Role of ADP-ribosylation factor 6 (ARF6) in gastric acid secretion

ADP-ribosylation factor (ARF) proteins are monomeric GTPases that are essential for membrane transport and exocytosis in a number of secretory cells. We investigated ARF6, the activation of which is insensitive to brefeldin A, to determine whether it regulates membrane traffic in gastric parietal cells. ARF6 translocated from cytosol to tubulovesicle in the presence of GTPgammaS, a potential inhibitor of acid secretion in permeabilized cells, whereas under the Mg2+-chelated condition where activity of ARF-GTPase activating protein is inhibited, ARF6 translocated to the apical secretory membrane. Immunohistochemical examination revealed that ARF6 mainly located in parietal cell within the gastric glands, and it translocated from the cytosol to the intracellular canaliculi when the glands were stimulated. These results indicated that the distribution of ARF6 between cytosol and the two different membranes was regulated by its GTPase activity. In cultured gastric glands infected with adenovirus expressing ARF6 Q67L, a mutant lacking GTP hydrolysis activity, gastric acid secretion was inhibited. These results suggest that ARF6 regulates gastric acid secretion in parietal cell and that the GTP hydrolysis cycle of ARF6 is essential for the activation pathway.     

ADP-ribosylation factor (ARF) interaction is not sufficient for yeast GGA protein function or localization

Golgi-localized gamma-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ~25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs and VPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.     

Synthesis and biological evaluation of (phenylpiperazinyl-propyl)arylsulfonamides as selective 5-HT2A receptor antagonists

A novel series of 5-HT_2A ligands that contain a (phenylpiperazinyl-propyl)arylsulfonamides skeleton was synthesized. Thirty-seven N-(cycloalkylmethyl)-4-methoxy-N-(3-(4-arylpiperazin-1-yl)propyl)-arylsulfonamide and N-(4-(4-arylpiperazin-1-yl)butan-2-yl)-arylsulfonamide compounds were obtained. The binding of these compounds to the 5-HT_2A, 5-HT_2C, and 5-HT₇ receptors was evaluated. Most of the compounds showed IC₅₀ values of less than 100 nM and exhibited high selectivity for the 5-HT_2A receptor. Among the synthesized compounds, 16a and 16d showed good affinity at 5-HT_2A (IC₅₀ = 0.7 nM and 0.5 nM) and good selectivity over 5-HT_2C (50–100 times) and 5-HT₇ (1500–3000 times).     

Novel C-terminal motif within Sec7 domain of guanine nucleotide exchange factors regulates ADP-ribosylation factor (ARF) binding and activation

ADP-ribosylation factors (ARFs) and their activating guanine nucleotide exchange factors (GEFs) play key roles in membrane traffic and signaling. All ARF GEFs share a ～200-residue Sec7 domain (Sec7d) that alone catalyzes the GDP to GTP exchange that activates ARF. We determined the crystal structure of human BIG2 Sec7d. A C-terminal loop immediately following helix J (loop>J) was predicted to form contacts with helix H and the switch I region of the cognate ARF, suggesting that loop>J may participate in the catalytic reaction. Indeed, we identified multiple alanine substitutions within loop>J of the full length and/or Sec7d of two large brefeldin A-sensitive GEFs (GBF1 and BIG2) and one small brefeldin A-resistant GEF (ARNO) that abrogated binding of ARF and a single alanine substitution that allowed ARF binding but inhibited GDP to GTP exchange. Loop>J sequences are highly conserved, suggesting that loop>J plays a crucial role in the catalytic activity of all ARF GEFs. Using GEF mutants unable to bind ARF, we showed that GEFs associate with membranes independently of ARF and catalyze ARF activation in vivo only when membrane-associated. Our structural, cell biological, and biochemical findings identify loop>J as a key regulatory motif essential for ARF binding and GDP to GTP exchange by GEFs and provide evidence for the requirement of membrane association during GEF activity.     

