Specificities for the small G proteins ARF1 and ARF6 of the guanine nucleotide exchange factors ARNO and EFA6.

ARF1 and ARF6 are distant members of the ADP-ribosylation factor (ARF) small G-protein subfamily. Their distinct cellular functions must result from specificity of interaction with different effectors and regulators, including guanine nucleotide exchange factors (GEFs). ARF nucleotide-binding site opener (ARNO), and EFA6 are analogous ARF-GEFs, both comprising a catalytic "Sec7" domain and a pleckstrin homology domain. In vivo ARNO, like ARF1, is mostly cytosolic, with minor localizations at the Golgi and plasma membrane; EFA6, like ARF6, is restricted to the plasma membrane. However, depending on conditions, ARNO appears active on ARF6 as well as on ARF1. Here we analyze the origin of these ARF-GEF selectivities. In vitro, in the presence of phospholipid membranes, ARNO activates ARF1 preferentially and ARF6 slightly, whereas EFA6 activates ARF6 exclusively; the stimulation efficiency of EFA6 on ARF6 is comparable with that of ARNO on ARF1. These selectivities are determined by the GEFs Sec7 domains alone, without the pleckstrin homology and N-terminal domains, and by the ARF core domains, without the myristoylated N-terminal helix; they are not modified upon permutation between ARF1 and ARF6 of the few amino acids that differ within the switch regions. Thus selectivity for ARF1 or ARF6 must depend on subtle folding differences between the ARFs switch regions that interact with the Sec7 domains.

Binding site of brefeldin A at the interface between the small G protein ADP-ribosylation factor 1 (ARF1) and the nucleotide-exchange factor Sec7 domain.

Sec7 domains (Sec7d) catalyze the exchange of guanine nucleotide on ARFs. Recent studies indicated that brefeldin A (BFA) inhibits Sec7d-catalyzed nucleotide exchange on ARF1 in an uncompetitive manner by trapping an early intermediate of the reaction: a complex between GDP-bound ARF1 and Sec7d. Using (3)H-labeled BFA, we show that BFA binds to neither isolated Sec7d nor isolated ARF1-GDP, but binds to the transitory Sec7d-ARF1-GDP complex and stabilizes it. Two pairs of residues at positions 190-191 and 198-208 (Arno numbering) in Sec7d contribute equally to the stability of BFA binding, which is also sensitive to mutation of H80 in ARF1. The catalytic glutamic (E156) residue of Sec7d is not necessary for BFA binding. In contrast, BFA does not bind to the intermediate catalytic complex between nucleotide-free ARF1 and Sec7d. These results suggest that, on initial docking steps between ARF1-GDP and Sec7d, BFA inserts like a wedge between the switch II region of ARF1-GDP and a surface encompassing residues 190-208, at the border of the characteristic hydrophobic groove of Sec7d. Bound BFA would prevent the switch regions of ARF1-GDP from reorganizing and forming tighter contacts with Sec7d and thereby would maintain the bound GDP of ARF1 at a distance from the catalytic glutamic finger of Sec7d.

Binding of the PH and polybasic C-terminal domains of ARNO to phosphoinositides and to acidic lipids.

The activity on ARF of the guanine nucleotide exchange factor ARNO depends on its membrane recruitment, induced by binding of its PH domain to phosphoinositides. A polycationic C-terminal extension to the PH domain might also contribute to its specific binding to phosphatidylinositol 4,5-bisphosphate [(4,5)PIP2] and to phosphatidylinositol 3,4,5-trisphosphate [(3,4,5)PIP3], and to ionic binding to other acidic lipids. We have analyzed in vitro the relative contributions to phospholipid binding of the PH domain and C-terminal extension by cosedimentation of "PH+C domain" and "nominal PH domain" protein constructs including or not including the polycationic C-terminus, with sucrose-loaded unilamellar vesicles made of equal proportions of the neutral lipids phosphatidylcholine and phosphatidylethanolamine, and supplemented or not with 30% acidic phosphatidylserine (PS) and 2% of various phosphoinositides. Binding was measured as a function of the vesicle concentration and of the medium ionic strength. Both proteins bound with higher affinity to (3,4,5)PIP3 than to (4,5)PIP2, the selectivity for (3,4,5)PIP3 being highest for the nominal PH domain. We observed also a clear selectivity of (3,4,5)PIP3 over (4,5)PIP2 for stimulating the activity of ARNO on ARF with vesicles containing 10% PS and 1% PIP2 or PIP3. Our data suggest that the PH domain provides the specific phosphoinositide binding site and some unspecific ionic interaction with acidic PS, whereas the polybasic C domain contributes to binding mainly by unspecific ionic interactions vith PS. Phosphorylation by protein kinase C of a serine in the C domain reduces the ionic affinity of the PH+C domain for PS, but does not affect the phosphoinositide specificity.

