ARL13B promotes angiogenesis and glioma growth by activating VEGFA-VEGFR2 signaling.

BACKGROUND: Tumor angiogenesis is essential for solid tumor progression, invasion and metastasis. The aim of this study was to identify potential signaling pathways involved in tumor angiogenesis. METHODS: Genetically engineered mouse models were used to investigate the effects of endothelial ARL13B(ADP-ribosylation factor-like GTPase 13B) over-expression and deficiency on retinal and cerebral vasculature. An intracranially transplanted glioma model and a subcutaneously implanted melanoma model were employed to examine the effects of ARL13B on tumor growth and angiogenesis. Immunohistochemistry was used to measure ARL13B in glioma tissues, and scRNA-seq was used to analyze glioma and endothelial ARL13B expression. GST-fusion protein-protein interaction and co-immunoprecipitation assays were used to determine the ARL13B-VEGFR2 interaction. Immunobloting, qPCR, dual-luciferase reporter assay and functional experiments were performed to evaluate the effects of ARL13B on VEGFR2 activation. RESULTS: Endothelial ARL13B regulated vascular development of both the retina and brain in mice. Also, ARL13B in endothelial cells regulated the growth of intracranially transplanted glioma cells and subcutaneously implanted melanoma cells by controlling tumor angiogenesis. Interestingly, this effect was attributed to ARL13B interaction with VEGFR2, through which ARL13B regulated the membrane and ciliary localization of VEGFR2 and consequently activated its downstream signaling in endothelial cells. Consistent with its oncogenic role, ARL13B was highly expressed in human gliomas, which was well correlated with the poor prognosis of glioma patients. Remarkably, ARL13B, transcriptionally regulated by ZEB1, enhanced the expression of VEGFA by activating Hedgehog signaling in glioma cells. CONCLUSIONS: ARL13B promotes angiogenesis and tumor growth by activating VEGFA-VEGFR2 signaling. Thus, targeting ARL13B might serve as a potential approach for developing an anti-glioma or anti-melanoma therapy.

Knockdown of ARL5B Induces Mitochondrial-mediated Apoptosis and Inhibits Glycolysis in Breast Cancer Cells by Activating MDA5 Signaling.

AIM: Mitochondria are essential for energy metabolism in the tumor microenvironment and the survival of cancer cells. BACKGROUND: ADP-ribosylation factor-like GTPase 5b (ARL5B) has been found to be associated with mitochondrial dysfunction and breast cancer (BC) progression, but the underlying mechanism needs to be further understood. OBJECTIVE: We investigated the effects of ARL5B on the apoptosis and glycolysis of breast cancer cells and its underlying mechanisms. METHODS: Quantitative reverse transcription-PCR (qRT-PCR) and western blot assays were used to detect the expression of ARL5B in breast cancer tissues and cells. An ARL5B loss-of-function assay was performed to verify its role in BC development. RESULTS: ARL5B was upregulated in breast cancer tissues and cell lines. ARL5B knockdown induced apoptosis and activated the mitochondrial pathway in breast cancer cells. Interestingly, the inhibition of ARL5B repressed the aerobic glycolysis of breast cancer cells. The role of ARL5B in breast cancer cells was exerted by mediating the activation of viral RNA sensor MDA5-evoked signaling. Silencing ARL5B triggered MDA5 signaling by upregulating the key proteins involved in the MDA5 pathway. Importantly, MDA5 silencing reversed the effects of ARL5B knockdown on mitochondrial-mediated apoptosis and glycolysis, whereas poly (I:C), as a ligand for MDA5, further enhanced ARL5B knockdown- facilitated mitochondrial apoptosis and the inhibition of glycolysis. CONCLUSION: The knockdown of ARL5B activated MDA5 signaling and thus led to the enhanced mitochondrial- mediated apoptosis and glycolysis inhibition in breast cancer cells. Our study suggested that ARL5B might be a potential therapy target for BC.

Cell Death Signaling Pathway Induced by Cholix Toxin, a Cytotoxin and eEF2 ADP-Ribosyltransferase Produced by Vibrio cholerae.