Tetrahydrothienopyridylbutyl-tetrahydrobenzindoles: new selective ligands of the 5-HT(7) receptor

The synthesis and the affinity for the 5-HT(7) receptor and other receptors of a novel series of fused-ring tetrahydropyridine derivatives are described. Some of the compounds showed high affinity for the 5-HT(7) receptor. Tetrahydrothienopyridylbutyl-tetrahydrobenzindoles and are potent ligands for the 5-HT(7) receptor, with high selectivity over the 5-HT(2) receptor and other receptors. These compounds should be useful tools for clarifying the biological role of the 5-HT(7) receptor.     

A regulatory role for ADP-ribosylation factor 6 (ARF6) in activation of the phagocyte NADPH oxidase

In activated neutrophils NADPH oxidase is regulated through various signaling intermediates, including heterotrimeric G proteins, kinases, GTPases, and phospholipases. ADP-ribosylation factor (ARF) describes a family of GTPases associated with phospholipase D (PLD) activation. PLD is implicated in NADPH oxidase activation, although it is unclear whether activation of PLD by ARF is linked to receptor-mediated oxidase activation. We explored whether ARF participates in NADPH oxidase activation by formyl-methionine-leucine-phenylalanine (fMLP) and whether this involves PLD. Using multicolor forward angle light scattering analyses to measure superoxide production in differentiated neutrophil-like PLB-985 cells, we tested enhanced green fluorescent fusion proteins of wild-type ARF1 or ARF6, or their mutant counterparts. The ARF6(Q67L) mutant defective in GTP hydrolysis caused increased superoxide production, whereas the ARF6(T27N) mutant defective in GTP binding caused diminished responses to fMLP. The ARF1 mutants had no effect on fMLP responses, and none of the ARF proteins affected phorbol 12-myristate 13-acetate-elicited oxidase activity. PLD inhibitors 1-butanol and 2, 3-diphosphoglycerate, or the ARF6(N48R) mutant assumed to be defective in PLD activation, blocked fMLP-elicited oxidase activity in transfected cells. The data suggest that ARF6 but not ARF1 modulates receptor-mediated NADPH oxidase activation in a PLD-dependent mechanism. Because PMA-elicited NADPH oxidase activation also appears to be PLD-dependent, but ARF-independent, ARF6 and protein kinase C may act through distinct pathways, both involving PLD.     

A new class of selective, non-basic 5-HT2A receptor antagonists

Based on an existing series of 5-HT2A receptor ligands containing a basic nitrogen, we designed a non-basic lead that had reduced affinity for both the 5-HT2A receptor and the IKr potassium channel. The present paper describes the development of this lead to a novel series of non-basic piperidine sulfonamides and amides that have high affinity for the 5-HT2A receptor, whilst maintaining excellent selectivity over off target activities such as the IKr channel. This work has shown that the proposed pharmacophore model for the 5-HT2A receptor which suggests that a basic nitrogen is required for the binding of ligands is questionable.     

Functional role of the NPxxY motif in internalization of the type 2 vasopressin receptor in LLC-PK1 cells

Interaction of the type 2 vasopressin receptor (V2R) with hormone causes desensitization and internalization. To study the role of the V2R NPxxY motif (which is involved in the clathrin-mediated endocytosis of several other receptors) in this process, we expressed FLAG-tagged wild-type V2R and a Y325F mutant V2R in LLC-PK1a epithelial cells that have low levels of endogenous V2R. Both proteins had a similar apical (35%) and basolateral (65%) membrane distribution. Substitution of Tyr³²⁵ with Phe³²⁵ prevented ligand-induced internalization of V2R determined by [³H]AVP binding and immunofluorescence but did not prevent ligand binding or signal transduction via adenylyl cyclase. Desensitization and resensitization of the V2R-Y325F mutation occurred independently of internalization. The involvement of clathrin in V2R downregulation was also shown by immunogold electron microscopy. We conclude that the NPxxY motif of the V2R is critically involved in receptor downregulation via clathrin-mediated internalization. However, this motif is not essential for the apical/basolateral sorting and polarized distribution of the V2R in LLC-PK1a cells or for adenylyl cyclase-mediated signal transduction. polarized cell culture; tyrosine motif; µ1b adaptor motif; protein traffic     

2-Aryl tryptamines: selective high-affinity antagonists for the h5-HT2A receptor

A series of 2-aryl tryptamines have been identified as high-affinity h5-HT_2A antagonists. Structure–activity relationship studies have shown that h5-HT_2A affinity can be attained via modifications to the tryptamine side chain and that selectivity over h5-HT_2C and hD₂ receptors can be controlled by suitable C-2 aryl groups.     

