GTP but not GDP analogues promote association of ADP-ribosylation factors, 20-kDa protein activators of cholera toxin, with phospholipids and PC-12 cell membranes.

ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance cholera toxin ADP-ribosyltransferase activity in the presence of GTP. ARFs have been purified from both membrane and cytosolic fractions. ARF purified from bovine brain cytosol requires phospholipid plus detergent for high affinity guanine nucleotide binding and for optimal enhancement of cholera toxin ADP-ribosyltransferase activity. The phospholipid requirements, combined with a putative role for ARF in vesicular transport, suggested that the soluble protein might interact reversibly with membranes. A polyclonal antibody against purified bovine ARF (sARF II) was used to detect ARF by immunoblot in membrane and soluble fractions from rat pheochromocytoma (PC-12) cell homogenates. ARF was predominantly cytosolic but increased in membranes during incubation of homogenates with nonhydrolyzable GTP analogues guanosine 5'-O-(3-thiotriphosphate), guanylyl-(beta gamma-imido)-diphosphate, and guanylyl-(beta gamma-methylene)-diphosphate, and to a lesser extent, adenosine 5'-O-(3-thiotriphosphate). GTP, GDP, GMP, and ATP were inactive. Cytosolic ARF similarly associated with added phosphatidylserine, phosphatidylinositol, or cardiolipin in GTP gamma S-dependent fashion. ARF binding to phosphatidylserine was reversible and coincident with stimulation of cholera toxin-catalyzed ADP-ribosylation. These observations may reflect a mechanism by which ARF could cycle between soluble and membrane compartments in vivo.     

Guanine nucleotide dependent formation of a complex between choleragen (cholera toxin) a subunit and bovine brain ADP-ribosylation factor

Cholera toxin activates adenylyl cyclase by catalyzing the ADP-ribosylation of Gs alpha, the stimulatory guanine nucleotide binding protein of the cyclase system. This toxin-catalyzed reaction, as well as the ADP-ribosylation of guanidino compounds and auto-ADP-ribosylation of the toxin A1 protein (CTA1), is stimulated, in the presence of GTP (or GTP analogue), by 19-21-kDa proteins, termed ADP-ribosylation factors or ARFs. These proteins directly activate CTA1 in a reaction enhanced by sodium dodecyl sulfate (SDS) or dimyristoylphosphatidylcholine (DMPC)/cholate. To determine whether ARF stimulation of ADP-ribosylation is associated with formation of a toxin-ARF complex, these proteins were incubated with guanine nucleotides and/or detergents and then subjected to gel permeation chromatography. An active ARF-toxin complex was observed in the presence of SDS and GTP gamma S [guanosine 5'-O-(3-thiotriphosphate)] but not GDP beta S [guanosine 5'-O-(2-thiodiphosphate)]. Only a fraction of the ARF was capable of complex formation. The substrate specificities of complexed and noncomplexed CTA differed; complexed CTA exhibited markedly enhanced auto-ADP-ribosylation. In the presence of GTP gamma S and DMPC/cholate, an ARF-CTA complex was not detected. A GTP gamma S-dependent ARF aggregate was observed, however, exhibiting a different substrate specificity from monomeric ARF. These studies support the hypothesis that in the presence of guanine nucleotide and either SDS or DMPC/cholate, ARF and toxin exist as multiple species which exhibit different substrate specificities.     

Mechanism of activation of cholera toxin by ADP-ribosylation factor (ARF): both low- and high-affinity interactions of ARF with guanine nucleotides promote toxin activation

Activation of adenylyl cyclase by cholera toxin A subunit (CT-A) results from the ADP-ribosylation of the stimulatory guanine nucleotide binding protein (GS alpha). This process requires GTP and an endogenous guanine nucleotide binding protein known as ADP-ribosylation factor (ARF). One membrane (mARF) and two soluble forms (sARF I and sARF II) of ARF have been purified from bovine brain. Because the conditions reported to enhance the binding of guanine nucleotides by ARF differ from those observed to promote optimal activity, we sought to characterize the determinants influencing the functional interaction of guanine nucleotides with ARF. High-affinity GTP binding by sARF II (apparent KD of approximately 70 nM) required Mg2+, DMPC, and sodium cholate. sARF II, in DMPC/cholate, also enhanced CT-A ADP-ribosyltransferase activity (apparent EC50 for GTP of approximately 50 nM), although there was a delay before achievement of a maximal rate of sARF II stimulated toxin activity. The delay was abolished by incubation of sARF II with GTP at 30 degrees C before initiation of the assay. In contrast, a maximal rate of activation of toxin by sARF II, in 0.003% SDS, occurred without delay (apparent EC50 for GTP of approximately 5 microM). High-affinity GTP binding by sARF II was not detectable in SDS. Enhancement of CT-A ADP-ribosyltransferase activity by sARF II, therefore, can occur under conditions in which sARF II exhibits either a relatively low affinity or a relatively high affinity for GTP. The interaction of GTP with ARF under these conditions may reflect ways in which intracellular membrane and cytosolic environments modulate GTP-mediated activation of ARF.     

