Protein prenylcysteine analog inhibits agonist-receptor-mediated signal transduction in human platelets.

Abstract

Signal transduction components, incuding the Ras superfamily of low molecular weight GTP-binding proteins and the y subunits of heterotrimeric G proteins, are reversibly carboxyl methylated at C-terminal prenylcysteine residues. We have previously shown that the prenylcysteine analog N-acetyl-S-tans,trans-farnesyl-L-cysteine (AFC) inhibits carboxyl methylation of these proteins in human platelets. Here we show that concentrations of AFC that inhibit Ras carboxyl methylation also block responses to agonists such as ADP, collagen, arachidonic acid, U46619 (a stable analog of prostaglandin H2), thrombin, and guanoine 5'-[y-thioltriphosphate. AFC does not inhibit aggregation induced by effectors such as ionomycin, phorbol 12,13-dibutyrate, and bacterial phospholipase C that bypass G proteins to activate platelets at the level of cytosolic Ca2+ concentration and protein kinase C. These findings indicate that AFC inhibits agonist-receptor-mediated signal transduction in human platelets.The finding that Ras proteins and the heterotrimeric G proteins are methylated at the carboxyl group of the C terminus has raised the possibility that this type of reversible covalent modification may play a role in signal transduction (1, 2). These GTP-binding proteins function to relay information from membrane receptors to control the cytoplasmic levels of second messengers, such as cAMP, cytosolic Ca2W, and diacylglycerol (DAG) (3,4), that act in turn to regulate protein kinases. Ras proteins and the y subunits of G proteins are initially synthesized with a characteristic C-terminal sequence, CXXX (a cysteine followed by three, generally aliphatic, residues) (2, 5). CXXX sequences provide a recognition site for cytosolic prenyltransferase enzymes, which catalyze the attachment of farnesyl or geranylgeranyl polyisoprenoid groups in thioether linkage to the cysteine (2, 6). The three residues distal to the cysteine in prenylated CXXX sequences are removed by membrane-associated protease activities (7), and the exposed prenylcysteine a-carboxylate undergoes methylesterificationbyamembrane-bound S-adenosylmethionine:prenylcysteine methyltransferase (8,9).Results with inhibitors ofpolyisoprenoid synthesis, such as compactin and mevinolin (lovastatin), indicate that prenylation is essential for the function of Ras proteins and 'y subunits ofthe heterotrimeric G proteins (10-13). Prenylation is required for all subsequent steps in CXXX processing (13,14), and without prenylation, these proteins cannot localize to their sites of action at the plasma membrane (15-17). Classical heterotrimeric G proteins as well as low molecular weight G proteins such as rapla, rapib, rap2, and rap2b are present in human platelets (18)(19)(20)(21)(22)(23)(24)(25)(26)(27). To specifically investigate the role of carboxyl methylation in G-protein-dependent signal transduction, we have examined the effects of a prenylcysteine methyltransferase inhibitor, N-acetyl-Strans,trans-farnesyl-L-cysteine (AFC), on agonist-receptormediated signal transduction in ...

Farnesylcysteine analogues inhibit store-regulated Ca2+ entry in human platelets: evidence for involvement of small GTP-binding proteins and actin cytoskeleton

“…In HEK cells, AFC significantly inhibited the Ca# + entry stimulated by TG at 50 µM [15]. In contrast, our results are different from those of Huzoor-Akbar et al [56], who concluded that AFC inhibits agonist-evoked Ca# + mobilization without having any effect on ionomycin-induced Ca# + entry. A possible explanation could be the different concentrations of the inhibitor used (as shown in Figures 2 and 3, the effects of FC analogues are clearly dependent on concentration).…”

Prenylcysteine Lyase Deficiency in Mice Results in the Accumulation of Farnesylcysteine and Geranylgeranylcysteine in Brain and Liver

“…However, we do not consider this to be likely, because the concentrations of the prenylcysteines in the Pcly-/-cells were in the picomolar range, significantly below the micromolar concentrations required to inhibit isoprenylcysteine carboxyl methyltransferase (6,20,23,24). In keeping with this prediction, we found that extracts from Pcly-/-cells and Pclyϩ/ϩ cells were equally effective in methylating recombinant farnesyl-K-Ras.…”

Tissue distribution of glycine N-methyltransferase, a major folate-binding protein of liver.

“…These cells are similar to those of the pancreas in that they are also (20). Recent studies have indicated that signal transduction in neutrophils (21) and in platelets (22) involves the carboxylmethylation ofthe ysubunit ofG proteins as an essential step. This methylation occurs on the carboxyl-terminal cysteine that has also been modified by the addition of a polyisoprene unit on the sulfur atom.…”