ADP-ribosylation Of Gs Alpha Research Articles

Effects of site-directed mutagenesis of Escherichia coli heat-labile enterotoxin on ADP-ribosyltransferase activity and interaction with ADP-ribosylation factors.

Escherichia coli heat-labile enterotoxin (LT), an oligomeric protein with one A subunit (LTA) and five B subunits, exerts its effects via the ADP-ribosylation of Gsalpha, a guanine nucleotide-binding (G) protein that activates adenylyl cyclase. LTA also ADP-ribosylates simple guanidino compounds (e.g., arginine) and catalyzes its own auto-ADP-ribosylation. All LTA-catalyzed reactions are enhanced by ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins. Replacement of arginine-7 (R7K), valine-53 (V53D), serine-63 (S63K), valine 97 (V97K), or tyrosine-104 (Y104K) in LTA resulted in fully assembled but nontoxic proteins. S63K, V53D, and R7K are catalytic-site mutations, whereas V97K and Y104K are amino acid replacements adjacent to and outside of the catalytic site, respectively. The effects of mutagenesis were quantified by measuring ADP-ribosyltransferase activity (i.e., auto-ADP-ribosylation and ADP-ribosylagmatine synthesis) and interaction with ARF (i.e., inhibition of ARF-stimulated cholera toxin ADP-ribosyltransferase activity and effects of ARF on mutant auto-ADP-ribosylation). All mutants were inactive in the ADP-ribosyltransferase assay; however, auto-ADP-ribosylation in the presence of recombinant human ARF6 was detected, albeit much less than that of native LT (Y104K > V53D > V97K > R7K, S63K). Based on the lack of inhibition by free ADP-ribose, the observed auto-ADP-ribosylation activity was enzymatic and not due to the nonenzymatic addition of free ADP-ribose. V53D, S63K, and R7K were more effective than Y104K or V97K in blocking ARF stimulation of cholera toxin ADP-ribosyltransferase. Based on these data, it appears that ARF-binding and catalytic sites are not identical and that a region outside the NAD cleft may participate in the LTA-ARF interaction.

Increased cyclic adenosine 3',5'-monophosphate inhibits G protein-coupled activation of phospholipase C in rat FRTL-5 thyroid cells.

Thyroid cell growth and function are regulated by several hormones and growth factors that bind to cell surface receptors coupled via G proteins, Gs and Gq, to stimulation of adenylyl cyclase and phospholipase C (PLC), respectively. We created a permanently transfected FRTL-5 cell line (TG8) in which the thyroglobulin gene promoter directs expression of the cholera toxin (CT) A1 subunit (CTA1). CTA1 catalyzes ADP ribosylation of Gs alpha, which results in persistent activation of Gs alpha. Activated Gs alpha causes constitutive stimulation of adenylyl cyclase and increases levels of intracellular cAMP. Because G protein-coupled signaling pathways exhibit cross-talk, we compared TG8 cells to FRTL-5 cells transfected with the neomycin resistance gene (TG4) to determine whether constitutive stimulation of adenylyl cyclase influences the PLC pathway. PLC activity was assessed by measuring levels of total inositol phosphates (IPs) in TG4 and TG8 cells that had been preincubated with myo-[3H]inositol for 2 days. Baseline values of [3H]IP production were similar for the two cell lines. Incubation of TG4 control cells with 10(-8) M TSH, 300 microM ATP, and 100 microM norepinephrine for 60 min stimulated 2.5-, 8.1-, and 3.4-fold increases, respectively, in [3H]IP production over the control value. By contrast, there was no [3H]IP response to any of these ligands in TG8 cells. TG8 cells exhibit a decrease in [35S]adenosine 5'-(gamma-thio)triphosphate binding to their cell surface compared to TG4 control cells counterparts, but no decrease in [125I]TSH binding. Treatment of TG4 cells with 100 ng/ml CT, 50 microM forskolin, or 1 mM 8-bromo-cAMP for 2 days reproduced the loss of ligand-stimulated [3H]IP synthesis present in TG8 cells. Although levels of immunoreactive Gq alpha and Gq alpha 11 were normal in TG8 cells, sodium fluoride-induced [3H]IP production was also inhibited. Levels of immunoreactive PLC beta 3, the dominant subtype of PLC beta in FRTL-5 cells, were not altered in TG8 cells or by CT treatment of TG4 cells. These data indicate that elevated levels of cAMP can inhibit the activity of G protein-coupled PLC. Further study of this model will elucidate our understanding of the exact mechanism responsible for this interaction.

