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At least 19 recordsLinked to original sources
PubMed1992

Distribution of smg p25A and smg p21s, ras p21-like guanine nucleotide-binding proteins, in the rat stomach.

Existence and distribution of small guanine nucleotide binding proteins (G proteins) such as smg p25A, smg p21s, and ras p21s were examined in the rat gastric mucosa. By immunoblot analysis using the specific monoclonal antibodies against smg p25A and ras p21 and the anti-smg p21 antiserum, smg p25A and smg p21s were detected in the gastric mucosa, while ras p21s were not detected. In immunocytochemical studies, moderate fluorescence of smg p25A was homogenously seen in the cytoplasmic portions of chief cells and surface epithelial cells. smg p21 immunoreactivity was detected in glandular cells and submucosal muscle layer in the mucosa. On the other hand, the cells constituting gastric glands were unstained with the anti-ras p21 monoclonal antibody, RASK-4. These results suggest that smg p25A and smg p21s exist as in other tissues and might exert their specific actions in the rat gastric mucosa.

Animals↗
PubMed1992

Microinjection of smg/rap1/Krev-1 p21 into Swiss 3T3 cells induces DNA synthesis and morphological changes.

Microinjection of either Ki-rasVal-12 p21 or the GDP-bound form of Ki-ras p21 plus smg GDP dissociation stimulator (GDS), a stimulatory GDP/GTP exchange protein for Ki-ras p21, smg/rap1/Krev-1 p21, and rho p21, into quiescent Swiss 3T3 cells induced DNA synthesis irrespective of the presence or absence of insulin. The guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of smg p21B or the GDP-bound form of smg p21B plus smg GDS also induced DNA synthesis but only in the presence of insulin. Either the GDP-bound form of Ki-ras p21 or the same form of smg p21B alone was inactive, but smg GDS alone was slightly active only in the presence of insulin. The morphology of the cells was analyzed by scanning electron, phase-contrast, and confocal laser scanning microscopies. Ki-rasVal-12 p21 induced membrane ruffling irrespective of the presence or absence of insulin. The GTP gamma S-bound form of smg p21B showed the same effect only in the presence of insulin. Either the GDP-bound form of Ki-ras p21, the same form of smg p21B, or smg GDS alone was inactive. Upon microinjection of Ki-rasVal-12 p21, stress fibers markedly decreased and the cells became round and piled up. In contrast, upon microinjection of the GTP gamma S-bound form of smg p21B, stress fibers did not markedly decrease and the cells neither became round nor piled up. These results indicate that both ras p21 and smg p21 are mitogenic in Swiss 3T3 cells but that their actions are slightly different.

3T3 Cells↗
PubMed1992

Role of rap1B and p21ras GTPase-activating protein in the regulation of phospholipase C-gamma 1 in human platelets.

Thrombin activates phospholipase C in human platelets, but the specific isoenzymes activated and the signal pathway used are unknown. Using specific antibodies, we found that phospholipase C-gamma 1 and the p21ras GTPase-activating protein, rasGAP, are present in human platelets. Furthermore, phospholipase C-gamma 1 was detectable, based on enzyme activity and Western blot analysis, in immunoprecipitates of rasGAP, suggesting that these two proteins form tight complexes. The pool of phospholipase C-gamma 1 associated with rasGAP was phosphorylated but not through tyrosine phosphorylation. Although thrombin stimulation had no effect on the level of phosphorylation of phospholipase C-gamma 1 and only slightly increased the tyrosine phosphorylation of rasGAP, the agonist induced the association of rasGAP with rap1B, as indicated by the appearance of rap1B on a Western blot of rasGAP immunoprecipitates. Our results suggest the formation of a signaling complex involving rasGAP, phospholipase C-gamma 1, and rap1B that might be important in the cascade leading to platelet activation.

Blood Platelets↗
PubMed1992

Localization of RAP1 and topoisomerase II in nuclei and meiotic chromosomes of yeast.