Role of the highly conserved Asp-Arg-Tyr motif in signal transduction of the CB2 cannabinoid receptor

The DRY motif, at the junction of transmembrane helix 3 and intracellular loop 2 of G protein-coupled receptors, is highly conserved. Mutations were introduced into the CB2 cannabinoid receptor to study the role of this motif in CB2 signaling. D mutations (DRY130-132AAA and D130A) markedly reduced binding of cannabinoid agonists, while no significant reduction was observed with R131A or Y132A. Mutating R (R131A) only partially reduced, and mutating Y (Y132A) more efficiently reduced the cannabinoid-induced inhibition of adenylyl cyclase. Thus, in CB2, D130 is involved in agonist binding, whereas Y seems to have a role in receptor downstream signaling.     

Differential subcellular localization of RIC-3 isoforms and their role in determining 5-HT3 receptor composition

RIC-3 has been identified as a chaperone molecule involved in promoting the functional expression of nicotinic acetylcholine and 5-HT(3) receptors in mammalian cells. In this study, we examined the effects of RIC-3a (isoform a) and a truncated isoform (isoform d) on RIC-3 localization, mobility, and aggregation and its effect on 5-HT3 receptor composition in mammalian cells. Human RIC-3a possesses an amino-terminal signal sequence that targets it to the endoplasmic reticulum where it is distributed within the reticular network, often forming large diffuse "slicks" and bright "halo" structures. RIC-3a is highly mobile within and between these compartments. Despite the propensity for RIC-3a to aggregate, its expression enhances the level of surface 5-HT3A (homomeric) receptors. In contrast, RIC-3a exerts an inhibitory action on the surface expression of heteromeric 5-HT3A/B receptors. RIC-3d exhibits an altered subcellular distribution, being localized to the endoplasmic reticulum, large diffuse slicks, tubulo-vesicular structures, and the Golgi. Bidirectional trafficking between the endoplasmic reticulum and Golgi suggests that RIC-3d constitutively cycles between these two compartments. In support of the large coiled-coil domain of RIC-3a being responsible for protein aggregation, RIC-3d, lacking this cytoplasmic domain, does not aggregate or induce the formation of bright aggregates. Regardless of these differences, isoform d is still capable of enhancing homomeric, and inhibiting heteromeric, 5-HT3 receptor expression. Thus, both isoforms of RIC-3 play a role in determining 5-HT3 receptor composition.     

A conserved peptide motif in Raver2 mediates its interaction with the polypyrimidine tract-binding protein

Raver2 was originally identified as a member of the hnRNP family through database searches revealing three N-terminal RNA recognition motifs (RRMs) bearing highest sequence identity in the RNP sequences to the related hnRNP Raver1. Outside the RRM region, both Raver proteins are quite divergent in sequence except for conserved peptide motifs of the [S/G][I/L]LGxxP consensus sequence. The latter have been implicated in Raver1 binding to the polypyrimidine tract-binding protein (PTB) a regulatory splicing repressor and common ligand of both Raver proteins. In the present study we investigated the association of Raver2 with RNA and PTB in more detail. The isolated RRM domain of Raver2 weakly interacted with ribonucleotides, but the full-length protein failed to directly bind to RNA in vitro. However, trimeric complexes with RNA were formed via binding to PTB. Raver2 harbors two putative PTB binding sequences in the C-terminal half of the protein, whose influence on Raver2-PTB complex formation was analyzed in a mutational approach, replacing critical leucine residues with alanines. While mutation of either sequence motif alone negatively affected Raver2 binding to PTB in vitro, only mutation of the more C-terminally located SLLGEPP motif significantly reduced the recruitment of Raver2 into perinucleolar compartments (PNCs) in HeLa cells. The latter observation was also confirmed for Raver1: out of four sequence motifs matching the PTB binding consensus, mutations in the SLLGEPP motif were the only ones attenuating the recruitment of Raver1 into PNCs. The conserved mode of PTB binding suggests that Raver2, like Raver1, may function as a modulator of PTB activity.     

