was completely blocked by a consensus GPR motif peptide, and placement of a phosphate moiety within a consensus GPR motif reduced the ability of the peptide to interact with G-proteins. These data suggest that phosphorylation of GPR domains may be a general mechanism regulating the interaction of GPR-containing proteins with G-proteins. Such a mechanism may be of particular note in regard to localized signal processing in the plasma membrane involving G-protein subunits and/or intracellular functions regulated by heterotrimeric G-proteins that occur independently of a typical G-protein-coupled receptor.     

Thermodynamic characterization of the binding of activator of G protein signaling 3 (AGS3) and peptides derived from AGS3 with G alpha i1

Activator of G protein signaling 3 (AGS3) is a guanine nucleotide dissociation inhibitor (GDI) that contains four G protein regulatory (GPR) or GoLoco motifs in its C-terminal domain. The entire C-terminal domain (AGS3-C) as well as certain peptides corresponding to individual GPR motifs of AGS3 bound to G alpha i1 and inhibited the binding of GTP by stabilizing the GDP-bound conformation of G alpha i1. The stoichiometry, free energy, enthalpy, and dissociation constant for binding of AGS3-C to G alpha i1 were determined using isothermal titration calorimetry. AGS3-C possesses two apparent high affinity (Kd approximately 20 nm) and two apparent low affinity (Kd approximately 300 nm) binding sites for G alpha i1. Upon deletion of the C-terminal GPR motif from AGS3-C, the remaining sites were approximately equivalent with respect to their affinity (Kd approximately 400 nm) for G alpha i1. Peptides corresponding to each of the four GPR motifs of AGS3 (referred to as GPR1, GPR2, GPR3, and GPR4, respectively, going from N to C terminus) bound to G alpha i1 with Kd values in the range of 1-8 microm. Although GPR1, GPR2, and GPR4 inhibited the binding of the fluorescent GTP analog BODIPY-FL-guanosine 5'-3-O-(thio)triphosphate to G alpha i1, GPR3 did not. However, addition of N- and C-terminal flanking residues to the GPR3 GoLoco core increased its affinity for G alpha i1 and conferred GDI activity similar to that of AGS3-C itself. Similar increases were observed for extended GPR2 and extended GPR1 peptides. Thus, while the tertiary structure of AGS3 may affect the affinity and activity of the GPR motifs contained within its sequence, residues outside of the GPR motifs strongly potentiate their binding and GDI activity toward G alpha i1 even though the amino acid sequences of these residues are not conserved among the GPR repeats.     

Differential G-alpha interaction capacities of the GoLoco motifs in Rap GTPase activating proteins

GoLoco motif proteins act as guanine nucleotide dissociation inhibitors (GDIs) for G-protein alpha subunits of the adenylyl cyclase-inhibitory (Galpha(i/o)) class. Rap1GAP2 is a newly identified GoLoco motif- and RapGAP domain-containing protein, and thus is considered a potential integrator of heterotrimeric and monomeric GTPase signaling. Primary sequence analysis indicated that the Rap1GAP2 GoLoco motif contains a lysine (Lys-75), rather than an arginine, at the crucial residue responsible for binding the alpha and beta phosphates of GDP and exerting GDI activity. To determine the functional outcome of this sequence variation we conducted a biophysical analysis of the human Rap1GAP2b/c GoLoco motif. We found that human Rap1GAP2b/c was deficient in GDI activity and Galpha interaction capability. Mutation of lysine-75 to arginine could not regain functional activity of the Rap1GAP2b/c GoLoco motif. Thus, the Rap1GAP2b/c GoLoco motif can be classed as inactive towards Galpha subunits. We also found that the Rap1GAP1a GoLoco motif, which lacks seven N-terminal amino acid residues present in canonical GoLoco motifs, does not interact with Galpha(i1). In contrast, the GoLoco motif of Rap1GAP1b, which is canonical in primary sequence, was found to interact with Galpha(i1).GDP.     