Expression of functional stimulatory guanine nucleotide binding protein in nonfunctioning thyroid adenomas is not correlated to adenylate cyclase activity and growth of these tumors.

In thyroid cells, the alpha-subunit of the stimulatory guanine nucleotide binding protein (Gs alpha) acts as signal transducer between the TSH receptor and the adenylate cyclase (AC), and it regulates both growth and function. In order to analyze Gs alpha expression by both Western blot analysis and in situ, we generated an antibody raised against a recombinant human Gs alpha protein. With this antibody, a strong cytoplasmic Gs alpha-immunostaining was detectable in cultured human thyroid cells and in TSH-stimulated rat thyroids, in contrast to normal human thyroids and to T4-treated rat thyroids, which showed only weak immunoreactivity. We obtained the following results by immunohistochemistry and Western blot analysis of 32 actively growing human thyroid adenomas: 1) strong Gs alpha expression in 11 adenomas, including 4 hyperfunctioning nodules, 1 of these with a point mutation in codon 201 of the Gs alpha gene; 2) no expression or only weak Gs alpha expression in 13 adenomas; and 3) a pattern of Gs alpha-positive and Gs alpha-negative cells in the remaining 8 adenomas. In addition, we analyzed ADP-ribosylation of Gs alpha and AC activity in 9 nonfunctioning adenomas and found a significant correlation between Gs alpha immunoreactivity and ADP-ribosylation and no correlation of both with basal and TSH-stimulated AC activity. In both types of adenomas, i.e. those with high as well as low Gs alpha, an indistinguishably high fraction of proliferating cells was detectable. We conclude that expression of functional Gs alpha protein in nonfunctioning thyroid adenomas is neither correlated to the basal or TSH-stimulated AC activity nor to the proliferation rate of these tumors.

Stimulation of rat parietal cell function by histamine and GLP-1-(7-36) amide is mediated by Gs alpha

It was the aim of the present study to determine in rat parietal cells whether Gs alpha, the stimulatory subunit of adenylate cyclase, mediates adenosine 3',5'-cyclic monophosphate (cAMP)-dependent parietal cell function in response to histamine and glucagon-like peptide 1 (GLP-1)-(7-36) amide. Cytoplasmic membrane from enriched (83 +/- 5%) rat parietal cells were incubated for 30 min with 30 microCi/ml [32P]NAD+ and 40 micrograms/ml preactivated cholera toxin (CT), a pharmacological tool for activation of Gs alpha. Subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography revealed [32P]ADP ribosylation of Gs alpha represented by three proteins with molecular masses ranging from 42 to 45 kDa. In intact parietal cells, CT (10(-12)-10(-8) M) caused marked stimulation of [14C]aminopyrine accumulation and cAMP production confirming the functional importance of Gs alpha in regulation of H+ production. Identical membrane preparations were preincubated (2 h, 4 degrees C) in parallel with and without RM/1, a rabbit polyclonal anti-Gs alpha-antibody. Subsequently, adenylate cyclase was stimulated by histamine, GLP-1-(7-36) amide, CT, or forskolin. At a 1:10 dilution, the antiserum completely abolished adenylate cyclase activity in response to maximal concentrations of histamine, GLP-1-(7-36) amide, and CT while reducing forskolin stimulation by only 22.0 +/- 4.9%. At 1:50, RM/1 reduced responses to histamine, GLP-1-(7-36) amide and CT by 20-30% but failed to inhibit forskolin-stimulated enzyme activity. At 1:100, the antiserum was ineffective versus all stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of adenylate cyclase and GTP-binding proteins in hepatic plasma membranes.

