Toxin-catalyzed ADP-ribosylation Research Articles

Homogeneous Dual-Parametric-Coupled Assay for Simultaneous Nucleotide Exchange and KRAS/RAF-RBD Interaction Monitoring.

We have developed a rapid and sensitive single-well dual-parametric method introduced in linked RAS nucleotide exchange and RAS/RAF-RBD interaction assays. RAS mutations are frequent drivers of multiple different human cancers, but the development of therapeutic strategies has been challenging. Traditionally, efforts to disrupt the RAS function have focused on nucleotide exchange inhibitors, GTP-RAS interaction inhibitors, and activators increasing GTPase activity of mutant RAS proteins. As the amount of biological knowledge grows, targeted biochemical assays enabling high-throughput screening have become increasingly interesting. We have previously introduced a homogeneous quenching resonance energy transfer (QRET) assay for nucleotide binding studies with RAS and heterotrimeric G proteins. Here, we introduce a novel homogeneous signaling technique called QTR-FRET, which combine QRET technology and time-resolved Förster resonance energy transfer (TR-FRET). The dual-parametric QTR-FRET technique enables the linking of guanine nucleotide exchange factor-induced Eu3+-GTP association to RAS, monitored at 615 nm, and subsequent Eu3+-GTP-loaded RAS interaction with RAF-RBD-Alexa680 monitored at 730 nm. Both reactions were monitored in a single-well assay applicable for inhibitor screening and real-time reaction monitoring. This homogeneous assay enables separable detection of both nucleotide exchange and RAS/RAF interaction inhibitors using low nanomolar protein concentrations. To demonstrate a wider applicability as a screening and real-time reaction monitoring method, the QTR-FRET technique was also applied for G(i)α GTP-loading and pertussis toxin-catalyzed ADP-ribosylation of G(i)α, for which we synthesized a novel γ-GTP-Eu3+ molecule. The study indicates that the QTR-FRET detection technique presented here can be readily applied to dual-parametric assays for various targets.

Ric-8A-mediated stabilization of the trimeric G protein subunit Gαi is inhibited by pertussis toxin-catalyzed ADP-ribosylation

The heterotrimeric G protein subunit Gαi can be activated by G protein-coupled receptors and the cytosolic protein Ric-8A, the latter of which is also known to prevent ubiquitin-dependent degradation of Gαi. Here we show that the amounts of the three Gαi-related proteins Gαi1, Gαi2, and Gαi3, but not that of Gαq, are rapidly decreased by cell treatment with pertussis toxin (PTX). The decrease appears to be due to ADP-ribosylation of Gαi, because PTX treatment does not affect the amount of a mutant Gαi2 carrying alanine substitution for Cys352, the residue that is ADP-ribosylated by the toxin. The presence of endogenous and exogenous Ric-8A increases Gαi stability as shown in cells treated with the protein synthesis inhibitor cycloheximide; however, Ric-8A fails to efficiently stabilize ADP-ribosylated Gαi. The failure agrees with the inability of Ric-8A to bind to ADP-ribosylated Gαi both in vitro and in vivo. Thus PTX appears to exert its pathological effects at least in part by converting Gαi to an unstable ADP-ribosylated form, in addition to the well-known inability of ADP-ribosylated Gαi to transduce signals triggered by G protein-coupled receptors.

Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin.

Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia.

Total glucosides of paeony inhibit the proliferation of fibroblast-like synoviocytes through the regulation of G proteins in rats with collagen-induced arthritis

The aim of this study was to investigate the expression of G proteins in fibroblast-like synoviocytes (FLSs) from rats with collagen-induced arthritis (CIA) and to determine the effect of total glucosides of paeony (TGP). CIA rats were induced with chicken type II collagen (CCII) in Freund's complete adjuvant. The rats with experimental arthritis were randomly separated into five groups and then treated with TGP (25, 50, and 100mg/kg) from days 14 to 35 after immunization. The secondary inflammatory reactions were evaluated through the polyarthritis index and histopathological changes. The level of cyclic adenosine monophosphate (cAMP) was measured by radioimmunoassay. The FLS proliferation response was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The toxin-catalyzed ADP-ribosylation of G proteins was performed through autoradiography. The results show that TGP (25, 50, and 100mg/kg) significantly decreased the arthritis scores of CIA rats and improved the histopathological changes. TGP inhibited the proliferation of FLSs and increased the level of cAMP. Moreover, the FLS proliferation and the level of Gαi expression were significantly increased, but the level of Gαs expression was decreased after stimulation with IL-1β (10ng/ml) in vitro. TGP (12.5 and 62.5μg/ml) significantly inhibited the FLS proliferation and regulated the balance between Gαi and Gαs. These results demonstrate that TGP may exert its anti-inflammatory effects through the suppression of FLS proliferation, which may be associated with its ability to regulate the balance of G proteins. Thus, TGP may have potential as a therapeutic agent for the treatment of rheumatoid arthritis.