Topoisomerase II (topoII) and RAP1 (Repressor Activator Protein 1) are two abundant nuclear proteins with proposed structural roles in the higher-order organization of chromosomes. Both proteins co-fractionate as components of nuclear scaffolds from vegetatively growing yeast cells, and both proteins are present as components of pachytene chromosome, co-fractionating with an insoluble subfraction of meiotic nuclei. Immunolocalization using antibodies specific for topoII shows staining of an axial core of the yeast meiotic chromosome, extending the length of the synaptonemal complex. RAP1, on the other hand, is located at the ends of the paired bivalent chromosomes, consistent with its ability to bind telomeric sequences in vitro. In interphase nuclei, again in contrast to anti-topoII, anti-RAP1 gives a distinctly punctate staining that is located primarily at the nuclear periphery. Approximately 16 brightly staining foci can be identified in a diploid nucleus stained with anti-RAP1 antibodies, suggesting that telomeres are grouped together, perhaps through interaction with the nuclear envelope.

Base Sequence↗
PubMed1992

Evidence for a role of rap1 protein in the regulation of human platelet Ca2+ fluxes.

The relationship between the 22-24 kDa cyclic AMP (cAMP)-dependent phosphoprotein previously described as being involved in the regulation of human platelet membrane Ca2+ transport and a GTP-binding protein of low molecular mass (ras-like protein) was investigated. After isolation of plasma membranes and intracellular membranes, it was found that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) bound to plasma membrane proteins ranging in molecular mass from 22 to 29 kDa, but not to intracellular membranes. The major GTP-binding protein appeared as a 24 kDa protein under reduced conditions and a 22 kDa protein under non-reduced conditions. A similar membrane location and electrophoretic mobility were found for both the cAMP phosphoprotein and the protein recognized by a specific anti-rap1 antibody. The identity between the cAMP phosphoprotein and the rap1 GTP-binding protein was further examined by studying the functional effect of GTP on plasma membrane Ca2+ transport. A maximal GTP[S] concentration of 40 microM was found to: (1) inhibit to the same degree (40%) both Ca(2+)-ATPase activity and the Ca2+ transport function mediated by the Ca(2+)-ATPase; (2) inhibit the phosphorylation of the 22-24 kDa protein by the catalytic subunit of the cAMP-dependent protein kinase (C.Sub.); and (3) abolish the stimulation of Ca2+ uptake induced by C.Sub. It is concluded that the platelet cAMP phosphoprotein is indeed the rap1 GTP-binding protein, and that it regulates plasma membrane Ca2+ transport, thus providing evidence for a new role of a ras-related protein.

Biological Transport↗
PubMed1992

The purification of a Rap1 GTPase-activating protein from bovine brain cytosol.

Two GTPase-activating proteins (GAPs) have been detected in extracts from bovine brain: GAP-1, which is specific for the activation of ras GTPases, and GAP-3, which is specific for the activation of the rap1 GTPases. We present a strategy for the purification to homogeneity of a cytosolic form of GAP-3 from bovine brain. The 100,000 x g supernatant from homogenized brains was chromatographed sequentially on DEAE Fast Flow, green H-E4BD Sepharose, Bio-Gel A1.5, hydroxyapatite, and phenyl-Sepharose prior to high resolution separation on Mono Q HR 5/5, phenyl-Superose HR 5/5, Mono Q PC 1.6/5, and Superose 12 PC 3.2/30. This procedure resulted in an approximately 18,000-fold purification, yielding 50 micrograms of GAP-3 from 1.6 kg of tissue. Purified cytosolic GAP-3 migrated as a single band of apparent Mr 55,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, on gel filtration cytosolic GAP-3 chromatographed as a dimer with an apparent Mr 92,000. Purified GAP-3 does not activate ras or rho GTPases and possesses no intrinsic GTPase activity. Amino acid sequence data indicated a proline-rich N terminus. The amino acid sequences of peptides generated by Staphylococcus aureus V8 digestion of reduced and pyridine-ethylated GAP-3 showed no similarity to the predicted primary structure of GAP-1 or any other proteins in the nucleic acid or protein data bases. By comparison with the data of Rubinfeld et al. (Rubinfeld, B., Munemitsu, S., Clark, R., Conroy, L., Watt, K., Crosier, W.J., McCormick, F., and Polakis, P. (1991) Cell 65, 1033-1042), it appears that the membrane-associated (Mr 85,000-95,000) and cytosolic forms of GAP-3 are derived from equivalent, or closely related, genes.

Amino Acid Sequence↗
PubMed1992

Subcellular distribution of the Rap1A protein in human neutrophils: colocalization and cotranslocation with cytochrome b559.