Pathogenic microorganisms produce various virulence factors, e.g., enzymes, cytotoxins, effectors, which trigger development of pathologies in infectious diseases. Cholera toxin (CT) produced by O1 and O139 serotypes of Vibrio cholerae (V. cholerae) is a major cytotoxin causing severe diarrhea. Cholix cytotoxin (Cholix) was identified as a novel eukaryotic elongation factor 2 (eEF2) adenosine-diphosphate (ADP)-ribosyltransferase produced mainly in non-O1/non-O139 V. cholerae. The function and role of Cholix in infectious disease caused by V. cholerae remain unknown. The crystal structure of Cholix is similar to Pseudomonas exotoxin A (PEA) which is composed of an N-terminal receptor-recognition domain and a C-terminal ADP-ribosyltransferase domain. The endocytosed Cholix catalyzes ADP-ribosylation of eEF2 in host cells and inhibits protein synthesis, resulting in cell death. In a mouse model, Cholix caused lethality with severe liver damage. In this review, we describe the mechanism underlying Cholix-induced cytotoxicity. Cholix-induced apoptosis was regulated by mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways, which dramatically enhanced tumor necrosis factor-α (TNF-α) production in human liver, as well as the amount of epithelial-like HepG2 cancer cells. In contrast, Cholix induced apoptosis in hepatocytes through a mitochondrial-dependent pathway, which was not stimulated by TNF-α. These findings suggest that sensitivity to Cholix depends on the target cell. A substantial amount of information on PEA is provided in order to compare/contrast this well-characterized mono-ADP-ribosyltransferase (mART) with Cholix.

BIG1 controls macrophage pro-inflammatory responses through ARF3-mediated PI(4,5)P2 synthesis.

Sepsis is caused by a dysregulated host inflammatory response to serious infections resulting in life-threatening organ dysfunction. The high morbidity and mortality make sepsis still a major clinical problem. Here, we investigated the roles of Brefeldin A-inhibited guanine nucleotide-exchange factor 1 (BIG1) in the pathogenesis process of sepsis and the underlying mechanisms. We found myeloid cell-specific BIG1 knockout (BIG1 cKO) significantly reduced the mortality and organ damage in LPS-induced and CLP-induced polymicrobial sepsis mouse model. The serum concentration and mRNA expression of pro-inflammatory cytokines including TNF-α, IL-6, IL-1ß, and IL-12 were obviously decreased in BIG1 cKO mice. In bone marrow-derived macrophages or THP-1 cells, BIG1 deficiency caused an inhibited ARF3 activation, which reduced PI(4,5)P2 synthesis and the recruitment of TIRAP to the plasma membrane through inhibiting the activation of PIP5K induced by LPS, and eventually resulted in the inhibitory activity of TLR4-MyD88 signaling pathway. These results reveal a crucial new role of BIG1 in regulating macrophage inflammation responses, and provide evidence for BIG1 as a potential promising therapeutic target in sepsis.

The GTPase ARFRP1 affects lipid droplet protein composition and triglyceride release from intracellular storage of intestinal Caco-2 cells.

Intestinal release of dietary triglycerides via chylomicrons is the major contributor to elevated postprandial triglyceride levels. Dietary lipids can be transiently stored in cytosolic lipid droplets (LDs) located in intestinal enterocytes for later release. ADP ribosylation factor-related protein 1 (ARFRP1) participates in processes of LD growth in adipocytes and in lipidation of lipoproteins in liver and intestine. This study aims to explore the impact of ARFRP1 on LD organization and its interplay with chylomicron-mediated triglyceride release in intestinal-like Caco-2 cells. Suppression of Arfrp1 reduced release of intracellularly derived triglycerides (0.69-fold) and increased the abundance of transitional endoplasmic reticulum ATPase TERA/VCP, fatty acid synthase-associated factor 2 (FAF2) and perilipin 2 (Plin2) at the LD surface. Furthermore, TERA/VCP and FAF2 co-occurred more frequently with ATGL at LDs, suggesting a reduced adipocyte triglyceride lipase (ATGL)-mediated lipolysis. Accordingly, inhibition of lipolysis reduced lipid release from intracellular storage pools by the same magnitude as Arfrp1 depletion. Thus, the lack of Arfrp1 increases the abundance of lipolysis-modulating enzymes TERA/VCP, FAF2 and Plin2 at LDs, which might decrease lipolysis and reduce availability of fatty acids for triglyceride synthesis and their release via chylomicrons.