Stabilization of the GDP-bound conformation of Gialpha by a peptide derived from the G-protein regulatory motif of AGS3

The G-protein regulatory (GPR) motif in AGS3 was recently identified as a region for protein binding to heterotrimeric G-protein alpha subunits. To define the properties of this approximately 20-amino acid motif, we designed a GPR consensus peptide and determined its influence on the activation state of G-protein and receptor coupling to G-protein. The GPR peptide sequence (28 amino acids) encompassed the consensus sequence defined by the four GPR motifs conserved in the family of AGS3 proteins. The GPR consensus peptide effectively prevented the binding of AGS3 to Gialpha1,2 in protein interaction assays, inhibited guanosine 5'-O-(3-thiotriphosphate) binding to Gialpha, and stabilized the GDP-bound conformation of Gialpha. The GPR peptide had little effect on nucleotide binding to Goalpha and brain G-protein indicating selective regulation of Gialpha. Thus, the GPR peptide functions as a guanine nucleotide dissociation inhibitor for Gialpha. The GPR consensus peptide also blocked receptor coupling to Gialphabetagamma indicating that although the AGS3-GPR peptide stabilized the GDP-bound conformation of Gialpha, this conformation of Gialpha(GDP) was not recognized by a G-protein coupled receptor. The AGS3-GPR motif presents an opportunity for selective control of Gialpha- and Gbetagamma-regulated effector systems, and the GPR motif allows for alternative modes of signal input to G-protein signaling systems.     

Motion of carboxyl terminus of Galpha is restricted upon G protein activation. A solution NMR study using semisynthetic Galpha subunits

The carboxyl terminus of the G protein alpha subunit plays a key role in interactions with G protein-coupled receptors. Previous studies that have incorporated covalently attached probes have demonstrated that the carboxyl terminus undergoes conformational changes upon G protein activation. To examine the conformational changes that occur at the carboxyl terminus of Galpha subunits upon G protein activation in a more native system, we generated a semisynthetic Galpha subunit, site-specifically labeled in its carboxyl terminus with 13C amino acids. Using expressed protein ligation, 9-mer peptides were ligated to recombinant Galpha(i1) subunits lacking the corresponding carboxyl-terminal residues. In a receptor-G protein reconstitution assay, the truncated Galpha(i1) subunit could not be activated by receptor; whereas the semisynthetic protein demonstrated functionality that was comparable with recombinant Galpha(i1). To study the conformation of the carboxyl terminus of the semisynthetic G protein, we applied high resolution solution NMR to Galpha subunits containing 13C labels at the corresponding sites in Galpha(i1): Leu-348 (uniform), Gly-352 (alpha carbon), and Phe-354 (ring). In the GDP-bound state, the spectra of the ligated carboxyl terminus appeared similar to the spectra obtained for 13C-labeled free peptide. Upon titration with increasing concentrations of AlF4-, the 13C resonances demonstrated a marked loss of signal intensity in the semisynthetic Galpha subunit but not in free peptide subjected to the same conditions. Because AlF4- complexes with GDP to stabilize an activated state of the Galpha subunit, these results suggest that the Galpha carboxyl terminus is highly mobile in its GDP-bound state but adopts an ordered conformation upon activation by AlF4-.     

RGS12 and RGS14 GoLoco motifs are G alpha(i) interaction sites with guanine nucleotide dissociation inhibitor Activity

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.     

Influence of cytosolic AGS3 on receptor--G protein coupling

Activator of G protein signaling 3 (AGS3) activates the Gbetagamma mating pathway in yeast in a manner that is independent of heptahelical receptors. It competes with Gbetagamma subunits to bind GDP-bound Gi/o(alpha) subunits via four repeated G protein regulatory (GPR) domains in the carboxyl-terminal half of the molecule. However, little is known about the functional role of AGS3 in cellular signaling. Here the effect of AGS3 on receptor-G protein coupling was examined in an Sf9 cell membrane-based reconstitution system. A GST-AGS3-GPR fusion protein containing the four individual AGS3-GPR domains inhibits receptor coupling to Galpha subunits as effectively as native AGS3 and more effectively than GST fusion proteins containing the individual AGS3-GPR domains. While none of the GPR domains distinguished among the three G(i)alpha subunits, both individual and full-length GPR domains interacted more weakly with G(o)alpha than with G(i)alpha. Cytosolic AGS3, but not membrane-associated AGS3, can interact with G(i)alpha subunits and disrupt their receptor coupling. Immunoblotting studies reveal that cytosolic AGS3 can remove G(i)alpha subunits from the membrane and sequester G(i)alpha subunits in the cytosol. These findings suggest that AGS3 may downregulate heterotrimeric G protein signaling by interfering with receptor coupling.     