Rap1A, a low molecular weight guanosine triphosphate-binding protein (LMWG), has been shown previously by us to be associated with purified cytochrome b from stimulated human neutrophils. In the present studies, we show that Rap1A is also associated with affinity-purified cytochrome b from unstimulated neutrophils and use specific anti-Rap1 peptide antibodies to biochemically and immunocytochemically determine the subcellular distribution of Rap1A in resting and activated human neutrophils. Analysis of the subcellular fractionation of unstimulated cells by Western blotting of isopycnic sucrose density gradient fractions with anti-Rap1 peptide antibodies indicated that Rap1A colocalized with cytochrome b in the plasma membrane as well as in the specific granule membranes and that it was translocated, along with cytochrome b, to the plasma membrane when the cells were stimulated with phorbol myristate acetate (PMA). No evidence for a cytosolic localization of Rap1A was found in our studies; however, if the cells were disrupted by sonication, rather than N2 cavitation, a fraction of the Rap1A was released from the membrane. Electron microscopy of thin sections of cryofixed, molecular-distillation dried neutrophils labeled with anti-Rap1 antibody alone or double-labeled with anti-Rap1 and anti-cytochrome b peptide antibodies confirmed our biochemical localization, and quantitation showed that more than half of the specific granule-associated Rap1A was translocated to the plasma membrane in PMA-stimulated cells. Ultrastructural analysis of neutrophils phagocytosing Staphylococcus aureus also demonstrated the translocation of Rap1A with cytochrome b. Approximately 70% of the total Rap1A labeling was associated with the phagolysosomal membrane, the site of assembly of the superoxide-generating system. The colocalization and cotranslocation of Rap1A with cytochrome b in resting and activated neutrophils is consistent with a functional association of these two molecules in the intact cell and provides further evidence for a role of this LMWG in the structure or function of the neutrophil superoxide-generating system.

Biological Transport↗
PubMed1992

Reconstitution of defective respiratory burst activity with partially purified human neutrophil cytochrome B in two genetic forms of chronic granulomatous disease: possible role of Rap1A.

Neutrophil plasma membranes from patients with the X-linked and autosomal recessive forms of chronic granulomatous disease (CGD) that lack cytochrome b are incapable of generating superoxide anion (O2-) in vivo and in vitro. The O2- generating activity of these defective membranes was reconstituted with the addition of partially purified human neutrophil cytochrome b in a detergent-based, cell-free activation system. Depending on the detergent system used, 50% to 100% of the activity of control membranes was recovered, and this activity was directly dependent on the cytochrome b concentration. However, when cytochrome b was purified to 99% homogeneity, the reconstitutive capacity of the cytochrome was lost, possibly because of subtle denaturation of the cytochrome or the removal of an additional required cofactor. Examination of the latter possibility with respect to a protein known to coassociate with the cytochrome, ie, Rap1A, indicated that this ras-like protein was present in the partially purified cytochrome preparation used to reconstitute activity in CGD membranes, but was missing in the highly purified preparation. However, the finding that Rap1A was present in normal amounts in the neutrophil membranes from all four major types of CGD (including those missing cytochrome b) suggested that the conditions required of the reconstitution assay did not favor the reassociation of the membrane-derived Rap1A with exogenously added cytochrome b or that another unidentified membrane component was lost during the final purification step. The normal expression of Rap1A in CGD cell membranes also indicates that this protein is not responsible for the absence of O2- production in the X-linked and autosomal recessive cytochrome b-negative forms of CGD. Finally, these results show that the expression of Rap1A in the plasma membrane is not dependent on the coordinate expression of cytochrome b, despite the close association shown for these two proteins in the normal cell membrane.

Cytochrome b Group↗
PubMed1992

Phosphorylation of smg p21B in rat peritoneal mast cells in association with histamine release inhibition by dibutyryl-cAMP.

IP3 formation and histamine release from rat peritoneal mast cells stimulated by compound 48/80 were dose-dependently inhibited by Bt2cAMP. These inhibitions were restored to the control level in the presence of H-8, a protein kinase A inhibitor. The 22 kDa protein in mast cells was revealed as a markedly phosphorylated protein by incubating with Bt2cAMP, and this phosphorylation was also diminished by H-8. The 22 kDa phosphoprotein of rat mast cells comigrated with phosphorylated smg p21B, purified from human platelets and phosphorylated by protein kinase A in cell-free system, in both one- and two-dimensional PAGE analysis. Moreover, 22 kDa protein in mast cells was identified as smg p21B by immunoblot analysis using an antibody against smg p21B. From the present study, it became clear that smg p21B is phosphorylated by means of protein kinase A system in rat peritoneal mast cells, and it was assumed that phosphorylated smg p21B plays some important role in the suppression of IP3 formation and histamine release from rat peritoneal mast cells.

