DEP domains: structurally similar but functionally different.

The Dishevelled, EGL-10 and pleckstrin (DEP) domain is a globular protein domain that is present in about ten human protein families with well-defined structural features. A picture is emerging that DEP domains mainly function in the spatial and temporal control of diverse signal transduction events by recruiting proteins to the plasma membrane. DEP domains can interact with various partners at the membrane, including phospholipids and membrane receptors, and their binding is subject to regulation.

Phosphorylation Dynamics in a flg22-Induced, G Protein-Dependent Network Reveals the AtRGS1 Phosphatase.

The microbe-associated molecular pattern flg22 is recognized in a flagellin-sensitive 2-dependent manner in root tip cells. Here, we show a rapid and massive change in protein abundance and phosphorylation state of the Arabidopsis root cell proteome in WT and a mutant deficient in heterotrimeric G-protein-coupled signaling. flg22-induced changes fall on proteins comprising a subset of this proteome, the heterotrimeric G protein interactome, and on highly-populated hubs of the immunity network. Approximately 95% of the phosphorylation changes in the heterotrimeric G-protein interactome depend, at least partially, on a functional G protein complex. One member of this interactome is ATBα, a substrate-recognition subunit of a protein phosphatase 2A complex and an interactor to Arabidopsis thaliana Regulator of G Signaling 1 protein (AtRGS1), a flg22-phosphorylated, 7-transmembrane spanning modulator of the nucleotide-binding state of the core G-protein complex. A null mutation of ATBα strongly increases basal endocytosis of AtRGS1. AtRGS1 steady-state protein level is lower in the atbα mutant in a proteasome-dependent manner. We propose that phosphorylation-dependent endocytosis of AtRGS1 is part of the mechanism to degrade AtRGS1, thus sustaining activation of the heterotrimeric G protein complex required for the regulation of system dynamics in innate immunity. The PP2A(ATBα) complex is a critical regulator of this signaling pathway.

The enzymatic oxygen sensor cysteamine dioxygenase binds its protein substrates through their N-termini.

The non-heme iron-dependent dioxygenase 2-aminoethanethiol (aka cysteamine) dioxygenase (ADO) has recently been identified as an enzymatic oxygen sensor that coordinates cellular changes to hypoxia by regulating the stability of proteins bearing an N-terminal cysteine (Nt-cys) through the N-degron pathway. It catalyzes O2-dependent Nt-cys sulfinylation, which promotes proteasomal degradation of the target. Only a few ADO substrates have been verified, including regulators of G-protein signaling (RGS) 4 and 5, and the proinflammatory cytokine interleukin-32, all of which exhibit cell and/or tissue specific expression patterns. ADO, in contrast, is ubiquitously expressed, suggesting it can regulate the stability of additional Nt-cys proteins in an O2-dependent manner. However, the role of individual chemical groups, active site metal, amino acid composition, and globular structure on protein substrate association remains elusive. To help identify new targets and examine the underlying biochemistry of the system, we conducted a series of biophysical experiments to investigate the binding requirements of established ADO substrates RGS5 and interleukin-32. We demonstrate, using surface plasmon response and enzyme assays, that a free, unmodified Nt-thiol and Nt-amine are vital for substrate engagement through active site metal coordination, with residues next to Nt-cys moderately impacting association and catalytic efficiency. Additionally, we show, through 1H-15N heteronuclear single quantum coherence nuclear magnetic resonance titrations, that the globular portion of RGS5 has limited impact on ADO association, with interactions restricted to the N-terminus. This work establishes key features involved in ADO substrate binding, which will help identify new protein targets and, subsequently, elucidate its role in hypoxic adaptation.

Structure-function analysis of plant G-protein regulatory mechanisms identifies key Gα-RGS protein interactions.

