A new GTP-binding protein in brain tissues serving as the specific substrate of islet-activating protein, pertussis toxin

A new GTP-binding protein serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, was purified from porcine brain membranes as an alpha beta gamma-heterotrimeric structure. The alpha-subunit of the purified protein (alpha 40 beta gamma) had a molecular mass of 40 kDa and differed from that of Gi (alpha 41 beta gamma) or Go (alpha 39 beta gamma) previously purified from brain tissues. The fragmentation patterns of limited tryptic digestion and immunological cross-reactivities among the three alpha were different from one another. However, the beta gamma-subunit resolved from the three IAP substrates similarly inhibited a membrane-bound adenylate cyclase and their beta-subunits were immunologically indistinguishable from one another. Thus, the alpha 40 beta gamma is a new IAP substrate protein different from Gi or Go, in the alpha-subunit only.     

Modification of the function of pertussis toxin substrate GTP-binding protein by cholera toxin-catalyzed ADP-ribosylation.

The alpha-subunit of Gi-2, in addition to that of Gs (GTP-binding proteins involved in adenylate cyclase inhibition and stimulation, respectively) was ADP-ribosylated by cholera toxin in HL-60 cell membranes when a chemotactic receptor was stimulated by formyl-Met-Leu-Phe (fMLP), and the sites modified by cholera and pertussis toxins on the alpha-subunit of Gi-2 were different (Iiri, T., Tohkin, M., Morishima, N., Ohoka, Y., Ui, M., and Katada, T. (1989) J. Biol. Chem. 264, 21394-21400). In order to investigate how the functions of Gi-2 were modified by cholera toxin, the ADP-ribosylated and unmodified proteins were purified from HL-60 cell membranes that had been incubated in the presence and absence of cholera toxin, respectively. The modified Gi-2 displayed unique properties as follows. 1) The ADP-ribosylated alpha-subunit had a more acidic pI than the unmodified one, leading to a partial resolution of the modified Gir2 trimer from the unmodified protein by an anion column chromatography. 2) When the purified proteins were incubated with [gamma-32P]GTP, the radioactivity was more greatly retained in the modified Gi-2 than in the unmodified protein. 3) The actual catalytic rate (kcat) of GTP hydrolysis was, indeed, markedly inhibited by cholera toxin-induced modification. 4) There was an increase in the apparent affinity of Gi-2 for GDP by cholera toxin-induced modification. 5) The modified Gi-2 exhibited a low substrate activity for pertussis toxin-catalyzed ADP-ribosylation. 6) A high-affinity fMLP binding to HL-60 cell membranes was more effectively reconstituted with the ADP-ribosylated Gi-2 than with the unmodified protein. These results suggested that the agonist-fMLP receptor complex was effectively coupled with the ADP-ribosylated Gi-2, resulting in the GTP-bound form, and that the hydrolysis of GTP on the modified alpha-subunit was selectively attenuated. Thus, cholera toxin ADP-ribosylated Gi-2 appeared to be not only a less sensitive pertussis toxin substrate but also an efficient signal transducer between receptors and effectors.     

Purification and characterization of a GTP-binding protein serving as pertussis toxin substrate in starfish oocytes

