ADP-ribosylation of G proteins

Carty, D. J. (1994). Pertussis toxin-catalyzed ADP-ribosylation of G proteins. Meth. Enzymol. 237, 63-70. 

For ADP-ribosylation of G proteins in cultured mammalian cells, we supplement the medium with pertussis toxin to a final concentration of at least 25 ng/ml, and not more than 200 ng/ml. The optimal concentration has to be found empirically. For pertussis toxin to be fully effective, we incubate at least overnight (24 h) although a few hours of treatment have been reported to be sufficient in most cases higher PT concentrations are required with a shorter time. It appeared to be very helpful to aliquot the batch of cells and treat one portion with the vehicle alone, omitting toxin, to allow comparison and quantification of the effect of pertussis toxin on cells. Under these conditions the toxin works well, e.g. G, proteins of HL-60 cells are more than 98% modified (25 ng/ml, 24 h) (Hageluken efal., 1995). 

Dissociation of A protomer and B oligomer, reduction, and activation of the enzymatically active A component prior to incubation with the substrate are not absolutely required but generally result in more extensive ADP-ribosylation of G proteins when studying cell-free systems. 

Kopf GS, Woolkalis MJ (1991) ADP-ribosylation of G proteins with pertussis toxin. In Methods Enzymol. 195 257-266... 

ADP-ribosylation completely blocks the actin ATPase activity and increases the rate of ATP exchange by about twofold (Geipel ef al., 1990 Geipel ef al., 1989). This effect is not due to inhibition of polymerization, because the basal ATPase activity of G-actin is also inhibited. Moreover, the ATPase activity of actin is blocked even in the quasi-monomeric actin-DNAse I complex after stimulation with the mycotoxin cytochalasin (Geipel ef al., 1990). Thus, by analogy with the ADP-ribosylation of G-proteins by cholera toxin, which inhibits G-protein-associated GTP hydrolysis, the ADP-ribosylation of actin inhibits its intrinsic ATPase activity. 

Current ideas regarding toxin-mediated ADP-ribosylation of G-proteins are also summarized in Figure 6 and the model enables us to understand how agonist action can be stimulated or inhibited by the action of various toxins. We can consider toxin-mediated modulation of adenylyl cyclase as an example. 

G-proteins. Studies have demonstrated endogenous ADP-ribosylation of G-proteins in various mammalian tissues (Duman et al., 1991), and recent experiments have shown that endogenous ADP-ribosylation is under the control of specific hormones and second messenger systems (Prune et al., 1990). These findings suggest that regulation of ADP-ribosylation may represent a mechanism by which receptor-coupled signal transduction systems modulate cell function. 

Figure 3. Proposed pleiotropic functions carried out by nuclear ADP-ribosylation reactions. Events such as cellular proliferation, differentiation, transformation, and DNA damage caused by external agents (e.g., ionizing radiation, drugs) involve changes in the integrity of DNA and/or chromatin architecture (a) which activate the poly(ADP-ribose) polymerase to catalyze the ADP-ribosylation of nuclear proteins predominantly at the expense of cytoplasmic NAD (b). The consequences of protein ADP-ribosylation are a decrease in cellular NAD content, alterations in chromatin structure, and possibly also the activity of various enzymes involved in chromatin function (c). This tripartite system operates, either wholly or partly, to ameliorate the activation of the polymerase by modulating the repair of DNA strand breaks, thereby affecting those processes which initially triggered the activation of the enzyme (d). Pr, protein NAm, nicotinamide (ADPR) , poly(ADP-ribose). (From Gaal and Pearson, 1986).

Several laboratories have reported that benzamide or benzamide derivatives, which are effective ii bitors of ADP-ribose transfer reactions, can inhibit differentiation of myeloid cells (3-5). The mechanism of this inhibition has not been investigated at the molecular level. We report here studies of the inhibition of granulocyte colony-stimulating factor (G-CSF)-induced differentiation by benzamide on a murine myelomonocytic leukemia cell line, WEHI-3BD+. We also report that ADP-ribosylation of membrane proteins in these cells is correlated with the inhibition of differentiation by benzamide. 