Heterotrimeric GTP-binding protein alpha subunit (Gα) and its cognate regulator of G-protein signaling (RGS) protein transduce signals in eukaryotes spanning protists, amoeba, animals, fungi, and plants. The core catalytic mechanisms of the GTPase activity of Gα and the interaction interface with RGS for the acceleration of GTP hydrolysis seem to be conserved across these groups; however, the RGS gene is under low selective pressure in plants, resulting in its frequent loss. Our current understanding of the structural basis of Gα:RGS regulation in plants has been shaped by Arabidopsis Gα, (AtGPA1), which has a cognate RGS protein. To gain a comprehensive understanding of this regulation beyond Arabidopsis, we obtained the x-ray crystal structures of Oryza sativa Gα, which has no RGS, and Selaginella moellendorffi (a lycophyte) Gα that has low sequence similarity with AtGPA1 but has an RGS. We show that the three-dimensional structure, protein-protein interaction with RGS, and the dynamic features of these Gα are similar to AtGPA1 and metazoan Gα. Molecular dynamic simulation of the Gα-RGS interaction identifies the contacts established by specific residues of the switch regions of GTP-bound Gα, crucial for this interaction, but finds no significant difference due to specific amino acid substitutions. Together, our data provide valuable insights into the regulatory mechanisms of plant G-proteins but do not support the hypothesis of adaptive co-evolution of Gα:RGS proteins in plants.

Lipid transport protein ORP2A promotes glucose signaling by facilitating RGS1 degradation.

Heterotrimeric GTP-binding proteins (G proteins) are a group of regulators essential for signal transmission into cells. Regulator of G protein signaling 1 (AtRGS1) possesses intrinsic GTPase-accelerating protein (GAP) activity and could suppress G protein and glucose signal transduction in Arabidopsis (Arabidopsis thaliana). However, how AtRGS1 activity is regulated is poorly understood. Here, we identified a knockout mutant of oxysterol binding protein-related protein 2A, orp2a-1, which exhibits similar phenotypes to the arabidopsis g-protein beta 1-2 (agb1-2) mutant. Transgenic lines overexpressing ORP2A displayed short hypocotyls, a hypersensitive response to sugar, and lower intracellular AtRGS1 levels than the control. Consistently, ORP2A interacted with AtRGS1 in vitro and in vivo. Tissue-specific expression of 2 ORP2A alternative splicing isoforms implied functions in controlling organ size and shape. Bioinformatic data and phenotypes of orp2a-1, agb1-2, and the orp2a-1 agb1-2 double mutant revealed the genetic interactions between ORP2A and Gß in the regulation of G protein signaling and sugar response. Both alternative protein isoforms of ORP2A localized in the endoplasmic reticulum (ER), plasma membrane (PM), and ER-PM contact sites and interacted with vesicle-associated membrane protein-associated protein 27-1 (VAP27-1) in vivo and in vitro through their two phenylalanines in an acidic track-like motif. ORP2A also displayed differential phosphatidyl phosphoinositide binding activity mediated by the pleckstrin homology domain in vitro. Taken together, the Arabidopsis membrane protein ORP2A interacts with AtRGS1 and VAP27-1 to positively regulate G protein and sugar signaling by facilitating AtRGS1 degradation.

In Silico Design of Novel RGS2-Galpha-q Interaction Inhibitors with Anticancer Activity.

Regulators of G-protein signaling (RGS) are a family of approximately 30 proteins that bind to and deactivate the alpha subunits of G-proteins (Gα) by accelerating their GTP hydrolysis rates, which terminates G-protein coupled receptor (GPCR) signaling. Thus, RGS proteins are essential in regulating GPCR signaling, and most members are implicated as critical nodes in human diseases such as hypertension, depression, and others. Regulator of G-protein signaling 2 (RGS2), a member of the R4 family of RGS proteins, is overexpressed in many solid breast cancers, and its levels in prostate cancer significantly correlate with the metastatic stage and poor prognosis. We sought to develop RGS2 inhibitors as potential chemotherapeutic agents utilizing structure-based drug design approaches. Available structures of the RGS2-Gα complex were used to extract a pharmacophore model for searching chemical databases. Docking of identified hits to RGS2 as well as other RGS structures was used to screen the hits for potent and selective RGS2 inhibitors. Whole cell assays showed the top 10 ranking compounds, AJ-1-AJ-10, to inhibit RGS2-Gαq interactions. Differential scanning fluorimetry showed AJ-3 to bind RGS2 but not Gαq. All 10 compounds inhibited the growth of several RGS2 expressing cancers in cell culture assays. In addition, AJ-3 inhibited the migration of LNCaP prostate cancer cells in wound healing assays. This is the first group of RGS2 inhibitors identified by structure-based approaches and that show anticancer activity. These results highlight the potential RGS2 inhibitors have to be a new class of chemotherapeutic agents.