In response to a meiosis-inducing hormone, 1-methyladenine (1-MA), starfish oocytes undergo reinitiation of meiosis with germinal vesicle breakdown. The 1-MA-initiated signal is, however, inhibited by prior microinjection of pertussis toxin into the oocytes (Shilling, F., Chiba, K., Hoshi, M., Kishimoto, T., and Jaffe, L.A. (1989) Dev. Biol. 133, 605-608), suggesting that a pertussis-toxin-sensitive guanine-nucleotide-binding protein (G protein) is involved in the 1-MA-induced signal transduction. Based on these findings, we purified a G protein serving as the substrate of pertussis toxin from the plasma membranes of starfish oocytes. The purified G protein had an alpha beta gamma-trimeric structure consisting of 39-kDa alpha, 37-kDa beta, and 8-kDa gamma subunits. The 39-kDa alpha subunit contained a site for ADP-ribosylation catalyzed by pertussis toxin. The alpha subunit was also recognized by antibodies specific for a common GTP-binding site of many mammalian alpha subunits or a carboxy-terminal ADP-ribosylation site of mammalian inhibitory G-alpha. An antibody raised against mammalian 36-/35-kDa beta subunits strongly reacted with the 37-kDa beta subunit of starfish G protein. The purified starfish G protein had a GTP-binding activity with a high affinity and displayed a low GTPase activity. The activity of the G protein serving as the substrate for pertussis-toxin-catalyzed ADP-ribosylation was inhibited by its association with a non-hydrolyzable GTP analogue. Thus, the starfish G protein appeared to be similar to mammalian G proteins at least in terms of its structure and properties of nucleotide binding and the pertussis toxin substrate. A possible role of the starfish G protein is also discussed in the signal transduction between 1-MA receptors and reinitiation of meiosis with germinal vesicle breakdown.     

Chemotactic peptide receptor-supported ADP-ribosylation of a pertussis toxin substrate GTP-binding protein by cholera toxin in neutrophil-type HL-60 cells

A 40-kDa protein, in addition to the alpha-subunits of Gs (a GTP-binding protein involved in adenylate cyclase stimulation), was [32P]ADP-ribosylated by cholera toxin (CT) in the membranes of neutrophil-like HL-60 cells, only if formyl Met-Leu-Phe (fMLP) was added to the ADP-ribosylation mixture. The 40-kDa protein proved to be the alpha-subunit of Gi serving as the substrate of pertussis toxin, islet-activating protein (IAP). No radioactivity was incorporated into this protein in membranes isolated from HL-60 cells that had been exposed to IAP. Gi-alpha purified from bovine brain and reconstituted into IAP-treated cell membranes was ADP-ribosylated by CT plus fMLP. Gi-alpha was ADP-ribosylated by IAP, but not by CT plus fMLP, in membranes from cells that had been pretreated with CT plus fMLP. When membrane Gi-alpha [32P]ADP-ribosylated by CT plus fMLP or IAP was digested with trypsin, the radiolabeled fragments arising from the two proteins were different from each other. These results suggest that CT ADP-ribosylates Gi-alpha in intact cells when coupled fMLP receptors are stimulated and that the sites modified by two toxins are not identical. CT-induced and fMLP-supported ADP-ribosylation of Gi-alpha was favored by Mg2+ and allow concentrations of GTP or its analogues but suppressed by GDP. The ADP-ribosylation did not occur at all, even in the presence of ADP-ribosylation factor that supported CT-induced modification of Gs, in phospholipid vesicles containing crude membrane extract in which Gi was functionally coupled to stimulated fMLP receptors. Thus, Gi activated via coupled receptors is the real substrate of CT-catalyzed ADP-ribosylation. This reaction may depend on additional factor(s) that are too labile to survive the process of membrane extraction.     

The formylpeptide chemoattractant receptor copurifies with a GTP-binding protein containing a distinct 40-kDa pertussis toxin substrate

Detergent extraction of plasma membranes from differentiated HL60 cells, specifically labeled with the chemoattractant, formyl-Nle-Leu-Phe-Nle-[125I-Tyr] Lys, resulted in the solubilization of a receptor-radioligand complex. GTP-binding activity coeluted with the radioligand when the sodium cholate extract was purified by chromatography on wheat germ agglutinin-Sepharose 6MB. A molecular size of approximately 59 A was estimated for the lectin-Sepharose-purified receptor complex by gel filtration chromatography on Ultrogel AcA 34. The isolated complex eluted from the gel filtration column exhibited an enhanced rate of ligand dissociation in response to GTP gamma S. Approximately 0.65 mol of pertussis toxin substrate/mol of receptor was estimated following partial purification of the receptor-ligand complex by sequential chromatography on wheat germ agglutinin-Sepharose, DEAE-Fractogel, and Ultrogel AcA 34. The pertussis toxin substrate which copurified with the receptor was compared with two distinct G proteins, containing alpha-subunits of 40 and 41 kDa, previously purified from HL60 cell plasma membranes. Approximately 86% of the pertussis toxin substrate identified in the receptor preparation consisted of the 40-kDa polypeptide. Differences in the peptide maps indicate that the predominant G protein which coelutes with the receptor is distinct from the purified G protein with an alpha-subunit of 41 kDa but homologous to the purified G protein with an alpha-subunit of 40 kDa.     