The Father, Son and Cholix Toxin: The Third Member of the DT Group Mono-ADP-Ribosyltransferase Toxin Family.

The cholix toxin gene (chxA) was first identified in V. cholerae strains in 2007, and the protein was identified by bioinformatics analysis in 2008. It was identified as the third member of the diphtheria toxin group of mono-ADP-ribosyltransferase toxins along with P. aeruginosa exotoxin A and C. diphtheriae diphtheria toxin. Our group determined the structure of the full-length, three-domain cholix toxin at 2.1 Å and its C-terminal catalytic domain (cholixc) at 1.25 Å resolution. We showed that cholix toxin is specific for elongation factor 2 (diphthamide residue), similar to exotoxin A and diphtheria toxin. Cholix toxin possesses molecular features required for infection of eukaryotes by receptor-mediated endocytosis, translocation to the host cytoplasm and inhibition of protein synthesis. More recently, we also solved the structure of full-length cholix toxin in complex with NAD+ and proposed a new kinetic model for cholix enzyme activity. In addition, we have taken a computational approach that revealed some important properties of the NAD+-binding pocket at the residue level, including the role of crystallographic water molecules in the NAD+ substrate interaction. We developed a pharmacophore model of cholix toxin, which revealed a cationic feature in the side chain of cholix toxin active-site inhibitors that may determine the active pose. Notably, several recent reports have been published on the role of cholix toxin as a major virulence factor in V. cholerae (non-O1/O139 strains). Additionally, FitzGerald and coworkers prepared an immunotoxin constructed from domains II and III as a cancer treatment strategy to complement successful immunotoxins derived from P. aeruginosa exotoxin A.

Characterization of Cholix toxin-induced apoptosis in HeLa cells.

Cholix toxin (Cholix) is a novel ADP-ribosylating cytotoxin produced by Vibrio cholerae, which utilizes eukaryotic elongation factor 2 as a substrate and acts by a mechanism similar to that of diphtheria toxin and Pseudomonas exotoxin A. First it was found that Cholix-treated HeLa cells exhibited caspase-dependent apoptosis, whereas intestinal cells such as Caco-2, HCT116, and RKO did not. Here we investigated Cholix-induced cell death signaling pathways in HeLa cells. Cholix-induced cytochrome c release into cytosol was initiated by specific conformational changes of pro-apoptotic Bak associated with Bax. Silencing of bak/bax genes or bak gene alone using siRNA significantly suppressed cytochrome c release and caspase-7 activation, but not activation of caspases-3 and -9. Although pretreatment with a caspase-8 inhibitor (Z-IETD-FMK) reduced Cholix-induced cytochrome c release and activation of caspases-3, -7, and -9, cytotoxicity was not decreased. Pretreatment with Z-YVAD-FMK, which inhibits caspase-1, -4, and -5, suppressed not only cytochrome c release, activation of caspase-3, -7, -8, or -9, and PARP cleavage, but also cytotoxicity, indicating that caspase-1, -4, and -5 activation is initiated at an early stage of Cholix-induced apoptosis and promotes caspase-8 activation. These results show that the inflammatory caspases (caspase-1, -4, and -5) and caspase-8 are responsible for both mitochondrial signals and other caspase activation. In conclusion, we showed that Cholix-induced caspase activation plays an essential role in generation of apoptotic signals, which are mediated by both mitochondria-dependent and -independent pathways.

Internalization of exogenous ADP-ribosylation factor 6 (Arf6) proteins into cells.