Residue-level determinants of RGS R4 subfamily GAP activity and specificity towards the Gi subfamily.

The structural basis for the GTPase-accelerating activity of regulators of G protein signaling (RGS) proteins, as well as the mechanistic basis for their specificity in interacting with the heterotrimeric (αßγ) G proteins they inactivate, is not sufficiently understood at the family level. Here, we used biochemical assays to compare RGS domains across the RGS family and map those individual residues that favorably contribute to GTPase-accelerating activity, and those residues responsible for attenuating RGS domain interactions with Gα subunits. We show that conserved interactions of RGS residues with both the Gα switch I and II regions are crucial for RGS activity, while the reciprocal effects of "modulatory" and "disruptor" residues selectively modulate RGS activity. Our results quantify how specific interactions between RGS domains and Gα subunits are set by a balance between favorable RGS residue interactions with particular Gα switch regions, and unfavorable interactions with the Gα helical domain.

Mixed-solvent molecular dynamics simulation-based discovery of a putative allosteric site on regulator of G protein signaling 4.

Regulator of G protein signaling 4 (RGS4) is an intracellular protein that binds to the Gα subunit ofheterotrimeric G proteins and aids in terminating G protein coupled receptor signaling. RGS4 has been implicated in pain, schizophrenia, and the control of cardiac contractility. Inhibitors of RGS4 have been developed but bind covalently to cysteine residues on the protein. Therefore, we sought to identify alternative druggable sites on RGS4 using mixed-solvent molecular dynamics simulations, which employ low concentrations of organic probes to identify druggable hotspots on the protein. Pseudo-ligands were placed in consensus hotspots, and perturbation with normal mode analysis led to the identification and characterization of a putative allosteric site, which would be invaluable for structure-based drug design of non-covalent, small molecule inhibitors. Future studies on the mechanism of this allostery will aid in the development of novel therapeutics targeting RGS4.

Regulator of G-protein signaling (RGS) proteins as drug targets: Progress and future potentials.

G protein-coupled receptors (GPCRs) play critical roles in regulating processes such as cellular homeostasis, responses to stimuli, and cell signaling. Accordingly, GPCRs have long served as extraordinarily successful drug targets. It is therefore not surprising that the discovery in the mid-1990s of a family of proteins that regulate processes downstream of GPCRs generated great excitement in the field. This finding enhanced the understanding of these critical signaling pathways and provided potentially new targets for pharmacological intervention. These regulators of G-protein signaling (RGS) proteins were viewed by many as nodes downstream of GPCRs that could be targeted with small molecules to tune signaling processes. In this review, we provide a brief overview of the discovery of RGS proteins and of the gradual and continuing discovery of their roles in disease states, focusing particularly on cancer and neurological disorders. We also discuss high-throughput screening efforts that have led to the discovery first of peptide-based and then of small-molecule inhibitors targeting a subset of the RGS proteins. We explore the unique mechanisms of RGS inhibition these chemical tools have revealed and highlight the most up-to-date studies using these tools in animal experiments. Finally, we discuss the future opportunities in the field, as there are clearly more avenues left to be explored and potentials to be realized.

High-resolution structure of RGS17 suggests a role for Ca2+ in promoting the GTPase-activating protein activity by RZ subfamily members.

Regulator of G protein signaling (RGS) proteins are negative regulators of G protein-coupled receptor (GPCR) signaling through their ability to act as GTPase-activating proteins (GAPs) for activated Gα subunits. Members of the RZ subfamily of RGS proteins bind to activated Gαo, Gαz, and Gαi1-3 proteins in the nervous system and thereby inhibit downstream pathways, including those involved in Ca2+-dependent signaling. In contrast to other RGS proteins, little is known about RZ subfamily structure and regulation. Herein, we present the 1.5-Å crystal structure of RGS17, the most complete and highest-resolution structure of an RZ subfamily member to date. RGS17 cocrystallized with Ca2+ bound to conserved positions on the predicted Gα-binding surface of the protein. Using NMR chemical shift perturbations, we confirmed that Ca2+ binds in solution to the same site. Furthermore, RGS17 had greater than 55-fold higher affinity for Ca2+ than for Mg2+ Finally, we found that Ca2+ promotes interactions between RGS17 and activated Gα and decreases the Km for GTP hydrolysis, potentially by altering the binding mechanism between these proteins. Taken together, these findings suggest that Ca2+ positively regulates RGS17, which may represent a general mechanism by which increased Ca2+ concentration promotes the GAP activity of the RZ subfamily, leading to RZ-mediated inhibition of Ca2+ signaling.