The Inhibitory G Protein G<sub>i</sub> Identified as Pertussis Toxin-Catalyzed ADP-Ribosylation

Pertussis toxin (PTX) produced by Bordetella pertussis was first introduced by Ui and his colleagues in research on signal transduction under the name islet-activating protein in 1979, when the mechanism of toxin-induced stimulation of insulin release from pancreatic islets was reported in the rat. The stimulatory effect of PTX in vivo results from the blockage of α(2)-adrenergic receptor-mediated inhibition of insulin release. The receptor-induced inhibition of cAMP formation was also abolished in pancreatic islets isolated from PTX-treated rats, suggesting that the toxin caused uncoupling of adenylyl cyclase inhibition from receptor stimulation. The action of PTX on isolated membranes required a cytosolic factor, nicotinamide adenine dinucleotide (NAD), and the uncoupling induced by PTX was shown to be due to the toxin-catalyzed ADP-ribosylation of a 41-kDa protein with NAD as another substrate. The 41-kDa PTX substrate was soon identified and purified as the α-subunit of the inhibitory G protein that transmits an inhibitory signal from membrane receptors to adenylyl cyclase. After demonstration of the molecular mechanism of PTX, the toxin was widely utilized as a probe for identifying and analyzing major αβγ-trimeric G proteins. Thus, PTX-sensitive G proteins appeared to carry positive and negative signals from many membrane receptors to a variety of effectors other than adenylyl cyclase.

ChemInform Abstract: Synthesis of Diphthamide: The Target of Diphtheria Toxin Catalyzed ADP-Ribosylation in Protein Synthesis Elongation Factor 2.

ChemInform Abstract: Synthesis of Diphthamide: The Target of Diphtheria Toxin Catalyzed ADP-Ribosylation in Protein Synthesis Elongation Factor 2.

ADP-ribosylation of arginine

Arginine adenosine-5′-diphosphoribosylation (ADP-ribosylation) is an enzyme-catalyzed, potentially reversible posttranslational modification, in which the ADP-ribose moiety is transferred from NAD+ to the guanidino moiety of arginine. At 540 Da, ADP-ribose has the size of approximately five amino acid residues. In contrast to arginine, which, at neutral pH, is positively charged, ADP-ribose carries two negatively charged phosphate moieties. Arginine ADP-ribosylation, thus, causes a notable change in size and chemical property at the ADP-ribosylation site of the target protein. Often, this causes steric interference of the interaction of the target protein with binding partners, e.g. toxin-catalyzed ADP-ribosylation of actin at R177 sterically blocks actin polymerization. In case of the nucleotide-gated P2X7 ion channel, ADP-ribosylation at R125 in the vicinity of the ligand-binding site causes channel gating. Arginine-specific ADP-ribosyltransferases (ARTs) carry a characteristic R-S-EXE motif that distinguishes these enzymes from structurally related enzymes which catalyze ADP-ribosylation of other amino acid side chains, DNA, or small molecules. Arginine-specific ADP-ribosylation can be inhibited by small molecule arginine analogues such as agmatine or meta-iodobenzylguanidine (MIBG), which themselves can serve as targets for arginine-specific ARTs. ADP-ribosylarginine specific hydrolases (ARHs) can restore target protein function by hydrolytic removal of the entire ADP-ribose moiety. In some cases, ADP-ribosylarginine is processed into secondary posttranslational modifications, e.g. phosphoribosylarginine or ornithine. This review summarizes current knowledge on arginine-specific ADP-ribosylation, focussing on the methods available for its detection, its biological consequences, and the enzymes responsible for this modification and its reversal, and discusses future perspectives for research in this field.