Small GTPases of the Rho family are ADP-ribosylated (e.g., at Asn4l of RhoA) and inactivated by C3-like toxins from Clostridium botulinum, Clostridium limosum, and Staphylococcus aureus. These proteins have a molecular mass of 23-30 kDa and consist only of the enzyme domain. Specific inhibition of Rho functions (Rho but not Rac or Cdc42 are targets) is the reason why C3 is widely used as a pharmacological tool [2]. 

Secondly, treatment of neutrophils with pertussis toxin, which ADP-ribosylates a neutrophil G protein and causes a loss of cell responsiveness via receptor-mediated pathways (40,41), has minimal effect on the response to HCH (Figure 11, lower panel). Thus it can be concluded that HCH activation of neutrophils is independent of receptor-mediated activation of G proteins. 

FIGURE 7.7 C-terminal residues of G-protein a subunits. The cysteine ADP-ribosylated by Pertussis toxin (PTx) is boxed. 

G proteins can be modified by ADP-ribosylation catalyzed by certain bacterial toxins. Among the tools that facilitated the discovery and characterization of G proteins were the bacterial toxins cholera and pertussis, which were known to influence adenylyl cyclase activity. Subsequently, it was shown that the actions of these toxins are achieved by their ability to catalyze the addition of an ADP-ribose group donated from nicotinamide adenine dinucleotide (NAD) to specific amino acid residues in certain heterotrimeric G protein a subunits [ 1 ]. 

Cholera toxin catalyzes the ADP-ribosylation of a specific arginine residue in G and Gat. This covalent modification inhibits the intrinsic GTPase activity of these a subunits and thereby freezes them in their activated, or free, state (Fig. 19-1C). By this mechanism, cholera toxin stimulates adenylyl cyclase activity and photoreceptor transduction mechanisms. The ability of cholera toxin to ADP-ribosylate G may require the presence of a distinct protein, ADP-ribosylation factor (ARF). ARF, which is itself a small G protein (Table 19-2), also is ADP-ribosylated by cholera toxin. ARF is implicated in controlling membrane vesicle trafficking (see Ch. 9). 

In contrast, pertussis toxin catalyzes the ADP-ribosyl-ation of a specific cysteine residue in Gai) G(m and Gal [1]. Only a subunits bound to their Py subunits can undergo this modification. Pertussis-toxin-mediated ADP-ribosylation inactivates these a subunits such that they cannot exchange GTP for GDP in response to receptor activation (Fig. 19-1B). By this mechanism, pertussis toxin blocks the ability of neurotransmitters to inhibit adenylyl cyclase or to influence the gating of K+ and Ca2+ channels in target neurons. However, since G is not a substrate for pertussis toxin, the toxin may not be able to block neurotransmitter-mediated inhibition of adenylyl cyclase in all cases. The Gq and Gn 16 types of G protein a subunit are not known to undergo ADP-ribosylation. 

A third type of bacterial toxin, diphtheria toxin, catalyzes the ADP-ribosylation of eukaryotic elongation factor (EFTU), a type of small G protein involved in protein synthesis (Table 19-2). The functional activity of the elongation factor is inhibitedby this reaction. Finally, a botulinum toxin ADP-ribosylates and disrupts the function of the small G protein Rho, which appears to be involved in assembly and rearrangement of the actin cytoskeleton (Table 19-2). These toxins maybe involved in neuropathy (see Ch. 36) and membrane trafficking (see Ch. 9). 

The receptors for fMet-Leu-Phe, C5a and PAF have all been cloned (see Chapter 3) and possess the predicted seven membrane-spanning domains present in other G-protein-linked receptors of the rhodopsin superfamily (see Fig. 3.2). The pertussis-toxin sensitivity of the G-proteins associated with these receptors arises from the ADP-ribosylation of a cysteine residue that is four amino acids from the COOH-terminus of the molecule. Some other pertussis-toxin-insensitive G-proteins that exist lack this critical cysteine residue. 

Pertussis toxin is a protein secreted by the bacterium Bordetella pertussis which causes whooping cough. The toxin enters a cell where it also catalyses ADP-ribosylation of the a-subunit of G-protein. 

Two bacterial toxins, namely pertussis toxin and cholera toxin, were of great importance in determining the function of G-proteins. Both toxins catalyze ADP ribosyla-tion of proteins. Dming ADP ribosylation, an ADP-ribose residue is transferred from NAD to an amino acid residue of a substrate protein (Fig. 5.15). 

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