An Interhelical Salt Bridge Controls Flexibility and Inhibitor Potency for Regulators of G-protein Signaling Proteins 4, 8, and 19.

Regulators of G-protein signaling (RGS) proteins modulate receptor signaling by binding to activated G-protein α-subunits, accelerating GTP hydrolysis. Selective inhibition of RGS proteins increases G-protein activity and may provide unique tissue specificity. Thiadiazolidinones (TDZDs) are covalent inhibitors that act on cysteine residues to inhibit RGS4, RGS8, and RGS19. There is a correlation between protein flexibility and potency of inhibition by the TDZD 4-[(4- fluorophenyl)methyl]-2-(4-methylphenyl)-1,2,4-thiadiazolidine-3,5-dione (CCG-50014). In the context of a single conserved cysteine residue on the α 4 helix, RGS19 is the most flexible and most potently inhibited by CCG-50014, followed by RGS4 and RGS8. In this work, we identify residues responsible for differences in both flexibility and potency of inhibition among RGS isoforms. RGS19 lacks a charged residue on the α 4 helix that is present in RGS4 and RGS8. Introducing a negative charge at this position (L118D) increased the thermal stability of RGS19 and decreased the potency of inhibition of CCG-50014 by 8-fold. Mutations eliminating salt bridge formation in RGS8 and RGS4 decreased thermal stability in RGS8 and increased potency of inhibition of both RGS4 and RGS8 by 4- and 2-fold, respectively. Molecular dynamics simulations with an added salt bridge in RGS19 (L118D) showed reduced RGS19 flexibility. Hydrogen-deuterium exchange studies showed striking differences in flexibility in the α 4 helix of RGS4, 8, and 19 with salt bridge-modifying mutations. These results show that the α 4 salt bridge-forming residue controls flexibility in several RGS isoforms and supports a causal relationship between RGS flexibility and the potency of TDZD inhibitors. SIGNIFICANCE STATEMENT: Inhibitor potency is often viewed in relation to the static structure of a target protein binding pocket. Using both experimental and computation studies we assess determinants of dynamics and inhibitor potency for three different RGS proteins. A single salt bridge-forming residue determines differences in flexibility between RGS isoforms; mutations either increase or decrease protein motion with correlated alterations in inhibitor potency. This strongly suggests a causal relationship between RGS protein flexibility and covalent inhibitor potency.

Loss-of-Function Mutations in Human Regulator of G Protein Signaling RGS2 Differentially Regulate Pharmacological Reactivity of Resistance Vasculature.