Dual interaction of ADP ribosylation factor 1 with Sec7 domain and with lipid membranes during catalysis of guanine nucleotide exchange.

Sec7 domains catalyze the replacement of GDP by GTP on the G protein ADP-ribosylation factor 1 (myrARF1) by interacting with its switch I and II regions and by destabilizing, through a glutamic finger, the beta-phosphate of the bound GDP. The myristoylated N-terminal helix that allows myrARF1 to interact with membrane lipids in a GTP-dependent manner is located some distance from the Sec7 domain-binding region. However, these two regions are connected. Measuring the binding to liposomes of functional or abortive complexes between myrARF1 and the Sec7 domain of ARNO demonstrates that myrARF1, in complex with the Sec7 domain, adopts a high affinity state for membrane lipids, similar to that of the free GTP-bound form. This tight membrane attachment does not depend on the release of GDP induced by the Sec7 domain but is partially inhibited by the uncompetitive inhibitor brefeldin A. These results suggest that the conformational switch of the N-terminal helix of myrARF1 to the membrane-bound form is an early event in the nucleotide exchange pathway and is a prerequisite for a structural rearrangement at the myrARF1-GDP/Sec7 domain interface that allows the glutamic finger to expel GDP from myrARF1.

RET-RGS, a retina-specific regulator of G-protein signaling, is located in synaptic regions of the rat retina.

RGS (regulators of G protein signaling) proteins negatively regulate the alpha subunit of G proteins by accelerating their intrinsic GTPase activity. In a previous work, we reported the cloning of a cDNA encoding for a new RGS protein, RET-RGS. We showed that it is specifically expressed in the retina, notably by photoreceptor cells and that it has an in vitro GAP activity on transducin. To understand the role of RET-RGS, and in particular to determine whether it regulates the phototransduction cascade in photoreceptor cells, RET-RGS was immunolocalized on rat retina sections. Whereas no labeling was detected in outer nor inner segments of photoreceptors cells, dense immunoreactive products were localized in the outer and inner plexiform layers which correspond to the regions of synaptic interplay between the different neurons of the retina including the photoreceptor cells. These results rule out a role of RET-RGS on the phototransduction cascade and suggest that it may participate in retina specific synaptic transductions.

Doppler study of mesenteric, hepatic, and portal circulation in alcoholic cirrhosis: relationship between quantitative Doppler measurements and the severity of portal hypertension and hepatic failure.

To determine the relationship between quantitative Doppler parameters of portal, hepatic, and splanchnic circulation and hepatic venous pressure gradient (HVPG), variceal size, and Child-Pugh class in patients with alcoholic cirrhosis, we studied forty patients with proved alcoholic cirrhosis who underwent Doppler ultrasonography, hepatic vein catheterization, and esophagoscopy. The following Doppler parameters were recorded: time-averaged mean blood velocity, volume flow of the main portal vein flow, and resistance index (RI) of the hepatic and of the superior mesenteric artery. Doppler findings were compared with HVPG, presence and size of esophageal varices, and Child-Pugh class. There was a significant inverse correlation between portal velocity and HVPG (r = -.69), as well as between portal vein flow and HVPG (r = -.58). No correlation was found between RI in the hepatic artery or superior mesenteric artery and HVPG. No correlation was found between portal vein measurements and presence and size of varices. Severe liver failure was associated with lower portal velocity and flow. In patients with alcoholic cirrhosis, only portal vein blood velocity and flow, but neither hepatic nor mesenteric artery RI, are correlated to the severity of portal hypertension and to the severity of liver failure.

A glutamic finger in the guanine nucleotide exchange factor ARNO displaces Mg2+ and the beta-phosphate to destabilize GDP on ARF1.