Identification of residues in human neuroglobin crucial for Guanine nucleotide dissociation inhibitor activity

Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. We previously demonstrated that ferric human Ngb binds to the alpha-subunits of heterotrimeric G proteins (Galpha) and acts as a guanine nucleotide dissociation inhibitor (GDI) for Galpha. Here we have investigated the interaction between Ngb and Galpha in more detail. We report that zebrafish Ngb, which shares about 50% amino acid sequence identity with human Ngb, does not have a GDI activity for Galpha. By carrying out exon swapping between zebrafish and human Ngb and site-directed mutagenesis, we have identified several residues that are crucial for the GDI activity of human Ngb.     

Molecular basis of guanine nucleotide dissociation inhibitor activity of human neuroglobin by chemical cross-linking and mass spectrometry

Oxidized human neuroglobin (Ngb), a heme protein expressed in the brain, has been proposed to act as a guanine nucleotide dissociation inhibitor (GDI) for the GDP-bound form of the heterotrimeric G protein alpha-subunit (Galpha(i)). Here, to elucidate the molecular mechanism underlying the GDI activity of Ngb, we used an glutathione-S-transferase pull-down assay to confirm that Ngb competes with G-protein betagamma-subunits (Gbetagamma) for binding to Galpha(i), and identified the Galpha(i)-binding site in Ngb by chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and sulfo-N-hydroxysuccinimide, coupled with mass spectrometry (MS). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis for tryptic peptides derived from the cross-linked Ngb-Galpha(i) complex revealed several binding regions in Ngb. Furthermore, MALDI-TOF/TOF MS analysis of the cross-linked Ngb and Galpha(i) peptides, together with the MS/MS scoring method, predicted cross-linking between Glu60 (Ngb) and Ser206 (Galpha(i)), and between Glu53 (Ngb) and Ser44 (Galpha(i)). Because Ser206 of Galpha(i) is located in the region that contacts Gbetagamma, binding of Ngb could facilitate the release of Gbetagamma from Galpha(i). Binding of Ngb to Galpha(i) would also inhibit the exchange of GDP for GTP, because Ser44 (Galpha(i)) is adjacent to the GDP-binding site and Glu53 (Ngb), which is cross-linked to Ser44 (Galpha(i)), could be located close to GDP. Thus, we have identified, for the first time, the sites of interaction between Ngb and Galpha(i), enabling us to discuss the functional significance of this binding on the GDI activity of Ngb.     

Real-time analysis of G protein-coupled receptor reconstitution in a solubilized system

Receptor based signaling mechanisms are the primary source of cellular regulation. The superfamily of G protein-coupled receptors is the largest and most ubiquitous of the receptor mediated processes. We describe here the analysis in real-time of the assembly and disassembly of soluble G protein-coupled receptor-G protein complexes. A fluorometric method was utilized to determine the dissociation of a fluorescent ligand from the receptor solubilized in detergent. The ligand dissociation rate differs between a receptor coupled to a G protein and the receptor alone. By observing the sensitivity of the dissociation of a fluorescent ligand to the presence of guanine nucleotide, we have shown a time- and concentration-dependent reconstitution of the N-formyl peptide receptor with endogenous G proteins. Furthermore, after the clearing of endogenous G proteins, purified Galpha subunits premixed with bovine brain Gbetagamma subunits were also able to reconstitute with the solubilized receptors. The solubilized N-formyl peptide receptor and Galpha(i3) protein interacted with an affinity of approximately 10(-6) m with other alpha subunits exhibiting lower affinities (Galpha(i3) > Galpha(i2) > Galpha(i1) Galpha(o)). The N-formyl peptide receptor-G protein interactions were inhibited by peptides corresponding to the Galpha(i) C-terminal regions, by Galpha(i) mAbs, and by a truncated form of arrestin-3. This system should prove useful for the analysis of the specificity of receptor-G protein interactions, as well as for the elucidation and characterization of receptor molecular assemblies and signal transduction complexes.     

Kinetics of ternary complex formation with fusion proteins composed of the A(1)-adenosine receptor and G protein alpha-subunits.

