Crystallization and preliminary X-ray diffraction studies of Drosophila melanogaster Gαo-subunit of heterotrimeric G protein in complex with the RGS domain of CG5036.

Regulator of G-protein signalling (RGS) proteins negatively regulate heterotrimeric G-protein signalling through their conserved RGS domains. RGS domains act as GTPase-activating proteins, accelerating the GTP hydrolysis rate of the activated form of Gα-subunits. Although omnipresent in eukaryotes, RGS proteins have not been adequately analysed in non-mammalian organisms. The Drosophila melanogaster Gαo-subunit and the RGS domain of its interacting partner CG5036 have been overproduced and purified; the crystallization of the complex of the two proteins using PEG 4000 as a crystallizing agent and preliminary X-ray crystallographic analysis are reported. Diffraction data were collected to 2.0 Šresolution using a synchrotron-radiation source.

Inhibition of RGS10 Aggravates Periapical Periodontitis via Upregulation of the NF-κB Pathway.

INTRODUCTION: Periapical periodontitis develops due to the interplay between root canal microorganisms and host defenses. The mechanism underlying the pathogenesis of periapical periodontitis remains unclear. Regulator of G protein signaling protein 10 (RGS10) has been suggested to play a role in regulating inflammation. This study explored the potential regulatory effects of RGS10 on periapical periodontitis and the proinflammatory pathway of nuclear factor (NF)-κB. METHODS: Disease models of periapical inflammation in mice were established, and adenovirus-associated virus (AAV) was used to inhibit RGS10 expression. Periapical lesions were detected using micro-computed tomography. Quantitative reverse transcriptase PCR (qRT-PCR), western blotting (WB), enzyme-linked immunosorbent assay (ELISA), enzyme activity staining of tartrate-resistant acid phosphatase, and immunohistochemistry were conducted to assess the role of RGS10 expression on NF-κB proinflammatory signaling, OPG, RANKL, and osteoclasts in the periapical regions of each group. TNFα was used to stimulate L929 cells alone or with small interfering RNA (siRNA). To assess the expression of associated molecules, WB, immunofluorescence, qRT-PCR, and ELISA were performed. RESULTS: RGS10 inhibition increased alveolar bone destruction in periapical periodontitis lesions and substantially enhanced the NF-κB proinflammatory signaling pathway activation level. Furthermore, RGS10 inhibition upregulated the ratio of OPG/RANKL and the maturation of osteoclasts during alveolar bone resorption. L929 cell TNFα stimulation and siRNA transfection confirmed these in vivo results. CONCLUSION: RGS10 negatively regulates NF-κB proinflammatory signaling in periapical periodontitis and participates in bone remodeling. Therefore, RGS10 is a promising treatment option for long-term chronic periapical inflammation and may be a new target for the artificial regulation of inflammation.

The RGS2 gene product from a candidate hypertension allele shows decreased plasma membrane association and inhibition of Gq.

Hypertension is a leading risk factor for the development of cardiovascular disease. Data from human and animal studies suggest that RGS2, a potent inhibitor of G(q) signaling, is important for blood pressure regulation. Several RGS2 mutations in the Japanese population have been found to be associated with hypertension. The product of one of these alleles, R44H, is mutated within the amino terminal amphipathic alpha-helix domain, the region responsible for plasma membrane-targeting. The functional consequence of this mutation and its potential link to the development of hypertension, however, are not known. In this study, we showed that R44H was a weaker inhibitor of receptor-mediated G(q) signaling than wild-type RGS2. Confocal microscopy revealed that YFP-tagged R44H bound to the plasma membrane less efficiently than wild-type RGS2. R44 is one of the basic residues positioned to stabilize lipid bilayer interaction of the RGS2 amphipathic helix domain. Tryptophan fluorescence and circular dichroism studies of this domain showed that the R44H mutation prevented proper entrenchment of hydrophobic residues into the lipid bilayer without disrupting helix-forming capacity. Together, these data suggest that decreasing the side-chain length and flexibility at R44 prevented proper lipid bilayer association and function of RGS2. Finally, the R44H protein did not behave as a dominant-negative interfering mutant. Thus, our data are consistent with the notion that a R44H missense mutation in human RGS2 produces a hypomorphic allele that may lead to altered receptor-mediated G(q) inhibition and contribute to the development of hypertension in affected subjects.

Assay of RGS protein activity in vitro using purified components.