Animals↗
PubMed1992

Epinephrine suppresses rap1B.GAP-activated GTPase activity in human platelets.

Lysate from quiescent platelets promotes rapid hydrolysis of [gamma-32P]GTP bound to rap1B. Various platelet agonists, including platelet-activating factor, phorbol 12,13-dibutyrate, alpha-thrombin, epinephrine, ADP, and iloprost, that affect platelet metabolism by different signal transduction pathways were used to stimulate intact platelets and study their effects on rap1B.GAP-activated GTPase activity (GAP, GTPase-activating protein). Only epinephrine was found to dramatically decrease not only the rate but also the amount of hydrolysis of rap1B-bound GTP activated by rap1B.GAP. This effect was dose dependent and occurred rapidly. The suppression of GTPase activity was specific for rap1B.GAP in that ras.GAP- and rap2B.GAP-activated GTPase activity were not affected by epinephrine stimulation. This effect appears to be mediated by the alpha 2-adrenergic receptor, as evidenced by a similar suppression of GTPase activity by stimulating platelets with the synthetic alpha 2-adrenergic receptor agonist UK14304 (bromoxidine). Furthermore, the selective alpha 2-adrenergic receptor antagonist yohimbine blocked the suppression of GTPase activity expressed in epinephrine-stimulated cell lysates. No apparent changes in the patterns of protein expression or tyrosine phosphorylation were observed. Although the migration characteristics upon anion-exchange chromatography of rap1B.GAP and ras.GAP activities were unaffected by epinephrine stimulation, the specific activity of rap1B.GAP was noticeably decreased with 250 and 500 microM epinephrine. These results suggest a possible role for rap1B and rap1B.GAP in epinephrine-stimulated signal transduction.

Blood Platelets↗
PubMed1992

Reversion of middle T antigen-transformed Rat-2 cells by Krev-1: implications for the role of p21c-ras in polyomavirus-mediated transformation.

Polyomavirus middle T antigen mediates transformation of cells, at least in part, by its association with and activation of the intrinsic protein tyrosine kinase activity of pp60c-src. pp60c-src, by analogy with pp60v-src, elicits cell proliferation through a signal transduction pathway that includes p21c-ras. Therefore, we tested the possibility that middle T antigen acts upstream of and in the same proliferative signaling pathway as p21c-ras. Co-transfection of Rat-2 cells with plasmids expressing human Krev-1, a dominant suppressor of Ki-ras transformation, and mT antigen resulted in a dose-dependent reduction of mT antigen-induced foci. Krev-1 did not affect the transforming activity of SV40 large T antigen, demonstrating that the transformation-suppressing activity of Krev-1 is specific. To determine the effect of Krev-1 on stably transformed cell lines, Krev-1 DNA was introduced into middle T antigen-transformed Rat-2 cells along with a G418 resistance marker. Of the G418-resistant colonies examined, 1% were morphologically untransformed. Characterization of several morphological revertants revealed that, with the exception of one cell line, all of the cell lines expressed middle T antigen, which was associated with pp60c-src, whose tyrosine kinase activity was similar to that found in the parental transformed cell lines. To determine whether other phenotypic traits associated with transformation were altered in these cell lines, their growth rates and ability to form colonies in agar suspension were examined. The majority of the revertants had longer doubling times, and grew less efficiently in agar suspension compared with their transformed parents. A direct correlation was observed between Krev-1 RNA and protein expression and the efficiency with which the revertants formed colonies in suspension. These results suggest that p21c-ras lies downstream of middle T antigen and pp60c-src in the same proliferative signal transduction pathway.

