G-proteins ADP ribosylation

Antonny, B., Beraud-Dufour, S., Chardin, P., and Chabre, M. (1997). N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipids upon GDP to GTP exchange. Biochemistry 36, 4675-4684. 

Robineau, S., Chabre, M., and Antonny, B. (2000). Binding site of brefeldin A at the interface between the small G protein ADP-ribosylation factor 1 (ARFl) and the nucleotide-exchange factor Sec7 domain. Proc. Nad. Acad. Sci. USA 97, 9913-9918. 

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). 

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).

Kun E, Romaschin AD, Blasdell RJ, Jackowski G (1981) ADP-ribosylation of nonhistone chromatin proteins in vivo and of actin in vitro and effects of normal and abnormal growth conditions and organ-specific hormonal influences. In Holzer H (ed) Proceedings of the international Titisee conference on metabolic interconversion of regulatory enzymes. Springer, Berlin Heidelberg New York, pp 280-293... 

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]. 

Pertussis toxin is produced by the bacterium Bordetella pertussis. It covalently modifies G-proteins of the G/Go family (transfer of a ADP-ribose moiety of NAD onto G-protein a-subunits). ADP-ribosylated G-proteins are arrested in their inactive state and, as a consequence, functionally uncoupled from their respective effectors. Examples for pertussis toxin-sensitive cellular responses include the hormonal inhibition of adenylyl cyclases, stimulation ofK+ channels, inhibition of Ca2+ channels and stimulation ofthe cGMP-phosphodiesterase in retinal rods. 

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. 

The first G-protein a subunit to be identified was Gs. The a subunit of Gs (as) is responsible for stimulating adenylate cyclase (hence, the subscript s ) and is ADP-ribosylated and activated by CTx. Gs has at least four molecular variants. Some evidence exists that as can also enhance the activity of cardiac L-type Ca2+ channels, independently of their phosphorylation by cAMP-stimu-lated protein kinase A. Golf is a cyclase-stimulating homolog in the olfactory epithelium, activated by the large family of olfactory receptors. 

G, is the G-protein responsible for inhibiting adenylate cyclase. The inhibition is mediated by the a subunit. Unlike Gs, G, is not affected by CTx but instead is ADP-ribosylated (and inhibited) by PTx. Of the three isoforms of G, (Gn 3), an is the most potent inhibitor of cyclase. G, also activates inward-rectifier (Kir3.1/3.2 and Kir 3.1/3.4) K+ channels (GIRK channels), and this activation is mediated by released f v subunits (see below). 

Gq and Gn are two closely related and widely distributed G-proteins whose a subunits stimulate PLC. They are not ADP-ribosylated by either PTx or CTx, so they are probably responsible for many instances of PTx-insensitive PLC stimulation. G14 and G15 are two more distantly related PTx-insensitive G-proteins that can stimulate PLC. G12 and G13 are other PTx-insensitive G-proteins related to Gq, while Gz is more closely related to G, the precise functions of these G-proteins are not yet clear. Though of restricted distribution (to hemopoietic-derived cells), G16 is interesting because it lacks receptor specificity and so acts as a universal PLC transducer. 

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

G proteins can be modified by ADP-ribosylation catalyzed by certain bacterial toxins 343... 

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 ]. 

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. 

Figure 2 The actin-ADP-ribosylating toxins, (a) Molecular mode of action. The actin-ADP-ribosylating toxins covalently transfer an ADP-ribose moiety from NAD+ onto Arg177 of G-actin in the cytosol of targeted cells. Mono-ADP-ribosylated G-actin acts as a capping protein and inhibits the assembly of nonmodified actin into filaments. Thus, actin polymerization is blocked at the fast-growing ends of actin filaments (plus or barbed ends) but not at the slow growing ends (minus or pointed ends). This effect ultimately increases the critical concentration necessary for actin polymerization and tends to depolymerize F-actin. Finally, all actin within an intoxicated cell becomes trapped as ADP-ribosylated G-actin.

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