Regulator of G protein signaling 2 (RGS2) plays a role in reducing vascular contraction and promoting relaxation due to its GTPase accelerating protein activity toward Gαq. Previously, we identified four human loss-of-function (LOF) mutations in RGS2 (Q2L, D40Y, R44H, and R188H). This study aimed to investigate whether those RGS2 LOF mutations disrupt the ability of RGS2 to regulate vascular reactivity. Isolated mesenteric arteries (MAs) from RGS2-/- mice showed an elevated contractile response to 5 nM angiotensin II and a loss of acetylcholine (ACh)-mediated vasodilation. Reintroduction of a wild-type (WT) RGS2-GFP plasmid into RGS2-/- MAs suppressed the vasoconstrictor response to angiotensin II. RGS2 LOF mutants failed to suppress the angiotensin II constriction response compared with RGS2 WT. In contrast, ACh-mediated vasoconstriction was restored by expression of RGS2 WT, D40Y, and R44H but not by RGS2 Q2L or R188H. Phosphorylation of RGS2 D40Y and R44H by protein kinase G (PKG) may explain their maintained function to support relaxation in MAs. This is supported by phosphomimetic mutants and suppression of vasorelaxation mediated by RGS2 D40Y by a PKG inhibitor. These results demonstrate that RGS2 attenuates vasoconstriction in MAs and that RGS2 LOF mutations cannot carry out this effect. Among them, the Q2L and R188H mutants supported less relaxation to ACh, whereas relaxation mediated by the D40Y and R44H mutant proteins was equal to that with WT protein. Phosphorylation of RGS2 by PKG appears to contribute to this vasorelaxation. These results provide insights for precision medicine targeting the rare individuals carrying these RGS2 mutations. SIGNIFICANCE STATEMENT: Regulator of G protein signaling 2 (RGS2) has been implicated in the control of blood pressure; rare mutations in the RGS2 gene have been identified in large-scale human gene sequencing studies. Four human mutations in RGS2 that cause loss of function (LOF) in cell-based assays were examined in isolated mouse arteries for effects on both vasoconstriction and vasodilation. All mutants showed the expected LOF effects in suppressing vasoconstriction. Surprisingly, the D40Y and R44H mutant RGS2 showed normal control of vasodilation. We propose that this is due to rescue of the mislocalization phenotype of these two mutants by nitric oxide-mediated/protein kinase G-dependent phosphorylation. These mechanisms may guide drug discovery or drug repurposing efforts for hypertension by enhancing RGS2 function.

Interplay of cysteine exposure and global protein dynamics in small-molecule recognition by a regulator of G-protein signaling protein.

Regulator of G protein signaling (RGS) proteins play a pivotal role in regulation of G protein-coupled receptor (GPCR) signaling and are therefore becoming an increasingly important therapeutic target. Recently discovered thiadiazolidinone (TDZD) compounds that target cysteine residues have shown different levels of specificities and potencies for the RGS4 protein, thereby suggesting intrinsic differences in dynamics of this protein upon binding of these compounds. In this work, we investigated using atomistic molecular dynamics (MD) simulations the effect of binding of several small-molecule inhibitors on perturbations and dynamical motions in RGS4. Specifically, we studied two conformational models of RGS4 in which a buried cysteine residue is solvent-exposed due to side-chain motions or due to flexibility in neighboring helices. We found that TDZD compounds with aromatic functional groups perturb the RGS4 structure more than compounds with aliphatic functional groups. Moreover, small-molecules with aromatic functional groups but lacking sulfur atoms only transiently reside within the protein and spontaneously dissociate to the solvent. We further measured inhibitory effects of TDZD compounds using a protein-protein interaction assay on a single-cysteine RGS4 protein showing trends in potencies of compounds consistent with our simulation studies. Thermodynamic analyses of RGS4 conformations in the apo-state and on binding to TDZD compounds revealed links between both conformational models of RGS4. The exposure of cysteine side-chains appears to facilitate initial binding of TDZD compounds followed by migration of the compound into a bundle of four helices, thereby causing allosteric perturbations in the RGS/Gα protein-protein interface.

Interplay between negative and positive design elements in Gα helical domains of G proteins determines interaction specificity toward RGS2.

Regulators of G protein signaling (RGS) proteins inactivate Gα subunits, thereby controlling G protein-coupled signaling networks. Among all RGS proteins, RGS2 is unique in interacting only with the Gαq but not with the Gαi subfamily. Previous studies suggested that this specificity is determined by the RGS domain and, in particular, by three RGS2-specific residues that lead to a unique mode of interaction with Gαq This interaction was further proposed to act through contacts with the Gα GTPase domain. Here, we combined energy calculations and GTPase activity measurements to determine which Gα residues dictate specificity toward RGS2. We identified putative specificity-determining residues in the Gα helical domain, which among G proteins is found only in Gα subunits. Replacing these helical domain residues in Gαi with their Gαq counterparts resulted in a dramatic specificity switch toward RGS2. We further show that Gα-RGS2 specificity is set by Gαi residues that perturb interactions with RGS2, and by Gαq residues that enhance these interactions. These results show, for the first time, that the Gα helical domain is central to dictating specificity toward RGS2, suggesting that this domain plays a general role in governing Gα-RGS specificity. Our insights provide new options for manipulating RGS-G protein interactions in vivo, for better understanding of their 'wiring' into signaling networks, and for devising novel drugs targeting such interactions.