Endogenous Arf6 is a myristoylated protein mainly involved in endosomal membrane traffic and structural organization at the plasma membrane. It has been shown that Arf6 mediates cancer cell invasion and shedding of plasma membrane microvesicles derived from tumor cells. In this article, we determined that Arf6 proteins both in the GDP and GTPγS bound forms can enter cells when simply added in the cell culture medium without requiring the myristoyl group. The GTPγS bound can enter cells at a faster rate than the GDP-bound Arf6. Despite the role of the endogenous Arf6 in endocytosis and membrane trafficking, the internalization of exogenous Arf6 may involve non-endocytic processes. As protein therapeutics is becoming important in medicine, we examined the effect of the uptake of Arf6 proteins on cellular functions and determined that exogenous Arf6 inhibits proliferation, invasion, and migration of cells. Future studies of the internalization of Arf6 mutants will reveal key residues that play a role in the internalization of Arf6 and its interaction and possible structural conformations bound to the plasma membrane.

ADP-ribosylation factors regulate the development of CT signaling in immature human enterocytes.

Diarrheal disease is a major cause of morbidity and mortality in infants and children worldwide. Evidence suggests that the interaction of immature human enterocytes with bacteria and their enterotoxins may account for the increased susceptibility of neonates to diarrheal diseases. However, the precise mechanisms that contribute to the excessive response to cholera toxin by the immature gut are largely unknown. Our aim was to characterize the cellular/molecular changes in Gs(alpha) during gut development. In this study, a colonic human epithelial cell line (T84) was used as representative of a mature enterocyte and a human fetal primary small intestinal cell line (H4) as representative of an immature enterocyte. Using our cell culture model of human intestinal development, we provide consistent evidence that cholera toxin (CT)-mediated Gs(alpha) activation in fetal enterocytes differs from that of mature enterocytes, and the difference may be related to ADP-ribosylation factor (ARF) interaction with the CT-signaling process. Here we demonstrated that ARF1 may play a critical role in clathrin-mediated CT trafficking through the endoplasmic reticulum and Golgi and that ARF6 may facilitate clathrin-mediated CT endocytosis that leads to enhanced Gs(alpha) activation by CT. Collectively, these findings support our hypothesis that there is a developmentally regulated intestinal cellular response to bacterial exotoxins involving complex cellular events that accounts for the increased incidence and severity of toxogenic diarrhea during infancy.

ARF6 and EFA6A regulate the development and maintenance of dendritic spines.

The cellular and molecular mechanisms underlying the development and maintenance of dendritic spines are not fully understood. ADP-ribosylation factor 6 (ARF6) is a small GTPase known to regulate actin remodeling and membrane traffic. Here, we report involvement of ARF6 and exchange factor for ARF6 (EFA6A) in the regulation of spine development and maintenance. An active form of ARF6 promotes the formation of dendritic spines at the expense of filopodia. EFA6A promotes spine formation in an ARF6 activation-dependent manner. Knockdown of ARF6 and EFA6A by small interfering RNA decreases spine formation. Live imaging indicates that ARF6 knockdown decreases the conversion of filopodia to spines and the stability of early spines. The spine-promoting effect of ARF6 is partially blocked by Rac1. ARF6 and EFA6A protect mature spines from inactivity-induced destabilization. These results suggest that ARF6 and EFA6A may regulate the conversion of filopodia to spines and the stability of both early and mature spines.

Growth factors mobilize multiple pools of KCa channels in developing parasympathetic neurons: role of ADP-ribosylation factors and related proteins.

