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ADP-ribosylation factors (ARFs) are members of a multigene family of 20-kDa guanine nucleotide-binding proteins that are regulatory components in several pathways of intracellular vesicular trafficking. The relatively small (∼180-amino acids) ARF proteins interact with a variety of molecules (in addition to GTP/GDP, of course). Cholera toxin was the first to be recognized, hence the name. Later it was shown that ARF also activates phospholipase D. Different parts of the molecule are responsible for activation of the two enzymes. In vesicular trafficking, ARF must interact with coatomer to recruit it to a membrane and thereby initiate vesicle budding. ARF function requires that it alternate between GTP- and GDP-bound forms, which involves interaction with regulatory proteins. Inactivation of ARF-GTP depends on a GTPase-activating protein or GAP. A guanine nucleotide-exchange protein or GEP accelerates release of bound GDP from inactive ARF-GDP to permit GTP binding. Inhibition of GEP by brefeldin A (BFA) blocks ARF activation and thereby vesicular transport. In cells, it causes apparent disintegration of Golgi structure. Both BFA-sensitive and insensitive GEPs are known. Sequences of peptides from a BFA-sensitive GEP purified in our laboratory revealed the presence of a Sec7 domain, a sequence of ∼200 amino acids that resembles a region in the yeast Sec7 gene product, which is involved in Golgi vesicular transport. Other proteins of unknown function also contain Sec7 domains, among them a lymphocyte protein called cytohesin-1. To determine whether it had GEP activity, recombinant cytohesin-1 was synthesized in E. coli. It preferentially activated class I ARFs 1 and 3 and was not inhibited by BFA but failed to activate ARF5 (class II). There are now five Sec7 domain proteins known to have GEP activity toward class I ARFs. It remains to be determined whether there are other Sec7 domain proteins that are GEPs for ARFs 4,5, or 6.

A membrane-associated arginine-specific mono-ADP-ribosyltransferase was purified 215,000-fold from rabbit skeletal muscle and its gene was isolated from a skeletal muscle cDNA library. The enzyme was a glycosylphosphatidyl-inositol-linked protein, present on the surface of differentiated skeletal muscle myoblasts (myotubes). Following incubation of cultured, intact myotubes with [adenylate-32P]NAD and analysis by SDS-PAGE, a major radiolabeled protein of 97/140 kDa (reduced/ nonreduced conditions) was observed. It was identified as integrin a 7 based on its size, binding to a laminin affinity column, immunoprecipitation with a monoclonal antibody, and partial amino acid sequencing.

ADP-ribosylation factors or ARFs are 20-kDa guanine nucleotide-binding proteins, initially identified as stimulators of cholera toxin-catalyzed ADP-ribosylation of Gsa. We now know that ARFs play a critical role in many vesicular trafficking events and ARF activation of a membrane-associated phospholipase D (PLD) has been recognized. ARF is active and associates with membranes when GTP is bound. The active state is terminated by hydrolysis of bound GTP, producing inactive ARF*GDP. The nucleotide effect on ARF association with membranes is related to alteration in orientation of the N-terminal myristoyl moiety that is important for ARF function. Cycling of ARF between active and inactive states involves guanine nucleotide-exchange proteins (GEPs) that accelerate replacement of bound GDP with GTP and GTPase-activating proteins (GAPS) that are responsible for ARF inactivation.

The RT6 alloantigenic system of the rat was discovered in the 1970s. It was originally designated Pta, AgF, A.R.T.-2, and RTLy-2 by the laboratories involved in its characterization. Exchange of reagents demonstrated that these laboratories had identified the same system, and the official designation RT6 was assigned in 1982 (1).