Enhanced Sensitivity to Cholera Toxin in ADP-Ribosylarginine Hydrolase-Deficient Mice

Cholera toxin (CT) produced by Vibrio cholerae causes the devastating diarrhea of cholera by catalyzing the ADP-ribosylation of the alpha subunit of the intestinal Gs protein (Gsalpha), leading to characteristic water and electrolyte losses. Mammalian cells contain ADP-ribosyltransferases similar to CT and an ADP-ribosyl(arginine)protein hydrolase (ADPRH), which cleaves the ADP-ribose-(arginine)protein bond, regenerating native protein and completing an ADP-ribosylation cycle. We hypothesized that ADPRH might counteract intoxication by reversing the ADP-ribosylation of Gsalpha. Effects of intoxication on murine ADPRH-/- cells were greater than those on wild-type cells and were significantly reduced by overexpression of wild-type ADPRH in ADPRH-/- cells, as evidenced by both ADP-ribose-arginine content and Gsalpha modification. Similarly, intestinal loops in the ADPRH-/- mouse were more sensitive than their wild-type counterparts to toxin effects on fluid accumulation, Gsalpha modification, and ADP-ribosylarginine content. Thus, CT-catalyzed ADP-ribosylation of cell proteins can be counteracted by ADPRH, which could function as a modifier gene in disease. Further, our study demonstrates that enzymatic cross talk exists between bacterial toxin ADP-ribosyltransferases and host ADP-ribosylation cycles. In disease, toxin-catalyzed ADP-ribosylation overwhelms this potential host defense system, resulting in persistence of ADP-ribosylation and intoxication of the cell.

Phosducin-like Protein Regulates G-Protein βγ Folding by Interaction with Tailless Complex Polypeptide-1α: DEPHOSPHORYLATION OR SPLICING OF PhLP TURNS THE SWITCH TOWARD REGULATION OF Gβγ FOLDING

Phosducin-like Protein Regulates G-Protein βγ Folding by Interaction with Tailless Complex Polypeptide-1α: DEPHOSPHORYLATION OR SPLICING OF PhLP TURNS THE SWITCH TOWARD REGULATION OF Gβγ FOLDING

Trimeric G proteins in crustacean (Callinectes sapidus) Y-organs: occurrence and functional link to protein synthesis

Crustacean Y-organs produce ecdysteroid molting hormones. Regulation of ecdysteroidogenesis appears to be complex, involving regulatory ligands (including but not limited to molt-inhibiting hormone, an eyestalk neurohormone) and the capacity of the Y-organs to respond to those ligands. Available data indicate cell signaling pathways involving cAMP, cGMP, or both may be involved in regulation of Y-organ function. Trimeric G proteins link receptor occupancy to regulation of intracellular cAMP levels. In studies reported here, we have assessed the occurrence of G proteins in blue crab (Callinectes sapidus) Y-organs, and the link of G proteins to Y-organ function. Bacterial toxin-catalyzed ADP-ribosylation revealed a PTX-sensitive (alpha i-like) protein in Y-organ membranes, but failed to reveal a CTX-sensitive (alpha s-like) protein in Y-organ membranes. Western blotting with primary antibodies raised against conserved regions of mammalian G proteins detected an alpha i-immunoreactive protein (approximately 40 kDa) and two alpha s-immunoreactive proteins (approximately 50 and approximately 57 kDa) in Y-organ membrane preparations. Incubation of Y-organ membrane fractions with cholera toxin significantly suppressed incorporation of [35S]-methionine into TCA-precipitable Y-organ proteins, but had no detectable effect on ecdysteroidogenesis in short-term (6 h) incubations. The combined results indicate that C. sapidus Y-organs possess both Gi and Gs proteins, and that alpha s is functionally linked to regulation of glandular protein synthesis.

Mutation of cysteine 214 in Gi1 alpha subunit abolishes its endogenous GTPase activity.

The functional consequences of the mutation of a conserved Cys-214 in Galpha(i1) have been investigated. We reported herein that substitutions of Cys-214 of Galpha(i1) to either alanine or tryptophan abolished the intrinsic GTPase activity. Free phosphate release from [32P]GTP-bound Galpha(i1) C214A or [32P]GTP-bound Galpha(i1) C214W was at least 30-fold lower than that of the wild-type Galpha(i1) in single-turnover GTPase assays. Consistently, tryptic proteolysis of C214A and C214W proteins showed that they were partially protected by GTP, further confirming that the GTPase activity in both mutant proteins was impaired. Expression of C214A or C214W mutants in Chinese hamster ovary K1 cells caused significant inhibition of forskolin-stimulated adenylate cyclase activity. However, the mutations did not significantly affect the GTP[S] (guanosine 5'-[gamma-[35S]thio]triphosphate)-binding activity. Both C214A and C214W mutants serve as good substrates for pertussis toxin-catalysed ADP ribosylation, indicating that they interact well with betagamma subunits. Moreover, RGS4 protein, a GTPase-activating protein for Galpha(i1), cannot interact with Cys-214 mutants even in the presence of AlF4-, which induces the transition state of Galpha. In summary, our findings suggest that C214A or C214W are GTPase-deficient mutants and can functionally serve as constitutively active forms of Galpha(i1) in cells.