Hepatic membrane subfractions prepared from control rats demonstrated forskolin (FSK)-stimulated adenylate cyclase activity in the basolateral (sinusoidal) but not apical (canalicular) plasma membrane. After bile duct ligation (BDL) for 12 or 24 h, there was an increase in FSK-stimulated adenylate cyclase activity in the apical membrane (54.2 +/- 3.9 pmol.mg-1 x min-1). The mechanism for this increase was explored further. ATP hydrolysis was found to be much higher in the apical than the basolateral membrane. Increasing the ATP levels in the assay enhanced apical membrane adenylate cyclase activity (10.5 +/- 0.2 pmol.mg-l.min-1); however, total adenosinetriphosphatase (ATPase) activity was not altered after BDL. Extraction of the apical membrane with bile acids or other detergents resulted in a two- to threefold increase in adenylate cyclase activity (30.6 +/- 3.6 pmol.mg-1 x min-1; detergent C12E8) This suggested that bile duct ligation was acting via the detergent-like action of bile acids to uncover latent adenylate cyclase activity on apical membranes. Further studies demonstrated that both BDL and detergent extraction also enhanced toxin-directed ADP-ribosylation of Gs alpha (cholera toxin) and Gi alpha (pertussis toxin) in the apical but not the basolateral membrane. After BDL, Gi alpha was found to be twofold greater in the apical membrane than the basolateral membrane. Immunoblotting using specific G protein antibodies further confirmed that apical membranes from control rats had a higher concentration of Gi1, 2 alpha and beta and slightly elevated levels of Gi3 alpha and Gs alpha compared with the basolateral membrane. The results demonstrate that adenylate cyclase and heterotrimeric GTP-binding proteins are present on the apical membrane, but measurement of their functional activity requires detergent permeabilization of apical membrane vesicles and is limited by the presence of high ATPase activity.

Effects of temperature on ADP-ribosylation factor stimulation of cholera toxin activity.

The effects of cholera toxin, a secretory product of Vibrio cholerae, result from ADP-ribosylation of the stimulatory guanine nucleotide-binding (Gs) protein of the adenylyl cyclase system. Cholera toxin A subunit (CTA) also uses agmatine, a simple guanidino compound, several proteins unrelated to Gs, and CTA itself as alternative ADP-ribose acceptors. The effects of toxin occur in the jejunum presumably at body core temperature. With agmatine as a model substrate, the optimal temperature for CTA-catalyzed ADP-ribosylation was 25-30 degrees C, and that for CTA-catalyzed auto-ADP-ribosylation was 20-25 degrees C. Both activities were significantly less at 37 degrees C, reflecting lower initial velocities, not heat-inactivation of the toxin. All the transferase activities of CTA are enhanced by ADP-ribosylation factors (ARFs), approximately 20-kDa guanine nucleotide-binding proteins that are ubiquitous in mammalian cells. Phospholipids and a soluble brain ARF, in a GTP-dependent manner, activated toxin NAD:agmatine ADP-ribosyltransferase activity; their simultaneous effect was maximal at physiological temperatures (approximately 37 degrees C). At lower temperatures, the stimulation by ARF was much less. There were similar effects on other toxin-catalyzed reactions, notably, the ADP-ribosylation of Gs alpha and the hydrolysis of NAD. Thus, host factors, such as ARF and phospholipid, synergistically increase cholera toxin activity at 37 degrees C and may be important in toxin action in the mammalian gut.

Human growth hormone enhances pertussis toxin-stimulated ADP-ribosylation of Gi in Nb2 cell membrane.

The Nb2 node lymphoma cell line has been widely used as a model for investigating lactogen cellular actions. Both pertussis (PTX) and cholera (CTX) toxins modulate lactogen-stimulated Nb2 cell mitogenesis, suggesting G protein involvement in lactogen signal transduction. The following studies were performed to further investigate this possibility. Both PTX-sensitive (41 kDa) and CTX-sensitive substrates (42 and 45 kDa) were identified in Nb2 cell membrane and recognized by specific anti-Gi and anti-Gs antibodies, respectively. Equal numbers of Nb2 cells were then incubated with the lactogen human growth hormone (hGH, 10 ng/ml) for 0-72 h. Membrane protein prepared from each time point (50 micrograms) was compared in toxin-stimulated ADP-ribosylation studies. CTX-stimulated ADP-ribosylation was unaffected by prior hGH incubation. PTX-stimulated ADP-ribosylation increased 237 +/- 69% (X +/- S.E.) compared with 0-h controls (n = 11; p less than 0.01) after 4-7 h of hGH incubation then decreased toward 0-h samples by 24 and 72 h. No change in Gi alpha concentration was observed, but beta subunit concentration increased (145 +/- 14% at 7 h; p less than 0.01; n = 3) in a time course that paralleled the changes in PTX-stimulated ADP-ribosylation. In summary, 1) both Gi and Gs were present in Nb2 cell membrane, 2) incubation of cells with a lactogen, hGH, for 4-7 h markedly enhanced PTX-stimulated ADP-ribosylation of Gi alpha in vitro, whereas CTX-stimulated ADP-ribosylation of Gs alpha was unchanged, and 3) although no change in Gi alpha concentration was observed, beta subunit concentration increased in parallel with the increase in PTX-stimulated ADP-ribosylation of Gi alpha. These results suggest that hGH may modify PTX-stimulated ADP-ribosylation of Gi not by changing Gi alpha concentration, perhaps by increasing beta subunit concentration, enhancing association of Gi alpha by beta gamma subunits, which, in turn, is preferentially ADP-ribosylated. This may represent a late signal transduction event and may also have implications for other effectors dependent on Gi-mediated events.