Mono-ADP-ribosylation, catalyzed by ADP-ribosyltransferases, is a posttranslational modification of proteins in which the ADP-ribose moiety of NAD is transferred to an acceptor protein(arginine). Several of the bacterial toxin ADP-ribosyltransferases have been well characterized in their ability to alter cellular metabolism. It has been postulated that these bacterial toxins mimic the actions of transferases from mammalian cells. We have cloned and characterized ADP-ribosyltransferases from rabbit and human skeletal muscle, and mouse lymphocytes. The muscle transferases are glycosylphosphatidylinositol (GPI)-anchored proteins that are conserved among species. Two distinct transferases, termed Yac-1 and Yac-2 were cloned from mouse lymphoma (Yac-1) cells. The Yac-1 transferase, like the muscle enzymes, is a GPI-linked exoenzyme. The Yac-2 transferase, on the other hand, is membrane-associated but appears not to be GPI-linked. In contrast to Yac-1, the Yac-2 enzyme had significant NAD glycohydrolase activity and may preferentially hydrolyze NAD.

We report that rat RT6.2 and recombinant mouse Rt6 locus 1 proteins possess autoADP-ribosyltransferase activity and that Rt6, but not RT6, catalyzes the ADP-ribosylation of exogenous histones. Based on NH2OH sensitivity, it appeared that the ADP-ribose was attached to arginine residues on proteins. We also observed that the NAD+ concentration in culture medium correlates inversely with the proliferation of rat RT6+ T cells. The data suggest that lymphocyte surface ADP-ribosyltransferases could be involved in signaling and immunoregulatory processes.

Mono-ADP-ribosylation is a reversible modification of arginine residues in proteins, with NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases constituting opposing arms of a putative ADP-ribosylation cycle. The enzymatic components of an ADP-ribosylation cycle have been identified in both prokaryotic and eukaryotic systems. The regulatory significance of the cycle has been best documented in prokaryotes. As shown by Ludden and coworkers, ADP-ribosylation controls the activity of dinitrogenase reductase in the phototropic bacterium Rhodospirillum rubrum. ADP-ribosylation of other amino acids, such as cysteine, has also been demonstrated, lending credence to the hypothesis that this modification is heterogeneous. In eukaryotes, the functional relationship between ADP-ribosyltransferases and ADP-ribosylarginine hydrolases is less well documented. The transferase-catalyzed reaction results in sterospecific formation of alpha-ADP-ribosylarginine from beta-NAD; ADP-ribosylarginine hydrolases specifically cleave the alpha-anomer, leading to release of ADP-ribose and regeneration of the free guanidino group of arginine. The two reactions can thus be coupled in vitro. Coupling in vivo is dependent on cellular localization. The deduced amino acid sequences of ADP-ribosyltransferases from avian and mammalian tissues have common consensus sequences involved in catalytic activity but, in some instances, enzyme-specific cellular localization signals. The presence of amino- and carboxy-terminal signal sequences is consistent with the glycosylphosphatidylinositol(GPI)-anchoring to the cell surface. The muscle and lymphocyte transferases ADP-ribosylate integrins. Some transferases lack the carboxy- terminal signal sequence needed for GPI-anchoring. Most ADP-ribosylarginine hydrolase activity is cytosolic, although perhaps some is located at the cell surface. Deduced amino acid sequences of hydrolases from a number of mammalian species are consistent with their cytoplasmic localization. Katada and coworkers have determined, however, that auto-ADP-ribosylated RT6, a GPI-linked protein, is metabolized by a hydrolase-like activity, consistent with the existence of an ADP-ribosylation cycle. ADP-ribosyl RT6 may be internalized, thereby coming in contact with the cytosolic hydrolase; alternatively, a novel form of the hydrolase may be located at the surface. The mechanism of coupling of ADP-ribosyltransferases and hydrolases in eukaryotic ADP-ribosylation cycles has yet to be clarified.