Identification of two novel GTP-binding protein alpha-subunits that lack apparent ADP-ribosylation sites for pertussis toxin

Molecular cloning of cDNAs encoding alpha-subunits of guanine nucleotide-binding regulatory proteins (G-proteins) has revealed the existence of nine species of alpha-subunits. We have identified two additional G-protein alpha-subunits, which we refer to as GL1 alpha and GL2 alpha, by isolating bovine liver cDNA clones that cross-hybridized at reduced stringency with bovine Gi1 alpha-subunit cDNA. The deduced amino acid sequences of GL1 alpha and GL2 alpha share 83% identity with each other and show 45-55% identity with those of other known G-protein alpha-subunits. Both GL1 alpha and GL2 alpha lack a consensus site for ADP-ribosylation by pertussis toxin. Messenger RNA corresponding to GL2 alpha was detected in all tissues examined, but GL1 alpha mRNA was detected only in liver, lung, and kidney. Antiserum prepared against a synthetic pentadecapeptide corresponding to the deduced carboxyl terminus of GL2 alpha specifically reacted with a 40-kDa protein in mouse liver, brain, lung, heart, kidney, and spleen. The amount of the 40-kDa protein was highest in brain and lung. We suggest that GL1 alpha and GL2 alpha are new members of a subfamily of pertussis toxin-insensitive G-proteins.     

A new GTP-binding protein in differentiated human leukemic (HL-60) cells serving as the specific substrate of islet-activating protein, pertussis toxin

A GTP-binding protein serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, was partially purified from human leukemic (HL-60) cells that had been differentiated into neutrophil type. The partially purified protein, referred to as GHL, predominantly consisted of at least two polypeptides with molecular masses of 40,000 daltons (alpha) and 36,000 or 35,000 daltons (beta). The structure was similar to Gi or Go previously purified from rat brain as an alpha beta gamma-heterotrimeric IAP substrate (Katada, T., Oinuma, M., and Ui, M. (1986) J. Biol. Chem. 261, 8182-8191), although the existence of the gamma of GHL was unclear. The 40,000-dalton polypeptide contained the site for IAP-catalyzed ADP-ribosylation and the binding site for guanine nucleotide with a high affinity. The 36,000- and 35,000-dalton polypeptides were cross-reacted with the affinity-purified antibody raised against the beta of brain Gi and Go. Limited proteolysis with trypsin and immunoblot analyses with the use of the affinity-purified antibodies raised against the alpha of brain Gi or Go indicated that the alpha of GHL was different from the alpha of Gi or Go. Kinetics of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding to GHL was also quite different from that to brain Gi or Go. Incubation of GHL with GTP gamma S resulted in a resolution into GTP gamma S-bound alpha and beta(gamma) thus purified had abilities to inhibit a membrane-bound adenylate cyclase activity and to associate with the alpha of brain IAP substrate in a fashion similar to the beta gamma of brain IAP substrates, suggesting that there were no significant differences in the biological activities between the beta(gamma) of GHL and those of Gi or Go. Physiological roles of the new GTP-binding protein, GHL, purified from the neutrophil-like cells in receptor-mediated signal transduction are discussed.     