Mechanism of the intrinsic arginine finger in heterotrimeric G proteins.

Heterotrimeric G proteins are crucial molecular switches that maintain a large number of physiological processes in cells. The signal is encoded into surface alterations of the Gα subunit that carries GTP in its active state and GDP in its inactive state. The ability of the Gα subunit to hydrolyze GTP is essential for signal termination. Regulator of G protein signaling (RGS) proteins accelerates this process. A key player in this catalyzed reaction is an arginine residue, Arg178 in Gαi1, which is already an intrinsic part of the catalytic center in Gα in contrast to small GTPases, at which the corresponding GTPase-activating protein (GAP) provides the arginine "finger." We applied time-resolved FTIR spectroscopy in combination with isotopic labeling and site-directed mutagenesis to reveal the molecular mechanism, especially of the role of Arg178 in the intrinsic Gαi1 mechanism and the RGS4-catalyzed mechanism. Complementary biomolecular simulations (molecular mechanics with molecular dynamics and coupled quantum mechanics/molecular mechanics) were performed. Our findings show that Arg178 is bound to γ-GTP for the intrinsic Gαi1 mechanism and pushed toward a bidentate α-γ-GTP coordination for the Gαi1·RGS4 mechanism. This movement induces a charge shift toward ß-GTP, increases the planarity of γ-GTP, and thereby catalyzes the hydrolysis.

Evaluation of the Selectivity and Cysteine Dependence of Inhibitors across the Regulator of G Protein-Signaling Family.

Since their discovery more than 20 years ago, regulators of G protein-signaling (RGS) proteins have received considerable attention as potential drug targets because of their ability to modulate Gα activity. Efforts to identify small molecules capable of inhibiting the protein-protein interactions between activated Gα subunits and RGS proteins have yielded a substantial number of inhibitors, especially toward the well studied RGS4. These efforts also determined that many of these small molecules inhibit the protein-protein interactions through covalent modification of cysteine residues within the RGS domain that are located distal to the Gα-binding interface. As some of these cysteine residues are highly conserved within the RGS family, many of these inhibitors display activity toward multiple RGS family members. In this work, we sought to determine the selectivity of these small-molecule inhibitors against 12 RGS proteins, as well as against the cysteine-null mutants for 10 of these proteins. Using both biochemical and cell-based methods to assess Gα-RGS complex formation and Gα enzymatic activity, we found that several previously identified RGS4 inhibitors were active against other RGS members, such as RGS14, with comparable or greater potency. Additionally, for every compound tested, activity was dependent on the presence of cysteine residues. This work defines the selectivity of commercially available RGS inhibitors and provides insight into the RGS family members for which drug discovery efforts may be most likely to succeed.

Regulation of neurite morphogenesis by interaction between R7 regulator of G protein signaling complexes and G protein subunit Gα13.

The R7 regulator of G protein signaling family (R7-RGS) critically regulates nervous system development and function. Mice lacking all R7-RGS subtypes exhibit diverse neurological phenotypes, and humans bearing mutations in the retinal R7-RGS isoform RGS9-1 have vision deficits. Although each R7-RGS subtype forms heterotrimeric complexes with Gß5 and R7-RGS-binding protein (R7BP) that regulate G protein-coupled receptor signaling by accelerating deactivation of Gi/o α-subunits, several neurological phenotypes of R7-RGS knock-out mice are not readily explained by dysregulated Gi/o signaling. Accordingly, we used tandem affinity purification and LC-MS/MS to search for novel proteins that interact with R7-RGS heterotrimers in the mouse brain. Among several proteins detected, we focused on Gα13 because it had not been linked to R7-RGS complexes before. Split-luciferase complementation assays indicated that Gα13 in its active or inactive state interacts with R7-RGS heterotrimers containing any R7-RGS isoform. LARG (leukemia-associated Rho guanine nucleotide exchange factor (GEF)), PDZ-RhoGEF, and p115RhoGEF augmented interaction between activated Gα13 and R7-RGS heterotrimers, indicating that these effector RhoGEFs can engage Gα13·R7-RGS complexes. Because Gα13/R7-RGS interaction required R7BP, we analyzed phenotypes of neuronal cell lines expressing RGS7 and Gß5 with or without R7BP. We found that neurite retraction evoked by Gα12/13-dependent lysophosphatidic acid receptors was augmented in R7BP-expressing cells. R7BP expression blunted neurite formation evoked by serum starvation by signaling mechanisms involving Gα12/13 but not Gαi/o These findings provide the first evidence that R7-RGS heterotrimers interact with Gα13 to augment signaling pathways that regulate neurite morphogenesis. This mechanism expands the diversity of functions whereby R7-RGS complexes regulate critical aspects of nervous system development and function.