ADP-ribosylation factors: a family of ∼20-kDa guanine nucleotide-binding proteins that activate cholera toxin

ADP-ribosylation factors (ARFs) comprise a family of 20 kDa guanine nucleotide-binding proteins that were discovered as one of several cofactors required in cholera toxin-catalyzed ADP-ribosylation of G_s, the guanine nucleotide-binding protein responsible for stimulation of adenylyl cyclase, and was subsequently found to enhance all cholera toxin-catalyzed reactions and to directly interact with, and activate the toxin. ARF is dependent on GTP or its analogues for activity, binds GTP with high affinity in the presence of dimyristoylphosphatidylcholine/cholate and contains consensus sequences for GTP-binding and hydrolysis. Six mammalian family members have been identified which have been classified into three groups (Class I, II, and III) based on size, deduced amino acid sequence identity, phylogenetic analysis and gene structure. ARFs are ubiquitous among eukaryotes, with a deduced amino acid sequence that is highly conserved across diverse species. They have recently been shown to associate with phospholipid and Golgi membranes in a GTP-dependent manner and are involved in regulating vesicular transport.Abbreviations ARF ADP-ribosylation factor - sARF I and sARF II soluble ADP-ribosylation factors purified from bovine brain - mARF purified membrane-associated ARF - hARF human ARF - bARF bovine ARF - yARF yeast ARF - ARF bacterially-expressed recombinant ARF - gARF Giardia ARF - dARF Drosophila ARF - G protein guanine nucleotide-binding protein - G_s G protein responsible for stimulation of adenylyl cyclase - GTPS guanosine-5-O-(3-thio-triphosphate) - CIAI cholera toxin A1 subunit - DMPC dimyristoylphosphatidylcholine - SDS sodium dodecyl sulfate     

Human and Giardia ADP-ribosylation factors (ARFs) complement ARF function in Saccharomyces cerevisiae.

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that stimulate the ADP-ribosyltransferase activity of cholera toxin in vitro. ARFs are highly conserved, ubiquitously expressed in eukaryotic cells and appear to be involved in vesicular protein transport. The two yeast ARFs are > 60% identical to mammalian ARFs and are essential for cell viability (Stearns, T., Kahn, R. A., Botstein, D., and Hoyt, M. A. (1990) Mol. Cell. Biol. 10, 6690-6699). Although the two yeast ARF proteins are 96% identical in amino acid sequence, the yeast ARF1 gene is constitutively expressed, whereas the ARF2 gene is repressed by glucose. Human ARF5 and ARF6 and a Giardia ARF differ substantially in size and amino acid identity from other mammalian and eukaryotic ARFs but will, as befits their designation, activate cholera toxin. Expression of human ARF5, ARF6, or Giardia ARF cDNA rescued the lethal yeast ARF double mutant (arf1, arf2). Strains rescued by human ARF5, ARF6, or Giardia ARF grew much more slowly than wild-type yeast or strains rescued with yeast ARF1. We infer from the impaired growth of these rescued strains that the homologous ARFs may have specific targeting information that does not interact effectively or efficiently with the yeast protein membrane trafficking system.     