Single-turnover and steady-state GTPase assays are an effective means to identify and characterize interactions between RGS and G alpha proteins in vitro. The advantage of the single turnover GTPase assay is that it permits simple and rapid assessment of RGS protein activity toward a putative G alpha-GTP substrate. Moreover, once an interaction between an RGS protein and a G alpha-GTP subunit has been identified, the single-turnover assay can be used to determine Michaelis-Menten constants and/or KI values for other competing G alpha substrates. A disadvantage of the single-turnover assay is that a negative result does not preclude the possibility of an interaction between given RGS and G alpha proteins in vivo. Inappropriate reaction conditions or the presence (or absence) of appropriate posttranslational modifications may result in small or undetectable increases in RGS protein-dependent GTPase activity. In these cases it may be tempting to examine RGS protein activity using steady-state GTPase assays in phospholipid vesicles reconstituted with receptors and heterotrimetric G proteins. The advantage to monitoring steady-state GTPase activity in reconstituted proteoliposomes is that ligand-dependent activation of the receptor facilitates GDP dissociation, such that effects of RGS proteins can be observed; multiple cycles of GTP binding and hydrolysis then amplify the GTPase signal. Additionally, the presence of the phospholipid membrane can increase the local RGS protein concentration approximately 10(4)-fold, permitting observation of interactions that are weak in solution. The primary disadvantage of the reconstituted system is the requirement for receptor purification, a technically demanding undertaking in comparison to the purification of G alpha, G beta gamma, and most RGS proteins.

Purification and in vitro functional analysis of R7 subfamily RGS proteins in complex with Gbeta5.

The study of purified regulator of G-protein signaling (RGS) proteins in steady-state GTPase assays using reconstituted proteoliposomes is a powerful approach to characterizing the RGS protein-mediated acceleration of intrinsic Galpha subunit GTPase activity in the context of various G-protein and G-protein-coupled receptor (GPCR) combinations. This approach has been applied successfully to the R7 subfamily of RGS proteins, RGS6, -7, -9, and -11, which form heterodimers with Gbeta5 subunits via the G-protein gamma-like domain of R7 proteins. This article describes the purification of heterodimers from Sf9 insect cells following the expression of recombinant R7 protein and histidine-tagged Gbeta5 using affinity and ion-exchange chromatography. The ability of the heterodimers to accelerate the intrinsic GTPase activity of Galpha subunits was assessed in steady-state GTPase assays performed on proteoliposomes consisting of phospholipids, purified G proteins, and purified GPCRs.

Characterization of R9AP, a membrane anchor for the photoreceptor GTPase-accelerating protein, RGS9-1.

The proper recovery of photoreceptor light responses requires timely inactivation of the G-protein transducin (Gt) by GTP hydrolysis. It is now well established that the GTPase-accelerating protein (GAP) RGS9-1 plays an important role in determining the recovery kinetics of photoresponses. RGS9-1 has been found to be anchored to photoreceptor disk membranes by a novel photoreceptor protein, R9AP. R9AP has a single transmembrane domain at its C-terminal region. Membrane tethering by R9AP enhances RGS9-1 GAP activity in vitro and has been hypothesized to be important for the regulation of RGS9-1 function in vivo. In addition, R9AP shows structural similarity to the SNARE complex protein syntaxin and has been shown to be required for the correct targeting and localization of the RGS9-1 protein in photoreceptors. Therefore, R9AP may have additional functions other than that in the phototransduction pathway. This article presents methods and protocols developed for the functional characterization of R9AP in phototransduction, including the immunoprecipitation of the endogenous protein, the expression and purification of recombinant proteins, the reconstitution of proteoliposomes, and assays for its interaction with RGS9-1 and its effects on RGS9-1 GAP activity. These methods may also be applied to the study of R9AP function in other pathways or other cell types or to the studies of other membrane proteins that are structurally similar to R9AP.

Rgs19 regulates mouse palatal fusion by modulating cell proliferation and apoptosis in the MEE.