The Sec7 domain of the guanine nucleotide exchange factor ARNO (ARNO-Sec7) is responsible for the exchange activity on the small GTP-binding protein ARF1. ARNO-Sec7 forms a stable complex with the nucleotide-free form of [Delta17]ARF1, a soluble truncated form of ARF1. The crystal structure of ARNO-Sec7 has been solved recently, and a site-directed mutagenesis approach identified a hydrophobic groove and an adjacent hydrophilic loop as the ARF1-binding site. We show that Glu156 in the hydrophilic loop of ARNO-Sec7 is involved in the destabilization of Mg2+ and GDP from ARF1. The conservative mutation E156D and the charge reversal mutation E156K reduce the exchange activity of ARNO-Sec7 by several orders of magnitude. Moreover, [E156K]ARNO-Sec7 forms a complex with the Mg2+-free form of [Delta17]ARF1-GDP without inducing the release of GDP. Other mutations in ARNO-Sec7 and in [Delta17]ARF1 suggest that prominent hydrophobic residues of the switch I region of ARF1 insert into the groove of the Sec7 domain, and that Lys73 of the switch II region of ARF1 forms an ion pair with Asp183 of ARNO-Sec7.

Role of protein-phospholipid interactions in the activation of ARF1 by the guanine nucleotide exchange factor Arno.

Arno is a 47-kDa human protein recently identified as a guanine nucleotide exchange factor for ADP ribosylation factor 1 (ARF1) with a central Sec7 domain responsible for the exchange activity and a carboxyl-terminal pleckstrin homology (PH) domain (Chardin, P., Paris, S., Antonny, B., Robineau, S., Béraud-Dufour, S., Jackson, C. L., and Chabre, M. (1996) Nature 384, 481-484). Binding of the PH domain to phosphatidylinositol 4,5-bisphosphate (PIP2) greatly enhances Arno-mediated activation of myristoylated ARF1. We show here that in the absence of phospholipids, Arno promotes nucleotide exchange on [Delta17]ARF1, a soluble mutant of ARF1 lacking the first 17 amino acids. This reaction is unaffected by PIP2, which suggests that the PIP2-PH domain interaction does not directly regulate the catalytic activity of Arno but rather serves to recruit Arno to membranes. Arno catalyzes the release of GDP more efficiently than that of GTP from [Delta17]ARF1, and a stable complex between Arno Sec7 domain and nucleotide-free [Delta17]ARF1 can be isolated. In contrast to [Delta17]ARF1, full-length unmyristoylated ARF1 is not readily activated by Arno in solution. Its activation requires the presence of phospholipids and a reduction of ionic strength and Mg2+ concentration. PIP2 is strongly stimulatory, indicating that binding of Arno to phospholipids is involved, but in addition, electrostatic interactions between phospholipids and the amino-terminal portion of unmyristoylated ARF1GDP seem to be important. We conclude that efficient activation of full-length ARF1 by Arno requires a membrane surface and two distinct protein-phospholipid interactions: one between the PH domain of Arno and PIP2, and the other between amino-terminal cationic residues of ARF1 and anionic phospholipids. The latter interaction is normally induced by insertion of the amino-terminal myristate into the bilayer but can also be artificially facilitated by decreasing Mg2+ and salt concentrations.

N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipids upon GDP to GTP exchange.

GDP/GTP exchange modulates the interaction of the small G-protein ADP-ribosylation factor-1 with membrane lipids: if ARF(GDP) is mostly soluble, ARF(GTP) binds tightly to lipid vesicles. Previous studies have shown that this GTP-dependent binding persists upon removal of the N-terminal myristate but is abolished following further deletion of the 17 N-terminal residues. This suggests a role for this amphipathic peptide in lipid membrane binding. In the ARF(GDP) crystal structure, the 2-13 peptide is helical, with its hydrophobic residues buried in the protein core. When ARF switches to the GTP state, these residues may insert into membrane lipids. We have studied the binding of ARF to model unilamellar vesicles of defined composition. ARF(GDP) binds weakly to vesicles through hydrophobic interaction of the myristate and electrostatic interaction of cationic residues with anionic lipids. Phosphatidylinositol 4,5-bis(phosphate) shows no specific effects other than strictly electrostatic. By using fluorescence energy transfer, the strength of the ARF(GTP)-lipid interaction is assessed via the dissociation rate of ARF(GTPgammaS) from labeled lipid vesicles. ARF(GTPgammaS) dissociates slowly (tau(off) approximately 75 s) from neutral PC vesicles. Including 30% anionic phospholipids increases tau(off) by only 3-fold. Reducing the N-terminal peptide hydrophobicity by point mutations had larger effects: F9A and L8A-F9A substitutions accelerate the dissociation of ARF(GTPgammaS) from vesicles by factors of 7 and 100, respectively. This strongly suggests that, upon GDP/GTP exchange, the N-terminal helix is released from the protein core so its hydrophobic residues can interact with membrane phospholipids.

Activation of ADP-ribosylation factor 1 GTPase-activating protein by phosphatidylcholine-derived diacylglycerols.