Interspecies relationships among ADP-ribosylation factors (ARFs): Evidence of evolutionary pressure to maintain individual identities

ADP-ribosylation factors (ARFs) are ～20-kDa guanine nucleotide-binding proteins that are allosteric activators of the NAD:arginine ADP-ribosyltransferase activity of cholera toxin and appear to play a role in intracellular vesicular trafficking. Although the physiological roles of these proteins have not been defined, it has been presumed that each has a specific intracellular function. To obtain genetic evidence that each ARF is under evolutionary pressure to maintain its structure, and presumably function, rat ARF cDNA clones were isolated and their nucleotide and deduced amino acid sequences were compared to those of other mammalian ARFs. Deduced amino acid sequences for rat ARFs 1, 2, 3, 5 and 6 were identical to those of the known cognate human and bovine ARFs; rat ARF4 was 96% identical to human ARF4. Nucleotide sequences of both the untranslated as well as the coding regions were highly conserved. These results indicate that the ARF proteins are, as a family, extraordinarily well conserved across mammalian species. The unusually high degree of conservation of the untranslated regions is consistent with these regions having important regulatory roles and that individual ARFs contain structurally unique elements required for specific functions.     

Microinjection of a 19-kDa guanine nucleotide-binding protein inhibits maturation of Xenopus oocytes

ADP-ribosylation factors (ARFs) are 19-21-kDa proteins purified from bovine brain that bind guanosine 5'-triphosphate (GTP). They exhibit GTP-dependent activity as activators of cholera toxin-catalyzed ADP-ribosylation of the alpha-subunit of the stimulatory guanine nucleotide-binding protein of the adenylyl cyclase system (Gs alpha). ARF, which interacts directly with the catalytic subunit of cholera toxin, has no known physiologic role. Intracellular microinjection of ARF was employed to investigate the effect of ARF on progesterone- and insulin-stimulated maturation of Xenopus oocytes. Maturation was inhibited by injection of ARF 3-8 h before exposure of oocytes to progesterone or insulin. ARF inhibition was dependent on progesterone concentration but not on insulin concentration. Inhibition was enhanced by concomitant injection of GTP and to a greater extent by guanosine 5'-O-(thiotriphosphate) (GTP gamma S) which, in the absence of ARF, inhibited somewhat at early time points. The demonstration of this effect of ARF on both progesterone- and insulin-stimulated oocyte maturation may provide a clue to the physiologic role of this guanine nucleotide-binding protein.     

Purification of a protein cofactor required for ADP-ribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin

A factor (ARF) that is required for the cholera toxin-dependent ADP-ribosylation of the stimulatory, GTP-binding regulatory component (Gs) of adenylate cyclase has been purified about 2000-fold from cholate extracts of rabbit liver membranes. ARF is an intrinsic membrane protein with Mr = 21,000. The final product can be resolved into two polypeptides with very similar molecular weights; each of these has ARF activity. The ADP-ribosylation of Gs can now be studied with defined components. GTP and ARF are both necessary cofactors. The data imply that the substrates for the activated toxin are NAD and a GTP X Gs X ARF complex, and the reaction proceeds in a lipid environment. The apparent ability of ARF to bind to the alpha subunit of Gs suggests that it may play another, unknown role in the regulation of adenylate cyclase activity.     

Differential expression during development of ADP-ribosylation factors, 20-kDa guanine nucleotide-binding protein activators of cholera toxin

Cholera toxin exerts its effects on cells in large part through the ADP-ribosylation of guanine nucleotide-binding proteins. Toxin-catalyzed ADP-ribosylation is enhanced by approximately 20-kDa guanine nucleotide-binding proteins termed ADP-ribosylation factors (ARFs), which are allosteric activators of the toxin catalytic unit. Rabbit antiserum against a purified bovine brain ARF (sARF II) reacted on immunoblots with two approximately 20-kDa ARF-like proteins (sARF I and II) in tissue extracts from bovine, rat, frog, and chicken. Levels of ARF were higher in brain than in non-neural tissues. In rat brain, on the second postnatal day, amounts of sARF I and II were similar. By the 10th postnatal day and thereafter, sARF II predominated. Relative levels of ARF determined by immunoreactivity were in agreement with levels assessed in functional assays of cholera toxin-catalyzed ADP-ribosylation. Based on nucleotide and deduced amino acid sequences of human and bovine cDNAs, there appear to be at least six different ARF-like genes. Northern blots of rat brain poly(A)+ RNA were hybridized with cDNA and oligonucleotide probes specific for each of the human and bovine ARF genes. From the second to the 27th postnatal day, ARF 3 mRNA increased, whereas mRNAs for ARFs 2 and 4 decreased; and those for ARFs 1, 5, and 6 were apparently unchanged. Partial amino acid sequence of sARF II is consistent with it being either the ARF 1 or 3 gene product. The developmental changes in rat brain ARF parallel neuronal maturation and synapse formation.     