Animals↗
PubMed1992

smg/rap1/Krev-1 p21s inhibit the signal pathway to the c-fos promoter/enhancer from c-Ki-ras p21 but not from c-raf-1 kinase in NIH3T3 cells.

smg/rap1A/Krev-1 p21 cDNA is known to inhibit v-Ki-ras p21-induced cell transformation in NIH3T3 cells, but the inhibitory mechanism is not clear at present. In the present study, we examined the effect of smg p21s on the c-fos promoter/enhancer linked to the luciferase reporter gene (c-fos-luciferase). After transfection of c-fos-luciferase into NIH3T3 cells constitutively expressing c-Ki-ras(val-12) p21 or activated c-raf-1 kinase, expression of c-fos-luciferase was much higher than after transfection into control NIH3T3 cells. Addition of platelet-derived growth factor (PDGF), 12-O-tetradecanoyl phorbol 13-acetate (TPA) or dibutyryl cyclic AMP (Bt2cAMP) to the control NIH3T3 cells stimulated c-fos-luciferase expression. Transfection of the smg p21 cDNAs inhibited the activated ras p21-, PDGF- or TPA-stimulated c-fos-luciferase expression, but did not inhibit the activated c-raf-1 kinase- or Bt2cAMP-stimulated reaction. These results indicate that smg p21s inhibit the signal pathways from the PDGF receptor, protein kinase C, and ras p21s to the c-fos promoter/enhancer, but not those from c-raf-1 kinase and cyclic AMP-dependent protein kinase to the c-fos promoter/enhancer.

3T3 Cells↗
PubMed1992

C-terminal truncation of RAP1 results in the deregulation of telomere size, stability, and function in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae DNA-binding protein RAP1 is capable of binding in vitro to sequences from a wide variety of genomic loci, including upstream activating sequence elements, the HML and HMR silencer regions, and the poly(G1-3T) tracts of telomeres. Recent biochemical and genetic studies have suggested that RAP1 physically and functionally interacts with the yeast telomere. To further investigate the role of RAP1 at the telomere, we have identified and characterized three intragenic suppressors of a temperature-sensitive allele of RAP1, rap1-5. These telomere deficiency (rap1t) alleles confer several novel phenotypes. First, telomere tract size elongates to up to 4 kb greater than sizes of wild-type or rap1-5 telomeres. Second, telomeres are highly unstable and are subject to rapid, but reversible, deletion of part or all of the increase in telomeric tract length. Telomeric deletion does not require the RAD52 or RAD1 gene product. Third, chromosome loss and nondisjunction rates are elevated 15- to 30-fold above wild-type levels. Sequencing analysis has shown that each rap1t allele contains a nonsense mutation within a discrete region between amino acids 663 and 684. Mobility shift and Western immunoblot analyses indicate that each allele produces a truncated RAP1 protein, lacking the C-terminal 144 to 165 amino acids but capable of efficient DNA binding. These data suggest that RAP1 is a central regulator of both telomere and chromosome stability and define a C-terminal domain that, while dispensable for viability, is required for these telomeric functions.

Alleles↗
PubMed1992

Effects of agonists on p21ras and ras-related proteins in rat pancreatic acinar cells.

This study shows the presence of seven different low-molecular-weight GTP binding proteins (smg proteins) with molecular masses between 18 and 27 kDa in subfractions of rat pancreatic acinar cells. After stimulation of isolated intact and permeabilized pancreatic acinar cells with cholecystokinin octapeptide (CCK-OP), the diacylglycerol (DG) analogue 12-O-tetradecanoylphorbol 13-acetate (TPA), vasoactive intestinal peptide (VIP), adenosine 3',5'-cyclic monophosphate (cAMP), or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), [alpha-32P]GTP binding to 21- to 22-kDa smg protein(s) in microsomal membranes (MM) was reduced, whereas the [alpha-32P]GTP binding to 23-kDa protein(s) was enhanced. In addition, prestimulation of permeabilized cells with GTP gamma S caused enhancement of [alpha-32P]GTP binding to a 19-kDa protein in MM [immunologically identified as the ADP-ribosylation factor (arf)]. In the presence of cytosol, direct addition of GTP gamma S to isolated MM resulted in an apparent translocation of the 19-kDa protein (arf) from the cytosol to membranes. This indicates increased association of arf with the membrane in its GTP-bound state. In CCK-OP-prestimulated acinar cells, [alpha-32P]GTP binding to plasma membrane-located 21- to 22-kDa proteins (immunologically identified as p21ras proteins) was enhanced, suggesting that there is an interrelationship between p21ras proteins and CCK receptors. Our results give evidence for a role of 19-kDa, 21- to 22-kDa, and 23-kDa smg proteins in cAMP-protein kinase A- and DG-protein kinase C-mediated stimulation of intracellular pathways in pancreatic acinar cells.