Alterations of adenylyl cyclase and G proteins in aortocaval shunt-induced heart failure.

Unlike most other experimental models of congestive heart failure, the volume overload model induced by aortocaval shunt (AVS) in rats was found to exhibit enhanced beta-adrenoceptor (beta-AR) signaling. To study whether the adenylyl cyclase (AC)-G protein system is involved in such a change, we examined cardiac AC activity and protein content as well as G(s)alpha and G(i)alpha activities, protein contents, and mRNA levels in both left (LV) and right (RV) ventricles at the failing stage (16 wk after surgery). Basal and forskolin-stimulated AC activities were significantly increased in both LV and RV from the failing hearts; this change was associated with an upregulation of type V/VI AC protein. In contrast to 5'-guanylyl imidodiphosphate and NaF, the stimulatory effect of isoproterenol on AC was increased in the failing heart. Although G(s)alpha and G(i)alpha protein contents in the failing hearts were not altered, the mRNA level for G(s)alpha was decreased by 20% and that for G(i)alpha was increased by 20%. In addition, the activity of G(s)alpha, but not G(i)alpha, as assessed by toxin-catalyzed ADP ribosylation, was significantly decreased in the failing heart. Losartan and imidapril treatments improved cardiac function and attenuated alterations in mRNA levels for G(s)alpha and G(i)alpha proteins, as well as G(s)alpha activity, without affecting changes in AC protein content or activities in heart failure due to volume overload. These data suggest that increased AC activity may contribute to the enhanced beta-AR signaling in the AVS model of heart failure, whereas alterations in gene expression for G proteins may be of an adaptive nature at this stage of heart failure.

Effect of chronic alcohol consumption on Go protein in rat brain

Almost all of the important pathophysiological targets for ethanol in the nervous system appear to be specific membrane proteins involved in signal transduction. In this paper we have examined levels and functionality of the α subunit of the Go protein (Goα) in cerebral cortex and cerebellum from rats that have chronically ingested ethanol, by using immunoblotting and pertussis toxin-catalyzed ADP-ribosylation experiments. Goα protein levels were increased in plasma membranes from the two brain areas, and this increase was shown to specifically affect Go 1α, one of the two isoforms of the Goα subunit. Results obtained here lead us to suggest that increased Go 1α in plasma membranes would counteract a modified and non-functional protein generated during chronic alcohol treatment.

The β6/α5 regions of Gαi2 and GαoA increase the promiscuity of Gα16 but are insufficient for pertussis toxin-catalyzed ADP-ribosylation

Replacement of β6/α5 region at the C-terminus on Gα16 with Gαz-specific residues has been shown to broaden the promiscuity of Gα16. Here, we substituted the last 44 residues of Gα16 with the corresponding region from either Gαi2 or GαoA (16i44 and 16o44). 16i44 and 16o44 chimeras were more effective than Gα16 at coupling to Gi-linked δ-opioid, μ-opioid, and Xenopus melatonin MT1c receptors when coexpressed in green monkey fibroblast (COS-7) cells. 16i44, but not 16o44, also enhanced the formyl peptide-induced stimulation of phospholipase C activity. Both chimeras were resistant to pertussis toxin-catalyzed [32P]ADP-ribosylation, despite the fact that pertussis toxin partially inhibited the chimera-mediated stimulation of phospholipase Cβ. The use of Gαt1 as a Gβγ scavenger revealed that the pertussis toxin-sensitivity can be attributed to endogenous Gβγ subunits released from Gi/o. Although incorporation of a Gαi-like β6/α5 region into the C-terminus of Gα16 increases its promiscuity, this region is not sufficient to support recognition by pertussis toxin.

Chronic morphine-induced changes in mu-opioid receptors and G proteins of different subcellular loci in rat brain.

