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.

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.

Thalamocortical Connections Drive Intracortical Activation of Functional Columns in the Mislaminated Reeler Somatosensory Cortex.

Neuronal wiring is key to proper neural information processing. Tactile information from the rodent's whiskers reaches the cortex via distinct anatomical pathways. The lemniscal pathway relays whisking and touch information from the ventral posteromedial thalamic nucleus to layer IV of the primary somatosensory "barrel" cortex. The disorganized neocortex of the reeler mouse is a model system that should severely compromise the ingrowth of thalamocortical axons (TCAs) into the cortex. Moreover, it could disrupt intracortical wiring. We found that neuronal intermingling within the reeler barrel cortex substantially exceeded previous descriptions, leading to the loss of layers. However, viral tracing revealed that TCAs still specifically targeted transgenically labeled spiny layer IV neurons. Slice electrophysiology and optogenetics proved that these connections represent functional synapses. In addition, we assessed intracortical activation via immediate-early-gene expression resulting from a behavioral exploration task. The cellular composition of activated neuronal ensembles suggests extensive similarities in intracolumnar information processing in the wild-type and reeler brains. We conclude that extensive ectopic positioning of neuronal partners can be compensated for by cell-autonomous mechanisms that allow for the establishment of proper connectivity. Thus, genetic neuronal fate seems to be of greater importance for correct cortical wiring than radial neuronal position.

Molecular mechanism of macrophage activation by red ginseng acidic polysaccharide from Korean red ginseng.

Red ginseng acidic polysaccharide (RGAP), isolated from Korean red ginseng, displays immunostimulatory and antitumor activities. Even though numerous studies have been reported, the mechanism as to how RGAP is able to stimulate the immune response is not clear. In this study, we aimed to explore the mechanism of molecular activation of RGAP in macrophages. RGAP treatment strongly induced NO production in RAW264.7 cells without altering morphological changes, although the activity was not strong compared to LPS-induced dendritic-like morphology in RAW264.7 cells. RGAP-induced NO production was accompanied with enhanced mRNA levels of iNOS and increases in nuclear transcription factors such as NF-κB, AP-1, STAT-1, ATF-2, and CREB. According to pharmacological evaluation with specific enzyme inhibitors, Western blot analysis of intracellular signaling proteins and inhibitory pattern using blocking antibodies, ERK, and JNK were found to be the most important signaling enzymes compared to LPS signaling cascade. Further, TLR2 seems to be a target surface receptor of RGAP. Lastly, macrophages isolated from RGS2 knockout mice or wortmannin exposure strongly upregulated RGAP-treated NO production. Therefore, our results suggest that RGAP can activate macrophage function through activation of transcription factors such as NF-κB and AP-1 and their upstream signaling enzymes such as ERK and JNK.

Attenuated food anticipatory activity and abnormal circadian locomotor rhythms in Rgs16 knockdown mice.

Regulators of G protein signaling (RGS) are a multi-functional protein family, which functions in part as GTPase-activating proteins (GAPs) of G protein α-subunits to terminate G protein signaling. Previous studies have demonstrated that the Rgs16 transcripts exhibit robust circadian rhythms both in the suprachiasmatic nucleus (SCN), the master circadian light-entrainable oscillator (LEO) of the hypothalamus, and in the liver. To investigate the role of RGS16 in the circadian clock in vivo, we generated two independent transgenic mouse lines using lentiviral vectors expressing short hairpin RNA (shRNA) targeting the Rgs16 mRNA. The knockdown mice demonstrated significantly shorter free-running period of locomotor activity rhythms and reduced total activity as compared to the wild-type siblings. In addition, when feeding was restricted during the daytime, food-entrainable oscillator (FEO)-driven elevated food-anticipatory activity (FAA) observed prior to the scheduled feeding time was significantly attenuated in the knockdown mice. Whereas the restricted feeding phase-advanced the rhythmic expression of the Per2 clock gene in liver and thalamus in the wild-type animals, the above phase shift was not observed in the knockdown mice. This is the first in vivo demonstration that a common regulator of G protein signaling is involved in the two separate, but interactive circadian timing systems, LEO and FEO. The present study also suggests that liver and/or thalamus regulate the food-entrained circadian behavior through G protein-mediated signal transduction pathway(s).

Evidence for enhanced M3 muscarinic receptor function and sensitivity to atrial arrhythmia in the RGS2-deficient mouse.