The protein cofactor necessary for ADP-ribosylation of Gs by cholera toxin is itself a GTP binding protein

A membrane-bound protein cofactor (ARF) is required for the cholera toxin-dependent ADP-ribosylation of the stimulatory regulatory component (Gs) of adenylate cyclase. Improved methods for the purification of ARF from bovine brain are described. ARF has a high-affinity binding site for guanine nucleotides. Binding of GTP or GTP gamma S to ARF is necessary for the activity of the cofactor; GDP X ARF does not support ADP-ribosylation of Gs. Although the protein as purified contains stoichiometric amounts of GDP, GTPase activity of isolated ARF was not detected. Cholera toxin-dependent activation of adenylate cyclase thus requires two guanine nucleotide binding proteins.     

Evidence for a rabbit luteal ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase.

1. An ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase was identified in a plasma membrane fraction from rabbit corpora lutea and partially characterized by comparing the properties of the luteal transferase with those of cholera toxin. 2. Incubation of luteal membranes in the presence of GTP and varying concentrations of NAD resulted in concentration-dependent increases in adenylyl cyclase activity. 3. Stimulation of adenylyl cyclase by NAD and cholera toxin plus NAD was observed in the presence of GTP but not in the presence of guanosine-5'-O-(2-thiodiphosphate) or guanyl-5'-yl imidodiphosphate. 4. NAD or cholera toxin plus NAD reduced the Kact values for luteinizing hormone to activate adenylyl cyclase 3- to 3.5-fold. 5. NAD or cholera toxin plus NAD increased the extent to which cholate extracts from luteal membranes were able to reconstitute adenylyl cyclase activity in S49 cyc- mouse lymphoma membranes. 6. It was necessary to add ADP-ribose and arginine to the incubation mixture in order to demonstrate cholera toxin-specific ADP-ribosylation of a protein corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (alpha Gs). 7. Treatment of luteal membranes with NAD prior to incubation in the presence of [32P]NAD plus cholera toxin resulted in reduced labeling of alpha Gs. 8. Endogenous ADP-ribosylation of alpha Gs was enhanced by Mg but was not altered by guanine nucleotide, NaF or luteinizing hormone and was inhibited by cAMP. 9. Incubation of luteal membranes in the presence of [32P]ADP-ribose in the absence and presence of cholera toxin did not result in the labeling of any membrane proteins.     

Activation of cholera toxin and Escherichia coli heat-labile enterotoxins by ADP-ribosylation factors, a family of 20 kDa guanine nucleotide-binding proteins

Cholera toxin and Escherichia coli heat-labile enterotoxins are responsible, in part, for the symptomatology of cholera and traveller's diarrhoea, respectively. Effects of the toxins result from ADP-ribosylation of regulatory guanine nucleotide-binding (G) proteins; the ADP-ribosylated G protein is stabilized in an activated state, resulting in prolonged effects on its target. Toxin-catalysed ADP-ribosylation is stimulated in vitro by a family of guanine nucleotide-binding proteins, c. 20 kDa, termed ADP-ribosylation factors or ARFs. In the presence of GTP, but not GDP or adenine analogues, ARFs serve as allosteric activators of the toxin. The effects are amplified by certain phospholipids and detergents which promote guanine nucleotide binding. Six different mammalian ARF genes have been identified. They encode highly conserved, ubiquitous proteins of 175 to 181 amino acids, containing consensus domains responsible for guanine nucleotide binding. Differences in amino acid sequences are localized near the amino terminus and in the carboxy half of the protein. Although the physiological functions of ARFs have not been precisely defined, their immunological localization to the Golgi is consistent with a role in the regulated orderly movement of newly synthesized proteins from the endoplasmic reticulum, through the Golgi system to their ultimate destination.     