Prolonged exposure to opioid agonists can induce adaptive changes resulting in tolerance and dependence. Here, rats were rendered tolerant by subcutaneous injections of increasing doses of morphine from 10 to 60 mg/kg for 3, 5, or 10 consecutive days. Binding parameters of the mu-opioid receptor in subcellular fractions were measured with [(3)H]DAMGO ([D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin). Although the density of surface mu-sites did not change after the 5-day morphine treatment, up-regulation of synaptic plasma membrane binding was detected after the 10-day drug administration. In contrast, the number of mu-binding sites in a light vesicle or microsomal fraction (MI) was elevated by 68 and 30% after 5 and 10 days of morphine exposure, respectively. The up-regulated MI mu-sites displayed enhanced coupling to G proteins compared with those detected in saline-treated controls. Pertussis toxin catalyzed ADP ribosylation, and Western blotting with specific antisera was used to quantitate chronic morphine-induced changes in levels of various G protein alpha-subunits. Morphine treatment of 5 days and longer induced significant increases in levels of Galpha(o), Galpha(i1), and Galpha(i2) in MI fractions that are part of an adaptation process. Up-regulation of intracellular mu-sites may be the result of post-translational changes and in part de novo synthesis. The results provide the first evidence that distinct regulation of intracellular mu-opioid receptor G protein coupling and G protein levels may accompany the development of morphine tolerance.

Dexras1/AGS-1 Inhibits Signal Transduction from the Gi-coupled Formyl Peptide Receptor to Erk-1/2 MAP Kinases

Dexras1 is a novel GTP-binding protein (G protein) that was recently discovered on the basis of rapid mRNA up-regulation by glucocorticoids in murine AtT-20 corticotroph cells and in several primary tissues. The human homologue of Dexras1, termed activator of G protein signaling-1 (AGS-1), has been reported to stimulate signaling by G(i) heterotrimeric G proteins independently of receptor activation. The effects of Dexras1/AGS-1 on receptor-initiated signaling by G(i) have not been examined. Here we report that Dexras1 inhibits ligand-dependent signaling by the G(i)-coupled N-formyl peptide receptor (FPR). Dexras1 and FPR were transiently co-expressed in both COS-7 and HEK-293 cells. Activation of FPR by ligand (N-formyl-methionine-leucine-phenylalanine (f-MLF)) caused phosphorylation of endogenous Erk-1/2 that was reduced by co-expression of Dexras1. Direct effects of Dexras1 on the activity of co-expressed, epitope-tagged Erk-2 (hemagglutinin (HA)-Erk-2) were measured by immune complex in vitro kinase assay. Expression of Dexras1 alone resulted in a 1.9- to 4.9-fold increase in HA-Erk-2 activity; expression of the unliganded FPR alone resulted in a 6.2- to 8.1-fold increase in HA-Erk-2 activity. Stimulation of FPR by f-MLF produced a further 8- to 10-fold increase in HA-Erk-2 activity over the basal (non-ligand-stimulated) state, and this ligand-dependent activity was attenuated at the time points of maximal activity by co-expression of Dexras1 (reduced 31 +/- 6.8% in COS-7 at 10 min and 86 +/- 9.2% in HEK-293 at 5 min, p < 0.01 for each). Expression of Dexras1 did not influence protein expression of FPR or Erk, suggesting that the inhibitory effects of Dexras1 reflect a functional alteration in the signaling cascade from FPR to Erk. Expression of Dexras1 had no effect on expression of G(i)alpha species, but significantly impaired pertussis toxin-catalyzed ADP-ribosylation of membrane-associated G(i)alpha. Expression of Dexras1 also significantly decreased in vitro binding of GTPgammaS in f-MLF-stimulated membranes of cells co-transfected with FPR. These data suggest that Dexras1 inhibits signal transduction from FPR to Erk-1/2 through an effect that is very proximal to receptor-G(i) coupling. While Dexras1 weakly activates Erk in the resting state, more potent effects are evident in the modulation of ligand-stimulated receptor signal transduction, where Dexras1 functions as an inhibitor rather than activator of the Erk mitogen-activated protein kinase signaling cascade.