Atrial fibrillation (AF) is the most common arrhythmia seen in general practice. Muscarinic ACh receptors (M2R, M3R) are involved in vagally induced AF. M2R and M3R activate the heterotrimeric G proteins, G(i) and G(q), respectively, by promoting GTP binding, and these in turn activate distinct K(+) channels. Signaling is terminated by GTP hydrolysis, a process accelerated by regulator of G protein signaling (RGS) proteins. RGS2 is selective for G(q) and thus may regulate atrial M3R signaling. We hypothesized that knockout of RGS2 (RGS2(-/-)) would render the atria more susceptible to electrically induced AF. One-month-old male RGS2(-/-) and C57BL/6 wild-type (WT) mice were instrumented for intracardiac electrophysiology. Atrial effective refractory periods (AERPs) were also determined in the absence and presence of carbachol, atropine, and/or the selective M3R antagonist darifenacin. Susceptibility to electrically induced AF used burst pacing and programmed electrical stimulation with one extrastimulus. Real-time RT-PCR measured atrial and ventricular content of RGS2, RGS4, M2R, M3R, and M4R mRNA. AERP was lower in RGS2(-/-) compared with WT mice in both the high right atrium (HRA) (30 +/- 1 vs. 34 +/- 1 ms, P < 0.05) and mid right atrium (MRA) (21 +/- 1 vs. 24 +/- 1 ms, P < 0.05). Darifenacin eliminated this difference (HRA: 37 +/- 2 vs. 39 +/- 2 ms, and MRA: 30 +/- 2 vs. 30 +/- 1, P > 0.4). RGS2(-/-) were more susceptible than WT mice to atrial tachycardia/fibrillation (AT/F) induction (11/22 vs. 1/25, respectively, P < 0.05). Muscarinic receptor expression did not differ between strains, whereas M2R expression was 70-fold higher than M3R (P < 0.01). These results suggest that RGS2 is an important cholinergic regulator in the atrium and that RGS2(-/-) mice have enhanced susceptibility to AT/F via enhanced M3 muscarinic receptor activity.

PCR differential display-based identification of regulator of G protein signaling 10 as the target gene in human colon cancer cells induced by black tea polyphenol theaflavin monogallate.

We have previously reported that black tea polyphenol theaflavin monogallate (TF-2) suppressed COX-2 and induced apoptosis in human colon cancer cells [Lu, J.B., Ho, C.-T., Ghai, G., Chen, K.Y., 2000. Differential effects of theaflavin monogallates on cell growth, apoptosis and Cox-2 gene expression in cancerous versus normal cells. Cancer Res. 60, 6465-6471.]. We now extended the study by using PCR-based differential display to search for genes that were selectively induced by TF-2. Here we report the identification of Regulator of G-binding protein signaling 10 (RGS10) as the target gene, which was induced as early as 4 h after the TF-2 treatment. We then examined the effect of TF-2 on several other RGS genes and found that, in addition to RGS10, TF-2 induced the expression of RGS14, but not RGS4. Other tea polyphenols, including theaflavin-3,3'-digallate (TF-3) and (-) epigallocatechin-3-gallate (EGCG), also induced the expression of RGS10 and RGS14, but not RGS4. However, theaflavin (TF-1), which does not contain the gallate moiety, was ineffective. These results showed for the first time that tea polyphenols can induce the expression of selective RGS genes and that the gallate moiety may be important in this induction. In view of the role of RGS in modulating G-protein mediated signal transduction pathways, our findings may be significant since dysregulation of G-signaling is prominently implicated in carcinogenesis.

Chemical genetics reveals an RGS/G-protein role in the action of a compound.

We report here on a chemical genetic screen designed to address the mechanism of action of a small molecule. Small molecules that were active in models of urinary incontinence were tested on the nematode Caenorhabditis elegans, and the resulting phenotypes were used as readouts in a genetic screen to identify possible molecular targets. The mutations giving resistance to compound were found to affect members of the RGS protein/G-protein complex. Studies in mammalian systems confirmed that the small molecules inhibit muscarinic G-protein coupled receptor (GPCR) signaling involving G-alphaq (G-protein alpha subunit). Our studies suggest that the small molecules act at the level of the RGS/G-alphaq signaling complex, and define new mutations in both RGS and G-alphaq, including a unique hypo-adapation allele of G-alphaq. These findings suggest that therapeutics targeted to downstream components of GPCR signaling may be effective for treatment of diseases involving inappropriate receptor activation.