GTP-binding proteins in human platelet membranes serving as the specific substrate of islet-activating protein, pertussis toxin

Two GTP-binding proteins serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, were purified from human platelet membranes as heterotrimers with an alpha beta gamma-subunit structure. The alpha of the major IAP substrate had a molecular mass of 40 kDa and differed from that of Gi 1 or Go previously purified from brain membranes. The partial amino acid sequences of the 40 kDa alpha completely matched with the sequences which were deduced from the nucleotide sequences of the human Gi 2 alpha gene. On the other hand, the alpha of the minor IAP substrate purified from human platelets was about 41 kDa and cross-reacted with an antibody raised against alpha of brain Gi 1 (Gi 1 alpha). These results indicate that the major IAP substrate present in human platelet membranes is a product of the Gi 2 alpha gene.     

Identification and purification of a novel Mr 43,000 tropomyosin-binding protein from human erythrocyte membranes

A new Mr 43,000 tropomyosin-binding protein (TMBP) has been identified in erythrocyte membranes by binding of 125I-labeled Bolton-Hunter tropomyosin to nitrocellulose blots of membrane proteins separated by sodium dodecyl sulfate-gel electrophoresis. This protein is not actin, because 125I-tropomyosin does not bind to purified actin on blots. Binding of 125I-tropomyosin to this protein is specific because it is inhibited by excess unlabeled tropomyosin but not by F-actin or muscle troponins. This protein has been purified to 95% homogeneity from a 1 M Tris extract of tropomyosin-depleted erythrocyte membranes by DEAE-cellulose and hydroxylapatite chromatography, followed by gel filtration on Ultrogel AcA 44. The purified protein has a Stokes radius of 3.9 nm and a sedimentation coefficient of 2.8 S, corresponding to a native molecular weight of 43,000. Binding of 125I-tropomyosin to the purified TMBP saturates at one tropomyosin molecule (Mr 60,000) to two Mr 43,000 TMBPs, with an affinity of about 5 X 10(-7) M. The TMBP is associated with the membrane skeleton after extraction of membranes with the non-ionic detergent, Triton X-100, and is present with respect to tropomyosin at a ratio of about one for every two tropomyosin molecules. Because there is enough tropomyosin for two tropomyosin molecules to be associated with each of the short actin filaments in the membrane skeleton, the erythrocyte membrane TMBP, together with tropomyosin, could function to restrict the number of spectrin molecules attached to each of the short actin filaments and thus specify the hexagonal symmetry of the spectrin-actin lattice. Alternatively, this TMBP could be homologous to one of the muscle troponins and might function with tropomyosin to regulate erythrocyte actomyosin-ATPase activity and influence erythrocyte shape.     

A GTP-binding protein in rat liver nuclei serving as the specific substrate of pertussis toxin-catalyzed ADP-ribosylation.

The ADP-ribosyl moiety of NAD was transferred to a 40-kDa protein when rat liver nuclei were incubated with pertussis toxin. The 40-kDa substrate in the nuclei displayed unique properties as follows, some of which were apparently distinct from those observed with the toxin-substrate GTP-binding protein (Gi) in the liver plasma membranes. 1) The nuclear 40-kDa protein was recognized with antibodies reacting with the alpha-subunits (alpha i-1 and alpha i-2) of Gi, but not with anti-Go-alpha-subunit antibody. 2) The nuclear protein had a higher mobility than alpha-subunit of the plasma membrane-bound Gi upon electrophoresis with a urea/sodium dodecyl sulfate-containing polyacrylamide gel. 3) The nuclear protein was not extracted from the nuclei with 1% Triton X-100, whereas Gi was easily solubilized from the plasma membranes. 4) There was a beta gamma-subunit-like activity in the nuclei, which was assayed by an ability to support pertussis toxin-catalyzed ADP-ribosylation of a purified alpha-subunit of Gi. Moreover, a 36-kDa protein in the nuclei was recognized with antibody raised against purified beta-subunits of Gi. 5) Pertussis toxin-induced ADP-ribosylation of the nuclear protein was selectively inhibited by the addition of a nonhydrolyzable GTP analogue, and its inhibitory action was competitively blocked by the simultaneous addition of GDP or its analogues, as had been observed with plasma membrane-bound Gi. It thus appeared that a novel form of alpha beta gamma-trimeric GTP-binding protein serving as the substrate of pertussis toxin was present in rat liver nuclei. In order to examine a possible role of the nuclear GTP-binding protein, rats were injected with carbon tetrachloride, a necrosis inducer of hepatocytes. There was a marked increase in the nuclear substrate activity from 3-6 days after the injection, without a significant change in the activity of Gi in the plasma membranes. The time course of the increase corresponded with a recovering stage from the hepatocyte necrosis. These results suggested that the nuclear GTP-binding protein found in the present study might be involved at some stages in the hepatocyte growth.     