ADP-ribosylation factors (ARFs) comprise a family of ~20 kDa guanine nucleotide-binding proteins that were discovered as one of several cofactors required in cholera toxin-catalyzed ADP-ribosylation of Gsα, the guanine nucleotide-binding protein responsible for stimulation of adenylyl cyclase, and was subsequently found to enhance all cholera toxin-catalyzed reactions and to directly interact with, and activate the toxin. ARF is dependent on GTP or its analogues for activity, binds GTP with high affinity in the presence of dimyristoylphosphatidylcholine/cholate and contains consensus sequences for GTP-binding and hydrolysis. Six mammalian family members have been identified which have been classified into three groups (Class I, II, and III) based on size, deduced amino acid sequence identity, phylogenetic analysis and gene structure. ARFs are ubiquitous among eukaryotes, with a deduced amino acid sequence that is highly conserved across diverse species. They have recently been shown to associate with phospholipid and Golgi membranes in a GTP-dependent manner and are involved in regulating vesicular transport. (Mol Cell Biochem 138: 157–166, 1994)

ADP-ribosylation is a reversible post-translational modification of proteins involving the addition of the ADP-ribose moiety of NAD to an acceptor protein or amino acid. NAD:arginine ADP-ribosyltransferase, purified from numerous animal tissues, catalyzes the transfer of ADP-ribose to an arginine residue in proteins. The reverse reaction, catalyzed by ADP-ribosylarginine hydrolase, removes ADP-ribose, regenerating free arginine. An ADP-ribosylarginine hydrolase, purified extensively from turkey erythrocytes, was a 39-kDa monomeric protein under denaturing and non-denaturing conditions, and was activated by Mg2+ and dithiothreitol. The ADP-ribose moiety was critical for substrate recognition; the enzyme hydrolyzed ADP-ribosylarginine and (2-phospho-ADP-ribosyl)arginine but not phosphoribosylarginine or ribosylarginine. The hydrolase cDNA was cloned from rat and subsequently from mouse and human brain. The rat hydrolase gene contained a 1086-base pair open reading frame, with deduced amino acid sequences identical to those obtained by amino terminal sequencing of the protein or of HPLC-purified tryptic peptides. Deduced amino acid sequences from the mouse and human hydrolase cDNAs were 94% and 83% identical, respectively to the rat. Anti-rat brain hydrolase polyclonal antibodies reacted with turkey erythrocyte, mouse and bovine brain hydrolase. The rat hydrolase, expressed in E. coli, demonstrated enhanced activity in the presence of Mg2+ and thiol, whereas the recombinant human hydrolase was stimulated by Mg2+ but was thiol-independent. In the rat and mouse enzymes, there are five cysteines in identical positions; four of the cysteines are conserved in the human hydrolase. Replacement of cysteine 108 in the rat hydrolase (not present in the human enzyme) resulted in a thiol-independent hydrolase without altering specific activity. Rabbit anti-rat brain hydrolase antibodies reacted on immunoblot with the wild-type rat hydrolase and only weakly with the mutant hydrolase. There was no immunoreactivity with either the wild-type or mutant human enzyme. Cysteine 108 in the rat and mouse hydrolase may be responsible in part for thiol-dependence as well as antibody recognition. Based on these studies, the mammalian and avian ADP-ribosylarginine hydrolases exhibit considerable conservation in structure and function. (Mol Cell Biochem 138: 119–122, 1994)

The amino acid sequences of several bacterial toxin ADP-ribosyltransferases, rabbit skeletal muscle transferases, and RT6.2, a rat T-cell NAD glycohydrolase, contain three separate regions of similarity, which can be aligned. Region I contains a critical histidine or arginine residue, region II, a group of closely spaced aromatic amino acids, and region III, an active-site glutamate which is at times seen as part of an acidic amino acid-rich sequence. In some of the bacterial ADP-ribosyltransferases, the nicotinamide moiety of NAD has been photo-crosslinked to this glutamate, consistent with its position in the active site. The similarities within these three regions, despite an absence of overall sequence similarity among the several transferases, are consistent with a common structure involved in NAD binding andADP-ribose transfer. (Mol Cell Biochem 138: 177–181, 1994)

Preface. I: Historical Perspective. II: Poly(ADP-ribosyl)ation. A. Structure and Enzymology of Poly(ADP-ribose) Polymerase. B. Polymer Regulation. C. Cellular Functions. D. Poly(ADP-ribose) Polymerase Gene Regulation. III: Mono(ADP-ribosylation). A. ADP-ribosylation Cycle. B. Cellular Mono-ADP-ribosylation. IV: Toxin Mono-ADP-Ribosylation. V: Inhibitors and Activators of ADP-Ribosylation. VI: Derivitization of Proteins with ADP-ribose, NAD and their Analogues. VII: Cyclic ADP-ribose, NAD Hydrolysis and ADP-ribose Synthesis.