Activation of Escherichia coli heat-labile enterotoxins by native and recombinant adenosine diphosphate-ribosylation factors, 20-kD guanine nucleotide-binding proteins.

Escherichia coli heat-labile enterotoxins (LT) are responsible in part for "traveler's diarrhea" and related diarrheal illnesses. The family of LTs comprises two serogroups termed LT-I and LT-II; each serogroup includes two or more antigenic variants. The effects of LTs result from ADP ribosylation of Gs alpha, a stimulatory component of adenylyl cyclase; the mechanism of action is identical to that of cholera toxin (CT). The ADP-ribosyltransferase activity of CT is enhanced by 20-kD guanine nucleotide-binding proteins, known as ADP-ribosylation factors or ARFs. These proteins directly activate the CTA1 catalytic unit and stimulate its ADP ribosylation of Gs alpha, other proteins, and simple guanidino compounds (e.g., agmatine). Because of the similarities between CT and LTs, we investigated the effects of purified bovine brain ARF and a recombinant form of bovine ARF synthesized in Escherichia coli on LT activity. ARF enhanced the LT-I-, LT-IIa-, and LT-IIb-catalyzed ADP ribosylation of agmatine, as well as the auto-ADP ribosylation of the toxin catalytic unit. Stimulation of ADP-ribosylagmatine formation by LTs and CT in the presence of ARF was GTP dependent and enhanced by sodium dodecyl sulfate. With agmatine as substrate, LT-IIa and LT-IIb exhibited less than 1% the activity of CT and LT-Ih. CT and LTs catalyzed ADP-ribosyl-Gs alpha formation in a reaction dependent on ARF, GTP, and dimyristoyl phosphatidylcholine/cholate. With Gs alpha as substrate, the ADP-ribosyltransferase activities of the toxins were similar, although CT and LT-Ih appeared to be slightly more active than LT-IIa and LT-IIb. Thus, LT-IIa and LT-IIb appear to differ somewhat from CT and LT-Ih in substrate specificity. Responsiveness to stimulation by ARF, GTP, and phospholipid/detergent as well as the specificity of ADP-ribosyltransferase activity are functions of LTs from serogroups LT-I and LT-II that are shared with CT.

Enhancement of choleragen ADP-ribosyltransferase activities by guanyl nucleotides and a 19-kDa membrane protein.

Choleragen activates adenylate cyclase by catalyzing, in the presence of NAD, the ADP-ribosylation of Gs alpha, the stimulatory guanyl nucleotide-binding protein of the cyclase system. Kahn and Gilman [Kahn, R. A. & Gilman, A. G. (1986) J. Biol. Chem. 261, 7906-7911] identified another guanyl nucleotide-binding protein termed ADP-ribosylation factor (ARF) that stimulated this reaction. It was proposed that the toxin substrate is an ARF-Gs alpha complex and that ARF may have a physiological role in regulation of Gs alpha activity. We have found that purified ARF from bovine brain enhances not only the ADP-ribosylation of Gs alpha but also Gs alpha-independent choleragen-catalyzed reactions. These are (i) ADP-ribosylation of agmatine, a low molecular weight guanidino compound; (ii) ADP-ribosylation of several proteins unrelated to Gs alpha; and (iii) auto-ADP-ribosylation of the toxin A1 peptide. These reactions, as well as the ADP-ribosylation of ARF itself, were stimulated by GTP or stable GTP analogues such as guanyl-5'-yl imido-beta gamma-diphosphate and guanosine 5'-O-[gamma-thio]triphosphate; GDP and guanosine 5'-O-[beta-thio]diphosphate were inactive. These observations are consistent with the conclusion that ARF interacts directly with the A subunit of choleragen in a GTP-dependent fashion thereby enhancing catalytic activity manifest as transfer of ADP-ribose to Gs alpha and other proteins, to the toxin A1 peptide, or to agmatine. It is tempting to speculate that ARF may be involved in regulating one or another of the ADP-ribosyltransferases found in animal cells.