3,4-Dihydro-2H-benzoxazinones are 5-HT(1A) receptor antagonists with potent 5-HT reuptake inhibitory activity

Starting from a high throughput screening hit, a series of 3,4-dihydro-2H-benzoxazinones has been identified with both high affinity for the 5-HT(1A) receptor and potent 5-HT reuptake inhibitory activity. The 5-(2-methyl)quinolinyloxy derivative combined high 5-HT(1A/1B/1D) receptor affinities with low intrinsic activity and potent inhibition of the 5-HT reuptake site (pK(i)8.2). This compound also had good oral bioavailability and brain penetration in the rat.     

Synthesis and evaluation of 5-HT(2A) and 5-HT(2C) receptor binding affinities of novel pyrimidine derivatives

In an effort to find potential anxiolytic and/or antipsychotic agents with selective 5-HT(2C) affinity a series of new pyrimidine derivatives was prepared and the binding affinities for 5-HT(2A) and 5-HT(2C) receptors were determined.     

Expression of ADP-ribosylation factor (ARF)-like protein 6 during mouse embryonic development

ADP-ribosylation factor (ARF)-like protein 6 (ARL6) is a member of the ARF-like protein (ARL) subfamily of small GTPases (Moss, 1995; Chavrier, 1999). ARLs are highly conserved through evolution and most of them possess the consensus sequence required for GTP binding and hydrolysis (Pasquallato, 2002). Among ARLs, ARL6 which was initially isolated from a J2E erythroleukemic cell line is divergent in its consensus sequences and its expression has been shown to be limited to the brain and kidney in adult mouse (Ingley, 1999). Recently, it was reported that mutations of the ARL6 gene cause type 3 Bardet-Biedl syndrome in humans and that ARL6 is involved in ciliary transport in C. elegans (Chiang, 2004; Fan, 2004). Here, we investigated the expression pattern of ARL6 during early mouse development by whole-mount in situ hybridization and found that interestingly, ARL6 mRNA was localized around the node at 7.0-7.5 days post coitum (dpc) embryos, while weak expression was also found in the ectoderm. At the later stage (8.5 dpc) ARL6 was expressed in the neural plate and probably in the somites. Based on these results, a possible role of ARL6 in early development is discussed in relation to the findings in human and C. elegans (Chiang, 2004; Fan, 2004).     

ADP-ribosylation factor 6 (ARF6) defines two insulin-regulated secretory pathways in adipocytes.

ADP-ribosylation factor 6 (ARF6) appears to play an essential role in the endocytic/recycling pathway in several cell types. To determine whether ARF6 is involved in insulin-regulated exocytosis, 3T3-L1 adipocytes were infected with recombinant adenovirus expressing wild-type ARF6 or an ARF6 dominant negative mutant (D125N) that encodes a protein with nucleotide specificity modified from guanine to xanthine. Overexpression of these ARF6 proteins affected neither basal nor insulin-regulated glucose uptake in 3T3-L1 adipocytes, nor did it affect the subcellular distribution of Glut1 or Glut4. In contrast, the secretion of adipsin, a serine protease specifically expressed in adipocytes, was increased by the expression of wild-type ARF6 and was inhibited by the expression of D125N. These results indicate a requirement for ARF6 in basal and insulin-regulated adipsin secretion but not in glucose transport. Our results suggest the existence of at least two distinct pathways that undergo insulin-stimulated exocytosis in 3T3-L1 adipocytes, one for adipsin release and one for glucose transporter translocation.     

Evidence for ADP-ribosylation factor (ARF) as a regulator of in vitro endosome-endosome fusion.