Differential Protein Dynamics of Regulators of G-Protein Signaling: Role in Specificity of Small-Molecule Inhibitors.

Small-molecule inhibitor selectivity may be influenced by variation in dynamics among members of a protein family. Regulator of G-protein Signaling (RGS) proteins are a family that plays a key role in G-Protein Coupled Receptor (GPCR) signaling by binding to active Gα subunits and accelerating GTP hydrolysis, thereby terminating activity. Thiadiazolidinones (TDZDs) inhibit the RGS-Gα interaction by covalent modification of cysteine residues in RGS proteins. Some differences in specificity may be explained by differences in the complement of cysteines among RGS proteins. However, key cysteines shared by RGS proteins inhibited by TDZDs are not exposed on the protein surface, and differences in potency exist among RGS proteins containing only buried cysteines. We hypothesize that differential exposure of buried cysteine residues among RGS proteins partially drives TDZD selectivity. Hydrogen-deuterium exchange (HDX) studies and molecular dynamics (MD) simulations were used to probe the dynamics of RGS4, RGS8, and RGS19, three RGS proteins inhibited at a range of potencies by TDZDs. When these proteins were mutated to contain a single, shared cysteine, RGS19 was found to be most potently inhibited. HDX studies revealed differences in α4 and α6 helix flexibility among RGS isoforms, with particularly high flexibility in RGS19. This could cause differences in cysteine exposure and lead to differences in potency of TDZD inhibition. MD simulations of RGS proteins revealed motions that correspond to solvent exposure observed in HDX, providing further evidence for a role of protein dynamics in TDZD selectivity.

Structural motifs in the RGS RZ subfamily combine to attenuate interactions with Gα subunits.

Regulators of G-protein Signaling (RGS) proteins inactivate heterotrimeric G proteins, thereby setting the duration of active signaling. In particular, the RGS RZ subfamily, which consists of RGS17, RGS19, and RGS20, mediates numerous physiological functions and human pathologies - mostly by functioning as GTPase Activating Proteins (GAPs) towards the Gαi subfamily. Yet, which RZ subfamily members mediate particular functions and how their GAP activity and specificity are governed at the amino acid level is not well understood. Here, we show that all RZ subfamily members have similar and relatively low GAP activity towards Gαo. We characterized four RZ-specific structural motifs that mediate this low activity, and suggest they perturb optimal interactions with the Gα subunit. Indeed, inserting these RZ-specific motifs into the representative high-activity RGS16 impaired GAP activity in a non-additive manner. Our results provide residue-level insights into the specificity determinants of the RZ subfamily, and enable to study their interactions in signaling cascades by using redesigned mutants such as those presented in this work.

Small-molecule binding of the axin RGS domain promotes ß-catenin and Ras degradation.

Both the Wnt/ß-catenin and Ras pathways are aberrantly activated in most human colorectal cancers (CRCs) and interact cooperatively in tumor promotion. Inhibition of these signaling may therefore be an ideal strategy for treating CRC. We identified KY1220, a compound that destabilizes both ß-catenin and Ras, via targeting the Wnt/ß-catenin pathway, and synthesized its derivative KYA1797K. KYA1797K bound directly to the regulators of G-protein signaling domain of axin, initiating ß-catenin and Ras degradation through enhancement of the ß-catenin destruction complex activating GSK3ß. KYA1797K effectively suppressed the growth of CRCs harboring APC and KRAS mutations, as shown by various in vitro studies and by in vivo studies using xenograft and transgenic mouse models of tumors induced by APC and KRAS mutations. Destabilization of both ß-catenin and Ras via targeting axin is a potential therapeutic strategy for treatment of CRC and other type cancers activated Wnt/ß-catenin and Ras pathways.