Comparative analysis of the expressed genome of the infective juvenile entomopathogenic nematode, Heterorhabditis bacteriophora.

We report the first cDNA-sequencing project of the entomopathogenic nematode, Heterorhabditis bacteriophora. A total of 1246 expressed sequence tags (ESTs) were generated by random sequencing of clones from a cDNA library of the infective juvenile stage. The ESTs were annotated resulting in 1072 useful ESTs that were categorized into functional categories according to Kyoto Encyclopedia of Genes and Genomes. Approximately 459 of 1072 ESTs (43%) had significant similarities to annotated sequences in GenBank. Of these, 417 had significant similarities to the free-living nematode Caenorhanditis elegans proteins. Most ESTs (18%) belonged to the genetic information processing category followed by metabolism (15% ESTs) and environmental information processing (15%) pathways. Several interesting ESTs were found that may have roles in the infectivity and survival of infective juveniles. These included proteases, dauer pathway genes (akt-1, pdk-1 & daf-7) and aging and stress resistance genes such as superoxide dismutase (sod-4), heat shock genes (hsp-4 & hsp-6), and eat genes, and signaling proteins like G-protein coupled receptors, regulators of G-protein signaling (rgs), and serine/threonine kinases. Other interesting ESTs include systemic RNAi defective protein (sid-1), ribonuclease III family members (rnh-2 &rnc) and transposase gene (Tc3A). About 67% of the ESTs did not find matches in any of the searched databases suggesting potentially novel genes in this enomopathogenic nematode. Note: Sequences described in this paper have been deposited in Genbank under the accessions DN 152655-DN 152999, and DN 153000-DN 153726.

In situ hybridization analysis of RGS mRNA regulation and behavioral phenotyping of RGS mutant mice.

To elucidate the functional role of regulators of G-protein signaling (RGS) in vivo, it will be critical to (i) determine how RGS activity is altered in response to a variety of manipulations and (ii) observe how the system is changed when RGS protein function is altered genetically. To facilitate studies of dynamic regulation of RGS protein activity, this article describes detailed methods for radioisotopic in situ hybridization for semiquantitative analyses of RGS mRNA abundances. Toward characterizing the functional differences in mice with genetically altered RGS activities, this article describes a subset of behavioral tests suitable for assaying sensitivities to drugs of abuse. These protocols should provide valuable guidance for investigators to establish these methodologies independently in their own laboratories and, over time, increase our understanding of RGS function in vivo.

Yeast-based screening for inhibitors of RGS proteins.

This article provides information on two screening platforms for the identification of regulators of G-protein signaling (RGS) protein modulators. Utilization of the yeast pheromone response pathway enabled the creation of a functional screen for RGS4 modulators. The RGSZ1-focused screen employs advances in yeast two-hybrid screening technologies and targets the protein-protein interface of the RGS domain/Galpha interaction. Moreover, the RGSZ1 screen provides the opportunity to multiplex the screening of two targets of interest, given the development of two different luciferase reporter genes that enabled sequential determination and intraassay controls. The screen formats were validated, implemented, and conducted as automated 384-well, liquid-based, high-throughput small molecule screens. Primary "hits" were confirmed using benchtop 96-well formats of these assays and advanced to in vitro functional evaluation assays. The yeast-based assay platforms provide robust cellular assays that result in the identification of small molecule modulators for both RGS targets. These molecules can serve both as tools with which to probe biological implications of RGS proteins and as potential starting points toward the development of novel modulators of G-protein signaling pathways. Such modulators may show potential for controlling and treating diseases resulting from inappropriate activity of G-protein signaling pathways.

RGS3 mediates a calcium-dependent termination of G protein signaling in sensory neurons.