Palatal development is one of the critical events in craniofacial morphogenesis. During fusion of the palatal shelves, removal of the midline epithelial seam (MES) is a fundamental process for achieving proper morphogenesis of the palate. The reported mechanisms for removing the MES are the processes of apoptosis, migration or general epithelial-to-mesenchymal transition (EMT) through modulations of various signaling molecules including Wnt signaling. RGS19, a regulator of the G protein signaling (RGS) family, interacts selectively with the specific α subunits of the G proteins (Gαi, Gαq) and enhances their GTPase activity. Rgs19 was reported to be a modulator of the Wnt signaling pathway. In mouse palatogenesis, the restricted epithelial expression pattern of Rgs19 was examined in the palatal shelves, where expression of Wnt11 was observed. Based on these specific expression patterns of Rgs19 in the palatal shelves, the present study examined the detailed developmental function of Rgs19 using AS-ODN treatments during in vitro palate organ cultivations as a loss-of-function study. After the knockdown of Rgs19, the morphological changes in the palatal shelves was examined carefully using a computer-aided three dimensional reconstruction method and the altered expression patterns of related signaling molecules were evaluated using genome wide screening methods. RT-qPCR and in situ hybridization methods were also used to confirm these array results. These morphological and molecular examinations suggested that Rgs19 plays important roles in palatal fusion through the degradation of MES via activation of the palatal fusion related and apoptotic related genes. Overall, inhibition of the proliferation related and Wnt responsive genes by Rgs19 are required for proper palatal fusion.

Role of regulator of G-protein signaling 2 (RGS2) in periodontal ligament cells under mechanical stress.

Mechanical stress is thought to regulate the expression of genes in the periodontal ligament (PDL) cells. Using a microarray approach, we recently identified a regulator of G-protein signaling 2 (RGS2) as an up-regulated gene in the PDL cells under compressive force. The RGS protein family is known to turn off G-protein signaling. G-protein signaling involves the production of cAMP, which is thought to be one of the biological mediators in response to mechanical stress. Here, we investigated the role of RGS2 in the PDL cells under mechanical stress. PDL cells derived from the ligament tissues of human premolar teeth were cultured in collagen gels and subjected to static compressive force. Compressive force application time-dependently enhanced RGS2 expression and intracellular cAMP levels. To examine the interrelationship between RGS2 and cAMP, the PDL cells were treated with 2',5'-dideoxyadenosine (DDA), an inhibitor of adenyl cyclase, or antisense S-oligonucleotide (S-ODN) to RGS2 under compressive force. DDA dose-dependently inhibited RGS2 stimulated by compressive force. Blockage of RGS2 by antisense S-ODN elevated the cAMP levels compared with controls. These results indicate that cAMP stimulates RGS2 expression, which in turn leads to a decrease in the cAMP production by inactivating the G-protein signaling in the mechanically stressed PDL cells.

RGS14 is a microtubule-associated protein.

Heterotrimeric G-proteins and their regulators are emerging as important players in modulating microtubule polymerization dynamics and in spindle force generation during cell division in C. elegans, D. melanogaster and mammals. We recently demonstrated that RGS14 is required for completion of the first mitotic division of the mouse embryo, and that it regulates microtubule organization in vivo. Here, we demonstrate that RGS14 is a microtubule-associated protein and a component of the mitotic spindle that may regulate microtubule polymerization and spindle organization. Taxol-stabilized tubulin, but not depolymerized tubulin coimmunoprecipitates with RGS14 from cell extracts. Furthermore, RGS14 copurifies with tubulin from porcine brain following multiple rounds of microtubule polymerization/depolymerization and binds directly to microtubules formed in vitro from pure tubulin (KD = 1.3 +/- 0.3 microM). Both RGS14 and Galpha(i1) in the presence of exogenous GTP promote tubulin polymerization, which is dependent on additional microtubule-associated proteins. However, preincubation of RGS14 with Galpha(i1)-GDP precludes either from promoting microtubule polymerization, suggesting that a functional GTP/GDP cycle is necessary. Finally, we show that RGS14 is a component of mitotic asters formed in vitro from HeLa cell extracts and that depletion of RGS14 from cell extracts blocks aster formation. Collectively, these results show that RGS14 is a microtubule-associated protein that may modulate microtubule dynamics and spindle formation.

Regulators of G-protein signaling (RGS) 4, insertion into model membranes and inhibition of activity by phosphatidic acid.