High affinity agonist binding to G protein-coupled receptors depends on the formation of a ternary complex between agonist, receptor, and G protein. This process is too slow to be accounted for by a simple diffusion-controlled mechanism. We have tested if the interaction between activated receptor and G protein is rate-limiting by fusing the coding sequence of the human A(1)-adenosine receptor to that of Galpha(i-1) (A(1)/Galpha(i-1)) and of Galpha(o) (A(1)/Galpha(o)). Fusion proteins of the expected molecular mass were detected following transfection of HEK293 cells. Ternary complex formation was monitored by determining the kinetics for binding of the high affinity agonist (-)-N(6)-3[(125)I](iodo-4-hydroxyphenylisopropyl)adenosine; these were similar in the wild-type receptor and the fusion proteins over the temperature range of 10 to 30 degrees C. Agonist dissociation may be limited by the stability of the ternary complex. This assumption was tested by creating fusion proteins in which the Cys(351) of Galpha(i-1) was replaced with glycine (A(1)/Galpha(i-1)C351G) or isoleucine (A(1)/Galpha(i-1)C351I) to lower the affinity of the receptor for the G protein. In these mutated fusion proteins, the dissociation rate of the ternary complex was accelerated; in contrast, the rate of the forward reaction was not affected. We therefore conclude that (i) receptor activation per se rather than its interaction with the G protein is rate-limiting in ternary complex formation; (ii) the stability of the ternary complex is determined by the dissociation rate of the G protein. These features provide for a kinetic proofreading mechanism that sustains the fidelity of receptor-G protein coupling.     

Signal transfer from GPCRs to G proteins: role of the G alpha N-terminal region in rhodopsin-transducin coupling

Catalysis of nucleotide exchange in heterotrimeric G proteins (Galphabetagamma) is a key step in cellular signal transduction mediated by G protein-coupled receptors. The Galpha N terminus with its helical stretch is thought to be crucial for G protein/activated receptor (R(*)) interaction. The N-terminal fatty acylation of Galpha is important for membrane targeting of G proteins. By applying biophysical techniques to the rhodopsin/transducin model system, we studied the effect of N-terminal truncations in Galpha. In Galphabetagamma, lack of the fatty acid and Galpha truncations up to 33 amino acids had little effect on R(*) binding and R(*)-catalyzed nucleotide exchange, implying that this region is not mandatory for R(*)/Galphabetagamma interaction. However, when the other hydrophobic modification of Galphabetagamma, the Ggamma C-terminal farnesyl moiety, is lacking, R(*) interaction requires the fatty acylated Galpha N terminus. This suggests that the two hydrophobic extensions can replace each other in the interaction of Galphabetagamma with R(*). We propose that in native Galphabetagamma, these two terminal regions are functionally redundant and form a microdomain that serves both to anchor the G protein to the membrane and to establish an initial docking complex with R(*). Accordingly, we find that the native fatty acylated Galpha is competent to interact with R(*) even in the absence of Gbetagamma, whereas nonacylated Galpha requires Gbetagamma for interaction. Experiments with N-terminally truncated Galpha subunits suggest that in the second step of the catalytic process, the receptor binds to the alphaN/beta1-loop region of Galpha to reduce nucleotide affinity and to make the Galpha C terminus available for subsequent interaction with R(*).     

G alpha selectivity and inhibitor function of the multiple GoLoco motif protein GPSM2/LGN

GPSM2 (G-protein signalling modulator 2; also known as LGN or mammalian Pins) is a protein that regulates mitotic spindle organization and cell division. GPSM2 contains seven tetratricopeptide repeats (TPR) and four Galpha(i/o)-Loco (GoLoco) motifs. GPSM2 has guanine nucleotide dissociation inhibitor (GDI) activity towards both Galpha(o)- and Galpha(i)-subunits; however, a systematic analysis of its individual GoLoco motifs has not been described. We analyzed each of the four individual GoLoco motifs from GPSM2, assessing their relative binding affinities and GDI potencies for Galpha(i1), Galpha(i2), and Galpha(i3) and Galpha(o). Each of the four GPSM2 GoLoco motifs (36-43 amino acids in length) was expressed in bacteria as a GST-fusion protein and purified to homogeneity. The binding of each of the four GST-GoLoco motifs to Galpha(i1)-, Galpha(o)-, and Galpha(s)-subunits was assessed by surface plasmon resonance; all of the motifs bound Galpha(i1), but exhibited low affinity towards Galpha(o). GDI activity was assessed by a fluorescence-based nucleotide-binding assay, revealing that all four GoLoco motifs are functional as GDIs for Galpha(i1), Galpha(i2), and Galpha(i3). Consistent with our binding studies, the GDI activity of GPSM2 GoLoco motifs on Galpha(o) was significantly lower than that toward Galpha(i1), suggesting that the in vivo targets of GPSM2 are most likely to be Galpha(i)-subunits.     