G proteins modulate synaptic transmission. Regulators of G protein signaling (RGS) proteins accelerate the intrinsic GTPase activity of Galpha subunits, and thus terminate G protein activation. Whether RGS proteins themselves are under cellular control is not well defined, particularly in native cells. In dorsal root ganglion neurons overexpressing RGS3, we find that G protein signaling is rapidly terminated (or "desensitized") by calcium influx through voltage-gated channels. This rapid desensitization is most likely mediated by direct binding of calcium to RGS3, as deletion of an EF-hand domain in RGS3 abolishes both the desensitization (observed physiologically) and a calcium-RGS3 interaction (observed in a gel-shift assay). A naturally occurring variant of RGS3 that lacks the EF hand neither binds calcium nor produces rapid desensitization, giving rise instead to a slower calcium-dependent desensitization that is attenuated by a calmodulin antagonist. Thus, activity-evoked calcium entry in sensory neurons may provide differential control of G protein signaling, depending on the isoform of RGS3 expressed in the cells. In complex neural circuits subjected to abundant synaptic inhibition by G proteins (as occurs in dorsal spinal cord), rapid termination of inhibition by electrical activity by EF hand-containing RGS3 may ensure the faithful transmission of information from the most active sensory inputs.

Chick RGS2L demonstrates concentration-dependent selectivity for pertussis toxin-sensitive and -insensitive pathways that inhibit L-type Ca2+ channels.

In neuronal cells, the influx of Ca2+ ions through voltage-dependent L-type calcium (L) channels couples excitation to multiple cellular functions. In addition to voltage, several neurotransmitters, hormones and cytokines regulate L channel gating via binding to G-protein-coupled receptors. Intracellular molecules that modify G-protein activity - such as regulator of G-protein-signalling (RGS) proteins - are therefore potential candidates for regulating Ca2+ influx through L channels. Here we show that a novel RGS2 splice variant from chick dorsal root ganglion (DRG) neurons, RGS2L, reduces bradykinin (BK)-mediated inhibition of neuronal L channels and accelerates recovery from inhibition. Chick RGS2 reduces the inhibition mediated by both the pertussis toxin (PTX)-sensitive (Gi/o-coupled) and the PTX-insensitive (presumably Gq/11-coupled) pathways. However, we demonstrate for the first time in a living cell that the extent of coupling to each pathway varies with RGS2L concentration. A low concentration of recombinant chick RGS2L (10 nM) preferentially reduces the inhibition mediated by the PTX-insensitive pathway, whereas a 100-fold higher concentration attenuates both PTX-sensitive- and PTX-insensitive-mediated components equally. Our data suggest that factors promoting RGS2L gene induction may regulate Ca2+ influx through L channels by recruiting low-affinity interactions with Gi/o that are absent at basal RGS2L levels.

Direct interactions between the heterotrimeric G protein subunit G beta 5 and the G protein gamma subunit-like domain-containing regulator of G protein signaling 11: gain of function of cyan fluorescent protein-tagged G gamma 3.

We used fluorescence resonance energy transfer imaging of enhanced cyan fluorescent protein (CFP)-tagged and enhanced yellow fluorescent protein (YFP)-tagged protein pairs to examine the hypothesis that G protein gamma subunit-like (GGL) domain-containing regulators of G protein signaling (RGS) can directly bind to the Gbeta5 subunit of heterotrimeric G proteins in vivo. We observed that Gbeta5 could interact with Ggamma2 and Ggamma13, after their expression in human embryonic kidney 293 cells. Interestingly, although untagged Ggamma3 did not interact with Gbeta5, CFP-tagged Ggamma3 strongly interacted with YFP-tagged Gbeta5 in FRET studies. Moreover, CFP-Ggamma3 supported Ca(2+) channel inhibition when paired with Gbeta5 or YFP-Gbeta5, indicating a "gain of function" for CFP-Ggamma3. Gbeta5 could also interact with RGS11 and its N-terminal, but not its C-terminal domain. On the other hand, RGS11 did not interact with Gbeta1. These studies demonstrate that the GGL domain-containing N terminus of RGS 11 can directly interact with Gbeta5 in vivo and supports the hypothesis that this interaction may contribute to the specificity of Gbeta5 interactions with cellular effector molecules.

Detailed localization of regulator of G protein signaling 2 messenger ribonucleic acid and protein in the rat brain.