Disassembly of the coatomer from Golgi vesicles requires that the small GTP-binding protein ADP-ribosylation factor 1 (ARF1) hydrolyzes its bound GTP by the action of a GTPase-activating protein. In vitro, the binding of the ARF1 GTPase-activating protein to lipid vesicles and its activity on membrane-bound ARF1GTP are increased by diacylglycerols with monounsaturated acyl chains, such as those arising in vivo as secondary products from the hydrolysis of phosphatidylcholine by ARF-activated phospholipase D. Thus, the phospholipase D pathway may provide a feedback mechanism that promotes GTP hydrolysis on ARF1 and the consequent uncoating of vesicles.

A human exchange factor for ARF contains Sec7- and pleckstrin-homology domains.

The small G protein ARF1 is involved in the coating of vesicles that bud from the Golgi compartments. Its activation is controlled by as-yet unidentified guanine-nucleotide exchange factors. Gea1, the first ARF exchange factor to be discovered in yeast, is a large protein containing a domain of homology with Sec7, another yeast protein that is also involved in secretion. Here we characterized a smaller human protein (relative molecular mass 47K) named ARNO, which contains a central Sec7 domain that promotes guanine-nucleotide exchange on ARF1. ARNO also contains an amino-terminal coiled-coil motif and a carboxy-terminal pleckstrin-homology (PH) domain. The PH domain mediates an enhancement of ARNO exchange activity by negatively charged phospholipid vesicles supplemented with phosphatidylinositol bisphosphate. The exchange activity of ARNO is not inhibited by brefeldin A, an agent known to block vesicular transport and inhibit the exchange activity on ARF1 in cell extracts. This suggests that a regulatory component which is sensitive to brefeldin A associates with ARNO in vivo, possibly through the amino-terminal coiled-coil. We propose that other proteins with a Sec7 domain regulate different members of the ARF family.

Myristoylation-facilitated binding of the G protein ARF1GDP to membrane phospholipids is required for its activation by a soluble nucleotide exchange factor.

We have investigated the role of N-myristoylation in the activation of bovine ADP-ribosylation factor 1 (ARF1). We previously showed that myristoylation allows some spontaneous GDP-to-GTP exchange to occur on ARF1 at physiological Mg2+ levels in the presence of phospholipid vesicles (Franco, M., Chardin, P., Chabre, M., and Paris, S. (1995) J. Biol. Chem. 270, 1337-1341). Here, we report that this basal nucleotide exchange can be accelerated (by up to 5-fold) by addition of a soluble fraction obtained from bovine retinas. This acceleration is totally abolished by brefeldin A (IC50 = 2 microM) and by trypsin treatment of the retinal extract, as expected for an ARF-specific guanine nucleotide exchange factor. To accelerate GDP release from ARF1, this soluble exchange factor absolutely requires myristoylation of ARF1 and the presence of phospholipid vesicles. The retinal extract also stimulates guanosine 5'-3-O-(thio)-triphosphate (GTP gamma S) release from ARF1 in the presence of phospholipids, but in this case myristoylation of ARF is not required. These observations, together with our previous findings that both myristoylated and non-myristoylated forms of ARF GTP-gamma S but only the myristoylated form of ARFGDP bind to membrane phospholipids, suggest that (i) the retinal exchange factor acts only on membrane-bound ARF, (ii) the myristate is not involved in the protein-protein interaction between ARF1 and the exchange factor, and (iii) N-myristoylation facilitates both spontaneous and catalyzed GDP-to-GTP exchange on ARF1 simply by facilitating the binding of ARFGDP to membrane phospholipids.

The small G-protein ARF1GDP binds to the Gt beta gamma subunit of transducin, but not to Gt alpha GDP-Gt beta gamma.

AlF4- activates heterotrimeric G-proteins G alpha subunits but not small GDP/GTP-binding proteins like ARF1. On retinal membranes containing holotransducin (Gt alpha GDP-Gt beta gamma) and incubated with ARFGDP, AlF4- induced Gt alpha GDP-AlF4 release and ARFGDP binding, probably to the remaining membrane-attached Gt beta gamma. On phospholipid vesicles reconstituted with Gt beta gamma, ARFGDP bound in proportion to Gt beta gamma, and was released upon subsequent Gt alpha GDP addition. Thus ARFGDP competes with Gt alpha GDP for binding to Gt beta gamma, probably through a conserved motif in the 'alpha 2 helix' of Gt alpha and ARF. This motif is found in the C-terminal helix of PH domains that bind to G beta gamma.

Myristoylation of ADP-ribosylation factor 1 facilitates nucleotide exchange at physiological Mg2+ levels.