In developing ciliary ganglion (CG) neurons, movement of functional large-conductance (BK type) Ca(2+)-activated K+ (K(Ca)) channels to the cell surface is stimulated by the endogenous growth factors TGF(beta)1 and beta-neuregulin-1 (NRG1). Here we show that a brief NRG1 treatment (0.5-1.5 h) mobilizes K(Ca) channels in a post-Golgi compartment, but longer treatments (>3.5 h) mobilize K(Ca) channels located in the endoplasmic reticulum or Golgi apparatus. Specifically, the effects of 3.5 h NRG1 treatment were completely blocked by treatments that disrupt Golgi apparatus function. These include inhibition of microtubules, or inhibition of the ADP-ribosylation factor-1 (ARF1) system by brefeldin A, by over-expression of dominant-negative ARF1, or over-expression of an ARF1 GTPase-activating protein that blocks ARF1 cycling between GTP- and GDP-bound states. These treatments had no effect on stimulation of K(Ca) evoked by 1.5 h treatment with NRG1, indicating that short-term responses to NRG1 do not require an intact Golgi apparatus. By contrast, both the acute and sustained effects of NRG1 were inhibited by treatments that block trafficking processes that occur close to the plasma membrane. Thus mobilization of K(Ca) was blocked by treatments than inhibit ADP-ribosylation factor-6 (ARF6) signaling, including overexpression of dominant-negative ARF6, dominant-negative ARNO, or dominant-negative phospholipase D1. TGF(beta)1, the effects of which on K(Ca) are much slower in onset, is unable to selectively mobilize channels in the post-Golgi pool, and its effects on K(Ca) are completely blocked by inhibition of microtubules, Golgi function and also by plasma membrane ARF6 and phospholipase D1 signaling.

Studies of the roles of ADP-ribosylation factors and phospholipase D in phorbol ester-induced membrane ruffling.

In this study, we have explored the roles of ADP-ribosylation factors (ARFs), phospholipase D (PLD) isozymes, and arfaptins in phorbol ester (PMA)-induced membrane ruffling in HeLa cells. PMA stimulation induced ruffling and translocated cortactin to the plasma membrane. The cortactin translocation was inhibited by dominant negative (DN)-ARF6, DN-ARF1, and DN-Rac1, but not by DN-RhoA and DN-Cdc42. The inability of DN-forms of ARF6, ARF1, and Rac1 to affect PLD activity in response to PMA indicated that this enzyme was not activated via these small G proteins and that its activation was not essential for the induction of ruffling. Endogenous-ARF1, -ARF6, and -Rac1 existed in the ruffling region along with cortactin after PMA stimulation. DN-ARF1 had no effect on the ruffling induced by DA-ARF6 or DA-Rac1, and DN-ARF6 had no effect on that induced by DA-ARF1 or DA-Rac1. On the other hand DN-Rac1 suppressed the effect of DA-ARF6 but not that of DA-ARF1. These results suggest that PMA causes membrane ruffling via an ARF6-Rac1 pathway and also an ARF1 pathway operating in parallel. Overexpression of PLD1 and PLD2 inhibited PMA-induced cortactin translocation and actin-cortactin complex formation, supporting the view that these enzymes are not required for ruffling, but actually suppress it. We conclude that PMA-induced membrane ruffling is caused via ARF6-Rac1 and ARF1 pathways operating in parallel and that PLD may be inhibitory.

Inactivation of enhanced expression of G(i) proteins by pertussis toxin attenuates the development of high blood pressure in spontaneously hypertensive rats.

We have previously shown that the enhanced expression of G(i) proteins in spontaneously hypertensive rats (SHR) that precedes the development of high blood pressure may be one of the contributing factors in the pathogenesis of hypertension. In the present study, we demonstrate that the inactivation of G(i) proteins by intraperitoneal injection of pertussis toxin (PT, 1.5 micro g/100 g body wt) into 2-week-old prehypertensive SHR prevented the development of hypertension up to 4 weeks and that, thereafter, it started to increase and reached the same level found in untreated SHR after 6 weeks. A second injection of PT after 4 weeks delayed the increase in blood pressure for another week. The PT-induced decrease in blood pressure in 6-week-old SHR was associated with a decreased level of G(i)alpha-2 and G(i)alpha-3 proteins in the heart, as determined by in vitro ADP ribosylation and immunoblotting. The decreased level of G(i) proteins was reflected in decreased G(i) functions. Furthermore, an augmentation of blood pressure to the same level in PT-treated SHR as found in untreated SHR was associated with enhanced expression and function of G(i). These results indicate that the inactivation of G(i) proteins by PT treatment in prehypertensive SHR attenuates the development of hypertension and suggest that the enhanced levels of G(i) proteins that result in the decreased levels of cAMP and associated impaired cellular functions may be contributing factors in the pathogenesis of hypertension in SHR.