Integration of G Protein α (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gα_i/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gα_i1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gα_i1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gα_o-AlF₄⁻. Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gα_i1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gα_o-AlF₄⁻ and an AlF₄⁻-insensitive mutant (G42R) of Gα_i1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gα_i1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gα_o-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity.     

A bifunctional Galphai/Galphas modulatory peptide that attenuates adenylyl cyclase activity

Signaling via G-protein coupled receptors is initiated by receptor-catalyzed nucleotide exchange on Galpha subunits normally bound to GDP and Gbetagamma. Activated Galpha . GTP then regulates effectors such as adenylyl cyclase. Except for Gbetagamma, no known regulators bind the adenylyl cyclase-stimulatory subunit Galphas in its GDP-bound state. We recently described a peptide, KB-752, that binds and enhances the nucleotide exchange rate of the adenylyl cyclase-inhibitory subunit Galpha(i). Herein, we report that KB-752 binds Galpha(s) . GDP yet slows its rate of nucleotide exchange. KB-752 inhibits GTPgammaS-stimulated adenylyl cyclase activity in cell membranes, reflecting its opposing effects on nucleotide exchange by Galpha(i) and Galpha(s).     

Ric-8A catalyzes guanine nucleotide exchange on G alphai1 bound to the GPR/GoLoco exchange inhibitor AGS3

Microtubule pulling forces that govern mitotic spindle movement of chromosomes are tightly regulated by G-proteins. A host of proteins, including Galpha subunits, Ric-8, AGS3, regulators of G-protein signalings, and scaffolding proteins, coordinate this vital cellular process. Ric-8A, acting as a guanine nucleotide exchange factor, catalyzes the release of GDP from various Galpha.GDP subunits and forms a stable nucleotide-free Ric-8A:Galpha complex. AGS3, a guanine nucleotide dissociation inhibitor (GDI), binds and stabilizes Galpha subunits in their GDP-bound state. Because Ric-8A and AGS3 may recognize and compete for Galpha.GDP in this pathway, we probed the interactions of a truncated AGS3 (AGS3-C; containing only the residues responsible for GDI activity), with Ric-8A:Galpha(il) and that of Ric-8A with the AGS3-C:Galpha(il).GDP complex. Pulldown assays, gel filtration, isothermal titration calorimetry, and rapid mixing stopped-flow fluorescence spectroscopy indicate that Ric-8A catalyzes the rapid release of GDP from AGS3-C:Galpha(i1).GDP. Thus, Ric-8A forms a transient ternary complex with AGS3-C:Galpha(i1).GDP. Subsequent dissociation of AGS3-C and GDP from Galpha(i1) yields a stable nucleotide free Ric-8A.Galpha(i1) complex that, in the presence of GTP, dissociates to yield Ric-8A and Galpha(i1).GTP. AGS3-C does not induce dissociation of the Ric-8A.Galpha(i1) complex, even when present at very high concentrations. The action of Ric-8A on AGS3:Galpha(i1).GDP ensures unidirectional activation of Galpha subunits that cannot be reversed by AGS3.     

Inhibition of GDP/GTP exchange on G alpha subunits by proteins containing G-protein regulatory motifs

A novel Galpha binding consensus sequence, termed G-protein regulatory (GPR) or GoLoco motif, has been identified in a growing number of proteins, which are thought to modulate G-protein signaling. Alternative roles of GPR proteins as nucleotide exchange factors or as GDP dissociation inhibitors for Galpha have been proposed. We investigated the modulation of the GDP/GTP exchange of Gialpha(1), Goalpha, and Gsalpha by three proteins containing GPR motifs (GPR proteins), LGN-585-642, Pcp2, and RapIGAPII-23-131, to elucidate the mechanisms of GPR protein function. The GPR proteins displayed similar patterns of interaction with Gialpha(1) with the following order of affinities: Gialpha(1)GDP > Gialpha(1)GDPAlF(4)(-) > or = Gialpha(1)GTPgammaS. No detectable binding of the GPR proteins to Gsalpha was observed. LGN-585-642, Pcp2, and RapIGAPII-23-131 inhibited the rates of spontaneous GTPgammaS binding and blocked GDP release from Gialpha(1) and Goalpha. The inhibitory effects of the GPR proteins on Gialpha(1) were significantly more potent, indicating that Gi might be a preferred target for these modulators. Our results suggest that GPR proteins are potent GDP dissociation inhibitors for Gialpha-like Galpha subunits in vitro, and in this capacity they may inhibit GPCR/Gi protein signaling in vivo.