GIT proteins, A novel family of phosphatidylinositol 3,4, 5-trisphosphate-stimulated GTPase-activating proteins for ARF6

ADP-ribosylation factor (ARF) proteins are key players in numerous vesicular trafficking events ranging from the formation and fusion of vesicles in the Golgi apparatus to exocytosis and endocytosis. To complete their GTPase cycle, ARFs require a guanine nucleotide-exchange protein to catalyze replacement of GDP by GTP and a GTPase-activating protein (GAP) to accelerate hydrolysis of bound GTP. Recently numerous guanine nucleotide-exchange proteins and GAP proteins have been identified and partially characterized. Every ARF GAP protein identified to date contains a characteristic zinc finger motif. GIT1 and GIT2, two members of a new family of G protein-coupled receptor kinase-interacting proteins, also contain a putative zinc finger motif and display ARF GAP activity. Truncation of the amino-terminal region containing the zinc finger motif prevented GAP activity of GIT1. One zinc molecule was found associated per molecule of purified recombinant ARF-GAP1, GIT1, and GIT2 proteins, suggesting the zinc finger motifs of ARF GAPs are functional and should play an important role in their GAP activity. Unlike ARF-GAP1, GIT1 and GIT2 stimulate hydrolysis of GTP bound to ARF6. Accordingly we found that the phospholipid dependence of the GAP activity of ARF-GAP1 and GIT proteins was quite different, as the GIT proteins are stimulated by phosphatidylinositol 3,4, 5-trisphosphate whereas ARF-GAP1 is stimulated by phosphatidylinositol 4,5-bisphosphate and diacylglycerol. These results suggest that although the mechanism of GTP hydrolysis is probably very similar in these two families of ARF GAPs, GIT proteins might specifically regulate the activity of ARF6 in cells in coordination with phosphatidylinositol 3-kinase signaling pathways.     

Molecular identification of ADP-ribosylation factor mRNAs and their expression in mammalian cells

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that serve as GTP-dependent allosteric activators of cholera toxin ADP-ribosyltransferase activity. Four species of mammalian ARF, termed ARF 1-4, have been identified by cloning. Hybridization of a bovine ARF 2 cDNA under low stringency with mammalian poly(A)+ RNA resulted in multiple bands that were subsequently assigned to the known ARF genes using ARF-specific oligonucleotide probes. The relative signal intensities of some bands (e.g. the 3.8- and 1.3-kilobase (kb) mRNAs) that hybridized with the cDNA were not, however, consistent with the intensities observed with the individual ARF-specific oligonucleotide probes. These inconsistencies suggested that other ARF-like mRNAs were comigrating with known ARF mRNAs. To explore this possibility, a cyclic AMP-differentiated HL-60 Lambda ZAP library was screened using the bovine ARF 2 cDNA. Clones corresponding to known ARF genes (1, 3, and 4) were identified by hybridization of positive clones with oligonucleotide probes specific for each ARF species; ARF 2 cDNA-positive, oligonucleotide-negative clones were sequenced. Two new ARF-like genes, ARF 5 and 6, encoding proteins of 180 and 175 amino acids, respectively, were identified. Both proteins contain consensus sequences believed to be involved in guanine nucleotide binding and GTP hydrolysis. ARF 5 was most similar in deduced amino acid sequence to ARF 4, which also has 180 amino acids. ARF 6, whose deduced amino acid sequence is identical with that of a putative chicken pseudogene (CPS1) except for a serine/threonine substitution, was different from other ARF species in size and deduced amino acid sequence. With mammalian poly(A)+ RNA from a variety of tissues and cultured cells, ARF 5 preferentially hybridized with a 1.3-kb mRNA, whereas ARF 6 hybridized with 1.8- and 4.2-kb mRNAs. The fact that the sizes of these mRNAs are similar to those of other ARFs (ARF 1, 1.9 kb; ARF 2, 2.6 kb; ARF 3, approximately 3.8 and 1.3 kb; ARF 4, 1.8 kb) explain the previously observed inconsistencies between the cDNA and ARF-specific oligonucleotide hybridization patterns. All six ARF cDNAs are more similar to each other than to other approximately 20-kDa guanine nucleotide-binding proteins.     

Effects of guanine nucleotides on cholera toxin catalyzed ADP-ribosylation in rat adipocyte plasma membranes

ADP-ribosylation of rat adipocyte plasma membrane proteins was investigated following incubation of membranes with [alpha-32P]NAD and cholera toxin in the presence and absence of various guanine nucleotides. In membranes incubated without guanine nucleotides, cholera toxin induced incorporation of 32P into three discrete proteins of 48, 45, and 41 kDa. In membranes containing 100 microM GTP or GDP, toxin-catalyzed incorporation of 32P into the 41-kDa protein was inhibited. GMP and Gpp(NH)p (100 microM) allowed moderate incorporation of 32P into the 41-kDa protein. Toxin-catalyzed labeling of all proteins was rapid, reaching maximal levels between 5 and 10 min. Toxin-catalyzed ADP-ribosylation of the 48- and 45-kDa proteins was stimulated by GTP, reaching maximal levels at 10(-5) M GTP. Inhibition of toxin-dependent labeling of the 41-kDa protein required GTP concentrations above 10(-7) M with complete inhibition occurring between 10(-5) and 10(-4) M GTP. Cholera toxin catalyzed ADP-ribosylation was increased up to 2-fold in membranes supplemented with adipocyte cytosol. These results indicate that cholera toxin catalyzes ADP-ribosylation of three distinct adipocyte plasma membrane proteins, each of which is regulated by the amount and type of added guanine nucleotides.     