We have used an in vitro endosome fusion assay, recombinant ARF, synthetic peptides, and guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) to study the role of ARF during endocytosis. Previous work has shown that GTP gamma S stimulates in vitro endosome fusion in dilute cytosol (less than 0.5 mg/ml) but inhibits fusion in concentrated cytosol (greater than 1.0 mg/ml). Two peptides corresponding to the NH2-terminal 16 amino acids of human ARF1 and ARF4 blocked GTP gamma S stimulation of fusion in dilute cytosol and reversed GTP gamma S inhibition of fusion in concentrated cytosol. The addition of recombinant human ARF1 to endosomes in dilute or concentrated cytosol resulted in GTP gamma S-dependent inhibition of fusion. Only the myristoylated form of ARF inhibited fusion. The NH2-terminal ARF1 peptide reversed inhibition by recombinant ARF1. Preincubation experiments showed that endosomes could form an ARF-resistant intermediate during the fusion process. Western blot analysis revealed clathrin-coated vesicles extracted with detergent retained ARF. The results suggest that ARF is involved in both the stimulatory and inhibitory effects of GTP gamma S in dilute and concentrated cytosol, respectively. Furthermore, myristoylation, the NH2-terminal domain, and binding to GTP appear to be critical for ARF activity during an early prefusion step required for endocytosis.     

Membrane association of the Arabidopsis ARF exchange factor GNOM involves interaction of conserved domains

The GNOM protein plays a fundamental role in Arabidopsis thaliana development by regulating endosome-to-plasma membrane trafficking required for polar localization of the auxin efflux carrier PIN1. GNOM is a family member of large ARF guanine nucleotide exchange factors (ARF-GEFs), which regulate vesicle formation by activating ARF GTPases on specific membranes in animals, plants, and fungi. However, apart from the catalytic exchange activity of the SEC7 domain, the functional significance of other conserved domains is virtually unknown. Here, we show that a distinct N-terminal domain of GNOM mediates dimerization and in addition interacts heterotypically with two other conserved domains in vivo. In contrast with N-terminal dimerization, the heterotypic interaction is essential for GNOM function, as mutations abolishing this interaction inactivate the GNOM protein and compromise its membrane association. Our results suggest a general model of large ARF-GEF function in which regulated changes in protein conformation control membrane association of the exchange factor and, thus, activation of ARFs.     

Serotonin 4 receptor (5-HT4R) internalization is isoform-specific: Effects of 5-HT and RS67333 on isoforms A and B

Serotonin 4 receptors (5-HT4Rs) are particularly abundant within the limbic system, where they constitute potential targets for the development of novel, rapid acting antidepressants. However, the population of limbic 5-HT4Rs is not homogenous, comprising various isoforms of which 5-HT4(a) and 5-HT4(b) are among the most abundant variants. Sequence divergence at their C-termini is predictive of specificity in isoform signalling and regulation, but the differences, if any, remain ill-defined. The present study compared isoforms 5-HT4(a) and 5-HT4(b) in their ability to undergo endocytic regulation following exposure to 5-HT and to the putatively fast acting antidepressant RS67333. Both ligands differed in their ability to induce internalization of either isoform, 5-HT being more effective than RS67333 in HEK293 cells and in neurons. In contrast, trafficking induced by 5-HT was isoform-specific. In particular, while PKC, GRK2 and βarrestin were necessary for 5-HT4(a)R internalization, sequestration of 5-HT4(b)Rs required PKC but not GRK2 and relied significantly less on βarrestin. After endocytosis, isoform (b) appeared scattered throughout the intracellular compartment and efficiently recycled to the membrane upon agonist removal. Isoform (a) accumulated in the perinuclear compartment and displayed little recycling. Isoform-specific subcellular distribution was present in HEK293 cells and in neurons. In neurons, where internalization by RS67333 was more pronounced than in HEK293 cells, receptors internalized by this ligand followed the same distribution pattern as observed with 5-HT. These results point to isoform-related differences in the way that 5-HTRs respond to different ligands. Such diversity should be taken into account when developing therapeutic agents that target 5-HT4Rs.