Recombinant N-myristoylated bovine ADP-ribosylation factor 1 (myr-rARF1) has been expressed in bacteria and purified to near homogeneity with a high (85%) myristoylation efficiency. Myr-rARF1 and nonmyristoylated rARF1 have been compared with respect to their kinetics of guanine nucleotide exchange and their interactions with phospholipids. Myristoylation is shown to allow the release of bound GDP at physiological (mM) concentrations of Mg2+. GDP dissociation is slow in the absence of phospholipids but is accelerated 2-fold in the presence of phospholipid vesicles. On the contrary, myristoylation decreases 10-fold the rate of dissociation of GTP or guanosine 5'-O-(thiotriphosphate) (GTP gamma S) in the presence of phospholipids. As a result, myr-ARF1 can be spontaneously activated by GTP or GTP gamma S (t1/2 approximately 30 min at 37 degrees C) at 1 mM Mg2+, in the sole presence of phospholipid membranes without the need for a nucleotide exchange factor. In contrast to the nonacylated protein, the GDP-bound form of myr-ARF1 interacts with phospholipids, as demonstrated by its cosedimentation with phospholipid vesicles and its comigration with phospholipid/cholate micelles on gel filtration. The interaction is, however, weaker than for the GTP-bound form, suggesting that only the myristate in myr-ARF1GDP interacts with phospholipids, whereas both the myristate and the amino-terminal hydrophobic residues in myr-ARF1GTP bind to phospholipids.

Roles of lipid modifications of transducin subunits in their GDP-dependent association and membrane binding.

Transducin is an unusually soluble and dissociable heterotrimeric G-protein, although its T alpha and T beta gamma subunits are N-acylated and farnesylated, respectively. These lipid modifications have been suggested to contribute directly to the GDP-dependent T alpha-T beta gamma association, through specific lipid recognition sites on both protein subunits. We studied the dependence of subunit association on their bound lipids and on the presence of different lipidic environments. Association of native N-acylated (nT alpha) or acyl-free recombinant (rT alpha) T alpha with farnesylated and carboxymethylated (fcT beta gamma), farnesylated (fT beta gamma), or farnesyl-free (dfT beta gamma) T beta gamma was analyzed by gradient centrifugation and gel filtration in the presence of detergent or phospholipid-cholate micelles and by cosedimentation with phospholipid vesicles. Without detergent, nT alpha GDP and fcT beta gamma associate only weakly in solution. The loss of T alpha acyl or T beta gamma farnesyl residues induces total dissociation. With detergent or lipids, isolated fcT beta gamma binds tightly to micelles or vesicles, while dfT beta gamma does not; nT alpha GDP binds weakly, while deacylated rT alpha GDP does not bind at all; and nT alpha GDP binds cooperatively with fcT beta gamma, while rT alpha GDP does not. Thus (i) the T alpha acyl chain binds weakly, whereas the T beta gamma farnesyl chain binds strongly to membrane lipids; (ii) there is no evidence for binding of the T alpha acyl chain to a polypeptide site in T beta gamma, nor for binding of the T beta gamma farnesyl chain to a polypeptidic site in T alpha, but the T alpha acyl chain seems to bind cooperatively with the T beta gamma farnesyl chain in the membrane lipids; (iii) the insertion of the two protein-attached lipids into the same membrane could contribute to the association of both subunits by favoring collision coupling of the properly oriented protein moieties on the membrane surface.

Myristoylation is not required for GTP-dependent binding of ADP-ribosylation factor ARF1 to phospholipids.

Membrane binding of ADP-ribosylation factors (ARFs) is GTP-dependent and seems to require amino-terminal myristoylation. Recently it has been proposed that myristoylation is needed not for the activation of ARF by GTP but for its subsequent association to membranes. Here we show that unmyristoylated bovine ARF1, expressed in bacteria, can be efficiently loaded with GTP gamma S (guanosine 5'-O-(thio)triphosphate) at 1 microM free Mg2+, in the presence of phospholipids. Unmyristoylated ARFGTP gamma S cosediments with phospholipid vesicles and totally binds to phospholipid-cholate micelles, as seen by gel filtration chromatography. We therefore propose that, in vivo, myristoylation is required for the interaction of ARFGDP with its membrane-bound exchange factor rather than for the association of ARFGTP with lipid membranes. Phospholipid-bound ARFGTP gamma S can also stably interact with and activate the catalytic subunit of cholera toxin, suggesting that ARFGTP provides a membrane anchor for cholera toxin and thereby facilitates its access to membrane-bound substrates.