Regulators of G-protein signaling (RGS) proteins are critical for attenuating G protein-coupled signaling pathways. The membrane association of RGS4 has been reported to be crucial for its regulatory activity in reconstituted vesicles and physiological roles in vivo. In this study, we report that RGS4 initially binds onto the surface of anionic phospholipid vesicles and subsequently inserts into, but not through, the membrane bilayer. Phosphatidic acid, one of anionic phospholipids, could dramatically inhibit the ability of RGS4 to accelerate GTPase activity in vitro. Phosphatidic acid is an effective and potent inhibitor of RGS4 in a G alpha(i1)-[gamma-(32)P]GTP single turnover assay with an IC(50) approximately 4 microm and maximum inhibition of over 90%. Furthermore, phosphatidic acid was the only phospholipid tested that inhibited RGS4 activity in a receptor-mediated, steady-state GTP hydrolysis assay. When phosphatidic acid (10 mol %) was incorporated into m1 acetylcholine receptor-G alpha(q) vesicles, RGS4 GAP activity was markedly inhibited by more than 70% and the EC(50) of RGS4 was increased from 1.5 to 7 nm. Phosphatidic acid also induced a conformational change in the RGS domain of RGS4 measured by acrylamide-quenching experiments. Truncation of the N terminus of RGS4 (residues 1-57) resulted in the loss of both phosphatidic acid binding and lipid-mediated functional inhibition. A single point mutation in RGS4 (Lys(20) to Glu) permitted its binding to phosphatidic acid-containing vesicles but prevented lipid-induced conformational changes in the RGS domain and abolished the inhibition of its GAP activity. We speculate that the activation of phospholipase D or diacylglycerol kinase via G protein-mediated signaling cascades will increase the local concentration of phosphatidic acid, which in turn block RGS4 GAP activity in vivo. Thus, RGS4 may represent a novel effector of phosphatidic acid, and this phospholipid may function as a feedback regulator in G protein-mediated signaling pathways.

RGS4 binds to membranes through an amphipathic alpha -helix.

RGS4, a mammalian GTPase-activating protein for G protein alpha subunits, requires its N-terminal 33 amino acids for plasma membrane localization and biological activity (Srinivasa, S. P., Bernstein, L. S., Blumer, K. J., and Linder, M. E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5584-5589). In this study, we tested the hypothesis that the N-terminal domain mediates membrane binding by forming an amphipathic alpha-helix. RGS4 bound to liposomes containing anionic phospholipids in a manner dependent on the first 33 amino acids. Circular dichroism spectroscopy of a peptide corresponding to amino acids 1-31 of RGS4 revealed that the peptide adopted an alpha-helical conformation in the presence of anionic phospholipids. Point mutations that either neutralized positive charges on the hydrophilic face or substituted polar residues on the hydrophobic face of the model helix disrupted plasma membrane targeting and biological activity of RGS4 expressed in yeast. Recombinant mutant proteins were active as GTPase-activating proteins in solution but exhibited diminished binding to anionic liposomes. Peptides corresponding to mutants with the most pronounced phenotypes were also defective in forming an alpha-helix as measured by circular dichroism spectroscopy. These results support a model for direct interaction of RGS4 with membranes through hydrophobic and electrostatic interactions of an N-terminal alpha-helix.

Determination of the contact energies between a regulator of G protein signaling and G protein subunits and phospholipase C beta 1.

Cell signaling proteins may form functional complexes that are capable of rapid signal turnover. These contacts may be stabilized by either scaffolding proteins or multiple interactions between members of the complex. In this study, we have determined the affinities between a regulator of G protein signaling protein, RGS4, and three members of the G protein-phospholipase Cbeta (PLC-beta) signaling cascade which may allow for rapid deactivation of intracellular Ca(2+) release and activation of protein kinase C. Specifically, using fluorescence methods, we have determined the interaction energies between the RGS4, PLC-beta, G-betagamma, and both deactivated (GDP-bound) and activated (GTPgammaS-bound) Galpha(q). We find that RGS4 not only binds to activated Galpha(q), as predicted, but also to Gbetagamma and PLCbeta(1). These interactions occur through protein-protein contacts since the intrinsic membrane affinity of RGS4 was found to be very weak in the absence of the protein partner PLCbeta(1) or a lipid regulator, phosphatidylinositol-3,4,5 trisphosphate. Ternary complexes between Galpha(q), Gbetagamma and phospholipase Cbeta(1) will form, but only at relatively high protein concentrations. We propose that these interactions allow RGS4 to remain anchored to the signaling complex even in the quiescent state and allow rapid transfer to activated Galpha(q) to shut down the signal. Comparison of the relative affinities between these interacting proteins will ultimately allow us to determine whether certain complexes can form and where signals will be directed.