Lymphocyte trafficking through the blood-brain barrier is dependent on endothelial cell heterotrimeric G-protein signaling.

We have previously shown that the engagement of ICAM-1 on brain endothelial cells (EC) results in the propagation of EC signaling pathways that are necessary for efficient lymphocyte migration across the tight vascular barriers of the brain. Signaling via this receptor alone, however, is unlikely to explain the differential recruitment of leukocytes at different vascular beds. In this study, we investigated the role of EC heterotrimeric G-protein-mediated signaling in supporting transendothelial migration of T lymphocytes. Treatment of brain EC monolayers with pertussis toxin (PTX) resulted in ADP-ribosylation of G-protein alpha subunits and inhibition (>80%) of lymphocyte migration without affecting lymphocyte adhesion. Aortic and high endothelial venule EC treated identically resulted in only partial inhibition of lymphocyte migration (<40%). Expression of ribosylation-resistant (PTX-insensitive) G-protein alpha subunits in brain EC restored their ability to support lymphocyte migration after pretreatment with PTX. Treatment of brain EC with PTX did not inhibit ICAM-1-stimulated tyrosine phosphorylation of focal adhesion kinase, suggesting the effects of PTX in inhibiting EC facilitation of lymphocyte migration are distinct from activation of EC through ICAM-1. We conclude that a heterotrimeric G-protein-mediated signaling pathway in brain EC is essential for efficient transendothelial migration of T lymphocytes into the brain.

Human ARF binds E2F1 and inhibits its transcriptional activity.

The INK4a/ARF locus which is frequently inactivated in human tumours encodes two different tumour suppressive proteins, p16(INK4a) and ARF. p16(INK4a) is a major component of the RB pathway. ARF is part of an ARF-mdm2-p53 network that exerts a negative control on hyperproliferative signals emanating from oncogenic stimuli. Among these is the transcription factor E2F1, a final effector of the RB pathway, that induces ARF expression. Recent data suggest that ARF function is not restricted to the p53 pathway. However, ARF target(s) implicated in this p53-independent function remains to be identified. We show that ARF is able to inhibit the proliferation of human cell lines independently of their p53 status. In this context, we demonstrate that ARF interacts physically with E2F1 and inhibits its transcriptional activity. Moreover, we show that mdm2 is required for the modulation of E2F1 activity by ARF. Beside the well-known p53 and mdm2 partners, these results identify E2F1 as a new ARF target. Thus, ARF can be viewed as a dual-acting tumour suppressor protein in both the p53 and RB pathways, further emphasizing its role in tumour surveillance.

Distinct roles for the N- and C-terminal regions in the cytotoxicity of pierisin-1, a putative ADP-ribosylating toxin from cabbage butterfly, against mammalian cells.

Pierisin-1 is an 850-aa cytotoxic protein found in the cabbage butterfly, Pieris rapae, and has been suggested to consist of an N-terminal region with ADP-ribosyltransferase domain and of a C-terminal region that might have a receptor-binding domain. To elucidate the role of each region, we investigated the functions of various fragments of pierisin-1. In vitro expressed polypeptide consisting of amino acid residues 1-233 or 234-850 of pierisin-1 alone did not show cytotoxicity against human cervical carcinoma HeLa cells. However, the presence of both polypeptides in the culture medium showed some of the original cytotoxic activity. Introduction of the N-terminal polypeptide alone by electroporation also induced cell death in HeLa cells, and even in the mouse melanoma MEB4 cells insensitive to pierisin-1. Thus, the N-terminal region has a principal role in the cytotoxicity of pierisin-1 inside mammalian cells. Analyses of incorporated pierisin-1 indicated that the entire protein, regardless of whether it consisted of a single polypeptide or two separate N- and C-terminal polypeptides, was incorporated into HeLa cells. However, neither of the terminal polypeptides was incorporated when each polypeptide was present separately. These findings indicate that the C-terminal region is important for the incorporation of pierisin-1. Moreover, presence of receptor for pierisin-1 in the lipid fraction of cell membrane was suggested. The cytotoxic effects of pierisin-1 were enhanced by previous treatment with trypsin, producing "nicked" pierisin-1. Generation of the N-terminal fragment in HeLa cells was detected after application of intact entire molecule of pierisin-1. From the above observations, it is suggested that after incorporation of pierisin-1 into the cell by interaction of its C-terminal region with the receptor in the cell membrane, the entire protein is cleaved into the N- and C-terminal fragments with intracellular protease, and the N-terminal fragment then exhibits cytotoxicity.