Stimulation of choleragen enzymatic activities by GTP and two soluble proteins purified from bovine brain

Choleragen (cholera toxin) activates adenylate cyclase by catalyzing ADP-ribosylation of Gs alpha, the stimulatory guanine nucleotide-binding protein. It was recently found (Tsai, S.-C., Noda, M., Adamik, R., Moss, J., and Vaughan, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5139-5142) that a bovine brain membrane protein known as ADP-ribosylation factor or ARF, which enhances ADP-ribosylation of Gs alpha, also increases the GTP-dependent NAD:arginine and NAD:protein ADP-ribosyltransferase, NAD glycohydrolase, and auto-ADP-ribosylation activities of choleragen. We report here the purification and characterization of two soluble proteins from bovine brain that similarly enhance the Gs alpha-dependent and independent ADP-ribose transfer reactions catalyzed by toxin. Like membrane ARF, both soluble factors are 19-kDA proteins dependent on GTP or GTP analogues for activity. Maximal ARF effects were observed at a molar ratio of less than 2:1, ARF/toxin A subunit. Dimyristoyl phosphatidylcholine was necessary for optimal ADP-ribosylation of Gs alpha but inhibited auto-ADP-ribosylation of the choleragen A1 subunit and NAD:agmatine ADP-ribosyltransferase activity. It appears that the soluble factors directly activate choleragen in a GTP-dependent fashion. The relationships of the ARF proteins to the ras oncogene products and to the family of guanine nucleotide-binding regulatory proteins that includes Gs alpha remains to be determined.     

Cholera toxin action on rabbit corpus luteum membranes: effects on adenylyl cyclase activity and adenosine diphospho-ribosylation of the stimulatory guanine nucleotide-binding regulatory component

Cholera toxin elicited 5- to 7-fold stimulation of adenylyl cyclase activity. Half-maximal activation was at 4.42 micrograms/ml cholera toxin. Cholera toxin-mediated activation was time dependent. At 0.1 mM ATP, both guanosine triphosphate (GTP) and nicotinamide adenine dinucleotide (NAD+) were required for cholera toxin activation of luteal adenylyl cyclase. The concentrations of GTP and NAD+ required for half-maximal activation were 1 and 200 microM, respectively. The GTP requirement could be eliminated by increasing the ATP concentration to 1.0 mM. Guanosine-5'-O-(2-thiodiphosphate) [GDP beta S] did not support cholera toxin activation of the luteal enzyme. Cholera toxin treatment increased GTP-stimulated activity, did not significantly alter guanyl-5'-yl imidodiphosphate [GMP-P(NH)P]-stimulated activity, and depressed NaF-stimulated activity. Furthermore, toxin treatment resulted in a 3.4-fold reduction in the Kact values for ovine luteinizing hormone (oLH) to activate adenylyl cyclase. A similar reduction in Kact values for oLH was obtained when concentration-effect curves performed in the presence of GMP-P(NH)P were compared to those performed in the presence of GTP. In addition, luteal membranes treated with cholera toxin and [32P]NAD+ were subjected to autoradiographic analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This treatment resulted in the [32P] adenosine diphospho (ADP)-ribosylation of a 45,000-dalton protein doublet, corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (Ns). As with activation of adenylyl cyclase activity, cholera toxin-specific [32P] ADP-ribosylation was time dependent and increased with increasing concentrations of cholera toxin. GTP, GMP-P(NH)P, and NaF, but not GDP beta S, were capable of supporting [32P] ADP-ribosylation of the protein doublet. oLH did not alter the ability of cholera toxin to ADP-ribosylate the protein activation of luteal adenylyl cyclase activity is due to the ADP-ribosylation of the alpha subunit of Ns and the concomitant inhibition of a GTPase associated with adenylyl cyclase.