Cholera, a devastating diarrheal disease characterized by abnormalities in fluid and electrolyte flux, results in part from the action of cholera toxin (choleragen), a secretory product of Vibrio cholerae, on the intestinal mucosa (Carpenter, 1980; Kelly, 1986). Cholera toxin exerts its effects by catalyzing the ADP-ribosylation of Gsα, the α subunit of the heterotrimeric stimulatory guanine nucleotide-regulatory protein that activates the adenylyl cyclase catalytic unit and is involved in the modulation of ion channels (Birnbaumer et al., 1987; Casey and Gilman, 1988; Moss and Vaughan, 1988). Activation of adenylyl cyclase, as well as other toxincatalyzed reactions, is enhanced by membrane and soluble factors that have been identified in preparations from numerous animal cells and tissues (Gill, 1976; Enomoto and Gill, 1980; Nakaya et al., 1980; Le Vine, III and Cuatrecasas, 1981; Pinkett and Anderson, 1982; Schleifer et al., 1982; Gill and Meren, 1983; Kahn and Gilman, 1984; Kahn and Gilman, 1986; Tsai et al., 1987, 1988; Gill and Coburn, 1987). A 21 kDa protein that stimulated the toxin-catalyzed ADP-ribosylation of Gsα was purified from liver membranes and termed ADP-ribosylation factor or ARF (Kahn and Gilman, 1984).

The majority of the mono(ADP-ribosyl) transferases identified to date in animal tissues (1–5) are characterized by their specific modification of the guanidinium group of arginine residues. In contrast, poly(ADP-ribose) polymerase is known only to modify carboxylate groups on protein acceptors, i.e., glutamate (6–9), carboxy-terminal lysine (8) and aspartate (10). Both classes of enzymes have identical substrate stereospecificity in which the β-configuration of the anomeric carbon of NAD+ is converted to the α-configuration in the product (11, 12). No major differences between these two classes of enzymes in substrate structural requirements have been documented. The present study identifies a difference in behavior of an NAD+:arginine mono(ADP-ribosyl) transferase from turkey erythrocytes (2) and poly(ADP-ribose) polymerase from calf thymus (13) toward 2′dNAD+ as a substrate.

Activation of adenylate cyclase by choleragen results from the toxincatalyzed ADP-ribosylation of a regulatory component of the cyclase system, Gsα, a guanine nucleotide-binding protein involved in stimulation of the cyclase catalytic unit (1). The ADP-ribosylation reaction and cyclase activation are enhanced by soluble and membrane components (2–9). One membrane protein, known as ADP-ribosylation factor or ARF, was extensively purified by Kahn and Gilman and shown to be a GTP-binding protein (8, 9).

There is a class of mono-ADP-ribosyltransferases that are distinguished by their ability to utilize as ADP-ribose acceptors the free amino acid arginine, other simple guanidino compounds, and proteins. Several transferases of this type were identified in and purified from turkey erythrocytes (1–3). The enzymes displayed different physical, kinetic and regulatory properties and were localized to the soluble, membrane and nuclear compartments (1–3). Similar NAD:arginine ADP-ribosyltransferases have been observed in other tissues and organ systems from a variety of species (1–5). The enzymes are found in viruses, bacteria and animal cells (1–7).

Choleragen (cholera toxin) exerts its effects on animal cells by activating adenylate cyclase, thereby increasing intracellular cAMP content (1). The A1 protein of choleragen, released from the holotoxin by reduction of a single disulfide bond linking the A1 and A2 proteins, catalyzes the mono- ADP-ribosylation of Gsα, a regulatory component of the adenylate cyclase system that is responsible for the GTP-dependent activation of the cyclase catalytic unit. ADP-ribosylation of Gsα apparently increases its sensitivity to GTP and its dissociation from the inhibitory Gβγ complex (1).