Sequential activation of heterotrimeric and monomeric G proteins mediates PLD activity in smooth muscle.

The identity of G proteins mediating CCK-stimulated phospholipase D (PLD) activity was determined in intestinal smooth muscle cells. CCK-8 activated G(q/11), G(13), and G(12), and the monomeric G proteins Ras-homology protein (RhoA) and ADP ribosylation factor (ARF). Activation of RhoA, but not ARF, was mediated by G(13) and inhibited by Galpha(13) antibody. CCK-stimulated PLD activity was partly mediated by RhoA and could be inhibited to the same extent (47 +/- 2% to 53 +/- 6%) by 1) a dominant negative RhoA mutant, 2) RhoA antibody or Galpha(13) antibody, and 3) Clostridium botulinum C3 exoenzyme. PLD activity was also inhibited by ARF antibody, and the effect was additive to that of RhoA antibody or C3 exoenzyme. PLD activity was inhibited by calphostin C, bisindolylmaleimide I, and a selective protein kinase C (PKC)-alpha inhibitor; the inhibition was additive to that of ARF and RhoA antibodies and C3 exoenzyme. In contrast, activated G(12) was not coupled to RhoA or ARF, and Galpha(12) antibody augmented PLD activity. Thus agonist-stimulated PLD activity is mediated additively by G(13)-dependent RhoA and by ARF and PKC-alpha and is modulated by an inhibitory G(12)-dependent pathway.

The ADP ribosylation factor nucleotide exchange factor ARNO promotes beta-arrestin release necessary for luteinizing hormone/choriogonadotropin receptor desensitization.

Desensitization of guanine nucleotide binding protein-coupled receptors is a ubiquitous phenomenon characterized by declining effector activity upon persistent agonist stimulation. The luteinizing hormone/choriogonadotropin receptor (LH/CGR) in ovarian follicles exhibits desensitization of effector adenylyl cyclase activity in response to the mid-cycle surge of LH. We have previously shown that uncoupling of the agonist-activated LH/CGR from the stimulatory G protein (G(s)) is dependent on GTP and attributable to binding of beta-arrestin present in adenylyl cyclase-rich follicular membrane fraction to the third intracellular (3i) loop of the receptor. Here, we report that LH/CGR-dependent desensitization is mimicked by ADP ribosylation factor nucleotide-binding site opener, a guanine nucleotide exchange factor of the small G proteins ADP ribosylation factors (Arfs) 1 and 6, and blocked by synthetic N-terminal Arf6 peptide, suggesting that the GTP-dependent step of LH/CGR desensitization is receptor-dependent Arf6 activation. Arf activation by GTP and ADP ribosylation factor nucelotide-binding site opener promotes the release of docked beta-arrestin from the membrane, making beta-arrestin available for LH/CGR; Arf6 but not Arf1 peptides block beta-arrestin release from the membrane. Thus, LH/CGR appears to activate two membrane delimited signaling cascades via two types of G proteins: heterotrimeric G(s) and small G protein Arf6. Arf6 activation releases docked beta-arrestin necessary for receptor desensitization, providing a feedback mechanism for receptor self-regulation.