Regulator of G protein signaling (RGS) proteins are a recently identified family of proteins which dampen G protein-coupled receptor-mediated signaling by accelerating the intrinsic GTPase activity of Galpha subunits of heterotrimeric G proteins. More than 20 different RGSs have been identified and at least 10 are expressed in the CNS. The present study describes in detail the localization in the rat brain of one member of this family, RGS2. The distribution of RGS2 mRNA and protein has been studied in parallel by performing in situ hybridization and immunoautoradiography on adjacent rat brain sections. Our localization study reveals that RGS2 mRNA and protein are widely expressed in the brain. Protein and mRNA are mostly colocalized such as in neocortex, piriform cortex, caudate-putamen, septum, hippocampus, locus coeruleus. Some mismatches were also observed such as presence of mRNA but not protein in the paraventricular nucleus, the substantia nigra pars compacta and the red nucleus, suggesting that RGS2 protein is present in neuronal projections. Previous reports describing an induction of RGS2 mRNA in the rat striatum after psychostimulants (amphetamine, cocaine) led us to focus on the distribution of RGS2 in the basal ganglia circuitry. The absence of RGS2 mRNA and protein in the globus pallidus suggests that RGS2 would play its regulatory role more in the direct (striatonigral) than in the indirect (striatopallidal) striatal output pathway. In addition, to delineate the implication of RGS2 in pre- and/or postsynaptic functions in the basal ganglia, we performed lesions of the nigrostriatal pathway by 6-hydroxydopamine (6-OHDA) and striatal quinolinic acid lesions. The 6-OHDA lesion did not modify RGS2 mRNA or protein levels in the caudate-putamen whereas the intrastriatal quinolinic acid infusion caused a marked reduction of RGS2 mRNA and protein in the lesioned zone. These data indicate that RGS2 is predominantly expressed in intrinsic striatal neurons. Moreover, the absence of detectable change in RGS2 expression after injections of 6-OHDA suggests also that RGS2 is not primarily involved in the hypersensitization of postsynaptic dopamine receptors observed after lesion of the nigrostriatal pathway.

Altered gene expression in cerebral capillaries of stroke-prone spontaneously hypertensive rats.

Stroke-prone spontaneously hypertensive rats (SHRSP) are a well-characterized, genetic model for stroke. We showed earlier that the structure and function of the tight junctions in SHRSP blood-brain barrier endothelial cells is disturbed prior to stroke. To investigate the molecular events leading to endothelial dysfunction in SHRSP cerebral capillaries, we carried out suppression subtractive hybridization (SSH) in combination with a cDNA filter screening step. We identified two cDNA fragments that were upregulated in SHRSP, compared to stroke-resistant spontaneously hypertensive rats (SHR), and found open reading frames of 133 and 138 amino acids, respectively. These peptides did not match any known proteins in public databases. A third upregulated SHRSP cDNA fragment was identified as the rat sulfonylurea receptor 2B (SUR2B). We also isolated and cloned the cDNA of the rat homologue for the mouse G-protein signaling 5 (RGS5) regulator. This regulator was downregulated in SHRSP. We used in situ hybridization to show that rat RGS5 is expressed in the brain capillary endothelium and in the choroid plexus. Our findings may lead to the identification of new stroke-related genes.

Regional distribution of regulators of G-protein signaling (RGS) 1, 2, 13, 14, 16, and GAIP messenger ribonucleic acids by in situ hybridization in rat brain.

Regulators of G-protein signaling (RGS) proteins are a novel family of GTPase-activating proteins that interact with Galpha subunits of the Gi/o, Gz, Gq and G(12/13) subfamilies to dampen G-protein-coupled receptor (GPCR)-mediated signaling by accelerating intrinsic Galpha-GTPase activity. In the present study, we report on messenger ribonucleic acid (mRNA) localization in rat brain of six RGS genes by in situ hybridization. The distribution patterns of RGS2, RGS13, RGS14 and GAIP (Galpha interacting protein) overlapped in most brain regions examined. Highest regional expression was observed for RGS2 in the cerebral cortical layers, striatum, hippocampal formation, several thalamic and hypothalamic nuclei and hindbrain regions such as the pontine, interpeduncular and dorsal raphe nuclei. Levels of RGS14 mRNA closely paralleled those of RGS2 expression levels throughout most brain regions. RGS13 mRNA was enriched in the hippocampal formation, amygdala, mammillary nuclei as well as the pontine and interpeduncular nuclei. GAIP expression levels were highest in the hippocampal formation with moderate to low levels present in all other regions studied. Of the six RGS genes probed, RGS16 mRNA displayed a discrete localization predominantly in the thalamic midline/intralaminar and principal relay nuclei, and the hypothalamic suprachiasmatic nucleus. RGS1 mRNA signal was not detected in brain. In conclusion, the in situ hybridization studies for RGS2, RGS13, RGS14, RGS16 and GAIP mRNAs extend our knowledge of the distribution of RGS genes expressed in the rat central nervous system, and indicate overlapping RGS-enriched regions that may be indicative of functional diversification in GPCR signaling pathway modulation.