Activation Mechanism of Gi and Go by Reactive Oxygen Species

Reactive oxygen species are proposed to work as intracellular mediators. One of their target proteins is the alpha subunit of heterotrimeric GTP-binding proteins (Galpha(i) and Galpha(o)), leading to activation. H(2)O(2) is one of the reactive oxygen species and activates purified Galpha(i2). However, the activation requires the presence of Fe(2+), suggesting that H(2)O(2) is converted to more reactive species such as c*OH. The analysis with mass spectrometry shows that seven cysteine residues (Cys(66), Cys(112), Cys(140), Cys(255), Cys(287), Cys(326), and Cys(352)) of Galpha(i2) are modified by the treatment with *OH. Among these cysteine residues, Cys(66), Cys(112), Cys(140), Cys(255), and Cys(352) are not involved in *OH-induced activation of Galpha(i2). Although the modification of Cys(287) but not Cys(326) is required for subunit dissociation, the modification of both Cys(287) and Cys(326) is necessary for the activation of Galpha(i2) as determined by pertussis toxin-catalyzed ADP-ribosylation, conformation-dependent change of trypsin digestion pattern or guanosine 5'-3-O-(thio)triphosphate binding. Wild type Galpha(i2) but not Cys(287)- or Cys(326)-substituted mutants are activated by UV light, singlet oxygen, superoxide anion, and nitric oxide, indicating that these oxidative stresses activate Galpha(i2) by the mechanism similar to *OH-induced activation. Because Cys(287) exists only in G(i) family, this study explains the selective activation of G(i)/G(o) by oxidative stresses.

Understanding salivary fluid and protein secretion.

Understanding salivary fluid and protein secretion.

Preparation of myristoylated Arf1 and Arf6 proteins.

ADP-ribosylation factor (Arf) proteins were first identified as cofactors for cholera toxin-catalyzed ADP-ribosylation of the heterotrimeric G-protein Gs. Subsequent cloning led to the discovery that Arfs were part of a group of GTP-binding proteins that is a subfamily of the ras superfamily. The Arf family is comprised of the Arf proteins and related Arf-like (Arl) proteins. Activity as cofactors for cholera toxin distinguishes the Arf proteins from the Arls. The Arf proteins can be further subdivided into class I (comprised of Arf 1 and Arf3 in humans), class II (comprised of Arf4 and Arf5), and class III (comprised of Arf6). Arfs have been found in all eukaryotes studied. Multiple Arf proteins occur within single organisms and within single cells, and Arf orthologs are highly conserved between species. The most extensively studied mammalian Arfs are Arf 1 and Arf6, which have been found to function as regulators of membrane traffic and the actin cytoskeleton (, , , , , , , , ). In biochemical studies, Arfs have also been found to activate phospholipase D (PLD) and phosphatidylinositol 4-phosphate 5-kinase, and to bind to several putative effectors such as arfaptin, arfophilin, and the GGAs (, , , , , , , , , , ). Unlike other ras family members which are prenylated at the C-terminus, Arfs are myristoylated on an N-terminal glycine in a cotranslational event catalyzed by N-myristoyl transferase (NMT) (, , , ). This lipid modification is crucial for activity.

Activation of the Luteinizing Hormone/Choriogonadotropin Hormone Receptor Promotes ADP Ribosylation Factor 6 Activation in Porcine Ovarian Follicular Membranes

Previously we demonstrated in a cell-free ovarian follicular plasma membrane model that agonist-dependent desensitization of the luteinizing hormone/choriogonadotropin receptor (LH/CG R) is GTP-dependent, mimicked by the addition of ADP-ribosylation factor (ARF) nucleotide binding site opener, which acts as a guanine nucleotide exchange factor for ARFs 1 and 6, and selectively inhibited by synthetic N-terminal ARF6 peptides. We therefore sought direct evidence that activation of the LH/CG R promotes activation of ARF1 and/or ARF6. Using a classic ARF activation assay, the cholera toxin-catalyzed ADP-ribosylation of G alpha(s), results show that LH/CG R activation stimulates an ARF protein by a brefeldin A-independent mechanism. Synthetic N-terminal inhibitory ARF6 but not ARF1 peptide blocks LH/CG R-stimulated ARF activity. LH/CG R activation also promotes the binding of a photoaffinity GTP analog to a protein that migrates on one- and two-dimensional polyacrylamide gel electrophoresis with ARF6. These results suggest that ARF6 is the predominant ARF activated by the LH/CG R. To activate ARF6, the LH/CG R does not appear to signal through the C-terminal regions of G alpha(i) or G alpha(q) or through the second or third intracellular loops or the N terminus of the cytoplasmic tail of the LH/CG R. Although exogenous recombinant ARNO promotes only a small increase in ARF6 activation in the presence of activated LH/CG R, hCG-stimulated ARF6 activation is reduced to basal levels by catalytically inactive ARF nucleotide binding-site opener. These results provide direct evidence that LH/CG R activation leads to the activation of membrane-delimited ARF6.