Animals↗
PubMed1992

Specific isoprenoid modification is required for function of normal, but not oncogenic, Ras protein.

While the Ras C-terminal CAAX sequence signals modification by a 15-carbon farnesyl isoprenoid, the majority of isoprenylated proteins in mammalian cells are modified instead by a 20-carbon geranylgeranyl moiety. To determine the structural and functional basis for modification of proteins by a specific isoprenoid group, we have generated chimeric Ras proteins containing C-terminal CAAX sequences (CVLL and CAIL) from geranylgeranyl-modified proteins and a chimeric Krev-1 protein containing the H-Ras C-terminal CAAX sequence (CVLS). Our results demonstrate that both oncogenic Ras transforming activity and Krev-1 antagonism of Ras transforming activity can be promoted by either farnesyl or geranylgeranyl modification. Similarly, geranylgeranyl-modified normal Ras [Ras(WT)CVLL], when overexpressed, exhibited the same level of transforming activity as the authentic farnesyl-modified normal Ras protein. Therefore, farnesyl and geranylgeranyl moieties are functionally interchangeable for these biological activities. In contrast, expression of moderate levels of geranylgeranyl-modified normal Ras inhibited the growth of untransformed NIH 3T3 cells. This growth inhibition was overcome by coexpression of the mutant protein with oncogenic Ras or Raf, but not with oncogenic Src or normal Ras. The similar growth-inhibiting activities of Ras(WT)CVLL and the previously described Ras(17N) dominant inhibitory mutant suggest that geranylgeranyl-modified normal Ras may exert its growth-inhibiting action by perturbing endogenous Ras function. These results suggest that normal Ras function may specifically require protein modification by a farnesyl, but not a geranylgeranyl, isoprenoid.

3T3 Cells↗
PubMed1992

Oncogenic ras induces an inhibitor of double-stranded RNA-dependent eukaryotic initiation factor 2 alpha-kinase activation.

The interferon-inducible 68-kDa dsRNA-dependent eIF2 alpha-kinase (dsI) is a potent cellular antiviral enzyme which is activated by autophosphorylation in response to double-stranded RNA (dsRNA). Activated dsI has also been implicated as a second messenger for gene induction by platelet-derived growth factor (PDGF) and interferon (IFN). We have shown previously that introduction of a transforming ras gene into BALB/c-3T3 fibroblasts blocks induction of responsive genes by PDGF and IFN. We therefore investigated the effect of transforming ras genes on dsI activity in these cells. We report here that dsRNA-mediated activation of dsI is blocked in v-ras-containing cells in a manner specific to ras and not attributable to the transformed phenotype since: 1) a dexamethasone-inducible v-Ha-ras gene produced the effect within 18 h of induction; 2) morphologic reversion of ras-transformed cells with cAMP or the Krev-1 gene restored potential for dsI activation; and 3) transformation by v-mos or v-abl had no effect on dsI activation. Latent dsI levels were unaffected by v-ras. A heat-sensitive dsI inhibitory activity could be demonstrated in v-ras-containing cells which functioned in trans when mixed with untransformed cell extracts prior to stimulation with dsRNA. The inhibitory activity, which was destroyed by phenol-chloroform extraction, did not bind dsRNA.

3T3 Cells↗
PubMed1992

Role of protein methylation in agonist-induced signal transduction in human platelets.

Possible role of methylation of proteins in platelet activation was examined in this study. Electropermeabilized platelets incorporated radioactivity in the presence of [methyl-3H]-S-adenosylmethionine. Thrombin, PDBu and GTP gamma S increased incorporation of radioactivity in a time-dependent manner. In other experiments, 23 kD membrane proteins incorporated radioactivity in the presence of [methyl-3H]-S-adenosylmethionine and platelet cytosol. Using rap specific antisera the 23 kD methylated proteins were characterized as low Mr G proteins, known as rap1 proteins. N-Acetyl-S-farnesyl-L-cysteine (AFC), an inhibitor of the methyltransferase, inhibited carboxyl methylation of platelet rap1 proteins and also inhibited platelet aggregation and mobilization of cytosolic calcium induced by a variety of agonists in a concentration-dependent manner. Inhibition of methylation of rap1 proteins as well as inhibition of platelet activation by AFC suggests that methylation and consequently translocation of rap1 proteins to plasma membrane may be important for agonist-induced signal transduction in human platelets.

Acetylcysteine↗