Control of the cystic fibrosis transmembrane conductance regulator by alphaG(i) and RGS proteins.

The cystic fibrosis transmembrane conductance regulator (CFTR) has been shown previously to be regulated by inhibitory G proteins. In the present study, we demonstrate inhibition of CFTR by alphaG(i2) and alphaG(i1), but not alphaG(0), in Xenopus oocytes. We further examined whether regulators of G protein signaling (RGS) proteins interfere with alphaG(i)-dependent inhibition of CFTR. Activation of CFTR by IBMX and forskolin was attenuated in the presence of alphaG(i2), indicating inhibition of CFTR by alphaG(i2) in Xenopus oocytes. Coexpression of the proteins RGS3 and RGS7 together with CFTR and alphaG(i2) partially recovered activation by IBMX/forskolin. 14-3-3, a protein that is known to interfere with RGS proteins, counteracted the effects of RGS3. These data demonstrate the regulation of CFTR by alphaG(i) in Xenopus oocytes. Because RGS proteins interfere with the G protein-dependent regulation of CFTR, this may offer new potential pathways for pharmacological intervention in cystic fibrosis.

RGS4 is arginylated and degraded by the N-end rule pathway in vitro.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. We used an expression-cloning screen to search for mouse proteins that are degraded by the ubiquitin/proteasome-dependent N-end rule pathway in a reticulocyte lysate. One substrate thus identified was RGS4, a member of the RGS family of GTPase-activating proteins that down-regulate specific G proteins. A determinant of the RGS4 degradation signal (degron) was located at the N terminus of RGS4, because converting cysteine 2 to either glycine, alanine, or valine completely stabilized RGS4. Radiochemical sequencing indicated that the N-terminal methionine of the lysate-produced RGS4 was replaced with arginine. Since N-terminal arginine is a destabilizing residue not encoded by RGS4 mRNA, we conclude that the degron of RGS4 is generated through the removal of N-terminal methionine and enzymatic arginylation of the resulting N-terminal cysteine. RGS16, another member of the RGS family, was also found to be an N-end rule substrate. RGS4 that was transiently expressed in mouse L cells was short-lived in these cells. However, the targeting of RGS4 for degradation in this in vivo setting involved primarily another degron, because N-terminal variants of RGS4 that were stable in reticulocyte lysate remained unstable in L cells.

Palmitoylation of a conserved cysteine in the regulator of G protein signaling (RGS) domain modulates the GTPase-activating activity of RGS4 and RGS10.

RGS4 and RGS10 expressed in Sf9 cells are palmitoylated at a conserved Cys residue (Cys(95) in RGS4, Cys(66) in RGS10) in the regulator of G protein signaling (RGS) domain that is also autopalmitoylated when the purified proteins are incubated with palmitoyl-CoA. RGS4 also autopalmitoylates at a previously identified cellular palmitoylation site, either Cys(2) or Cys(12). The C2A/C12A mutation essentially eliminates both autopalmitoylation and cellular [(3)H]palmitate labeling of Cys(95). Membrane-bound RGS4 is palmitoylated both at Cys(95) and Cys(2/12), but cytosolic RGS4 is not palmitoylated. RGS4 and RGS10 are GTPase-activating proteins (GAPs) for the G(i) and G(q) families of G proteins. Palmitoylation of Cys(95) on RGS4 or Cys(66) on RGS10 inhibits GAP activity 80-100% toward either Galpha(i) or Galpha(z) in a single-turnover, solution-based assay. In contrast, when GAP activity was assayed as acceleration of steady-state GTPase in receptor-G protein proteoliposomes, palmitoylation of RGS10 potentiated GAP activity >/=20-fold. Palmitoylation near the N terminus of C95V RGS4 did not alter GAP activity toward soluble Galpha(z) and increased G(z) GAP activity about 2-fold in the vesicle-based assay. Dual palmitoylation of wild-type RGS4 remained inhibitory. RGS protein palmitoylation is thus multi-site, complex in its control, and either inhibitory or stimulatory depending on the RGS protein and its sites of palmitoylation.