Isolation of GTP-binding proteins from myeloid HL-60 cells. Identification of two pertussis toxin substrates

We have isolated the major GTP-binding proteins from myeloid HL-60 cell plasma membranes. Two pertussis toxin substrates with similar apparent molecular masses of 40 and 41 kDa, respectively, are contained in these preparations, with both proteins being ADP-ribosylated to a similar extent. Partial chymotryptic proteolysis of fractions containing the [32P]ADP-ribosylated 40-kDa GTP-binding protein alpha subunit demonstrated production of 32P-labeled peptides of 28 and 16 kDa which were not observed after partial proteolysis of fractions containing solely the 41-kDa protein. Similarly, mild acid hydrolysis produced an additional 28-kDa fragment only from fractions containing the 40-kDa protein. The results presented here indicate the presence of two distinct pertussis toxin substrates in myeloid cells. The 41-kDa pertussis toxin substrate is likely to represent the alpha subunit of the inhibitory GTP-binding regulatory protein of adenylate cyclase, whereas the 40-kDa substrate may represent the alpha subunit of the GTP-binding protein which is coupled to chemoattractant receptors. In addition to the pertussis toxin substrates, an additional major peak of guanosine 5'-(3-O-thio)triphosphate-binding activity closely corresponded to the appearance of a 23-kDa protein.     

Purification and characterization of Mr 43,000 protein similar to Mr 25,000 protein, a substrate for protein Ser/Thr kinases, identified as a part of Xenopus laevis vitellogenin B1

Mr 25,000 protein (pp25), a substrate for protein Ser/Thr kinases, was recently shown to consist of a portion of the Xenopus laevis vitellogenin B1 protein. By Western blot analyses using antibodies against pp25, a minor protein band with Mr 43,000 (pp43) was detected in purified preparations of pp25. In this study, pp43 was highly purified through several column chromatography steps and its protein structure was analyzed. The amino acid sequence of the amino-terminal region of pp43 was the same as that of pp25. pp43 contained about two times more phosphates than pp25. These phosphates in pp43 were more resistant to acid phosphatase attack than those of pp25. pp43 was able to bind to pNiXa, a binding protein of pp25. Alpha-chymotryptic digestion generated a common fragment with molecular mass of 23,000 from both pp43 and pp25. These results suggest that pp43 may be a precursor of pp25 generated during processing of vitellogenin B1.     

Immunochemical evidence for a novel pertussis toxin substrate in human neutrophils

Pertussis toxin catalyzes incorporation of 20.2 pmol of ADP-ribose/mg of protein into approximately 40-kDa protein(s) in human neutrophil membranes compared with 14.1 pmol/mg in bovine brain membranes. Based on these measurements we estimate that pertussis toxin substrate(s) should represent at least 0.085% of total membrane protein in neutrophils. Both brain and neutrophil membranes show high concentrations (0.34 versus 0.16% of total membrane protein, respectively) of the common beta subunit of guanine nucleotide binding proteins. Affinity purified antibodies specific for Go-alpha fail to detect any protein in immunoblots of neutrophil membranes (150 micrograms) under conditions where as little as 10 ng of purified Go-alpha is detectable, and Go-alpha is readily detected in brain membranes (100 micrograms). An antiserum against transducin that cross-reacts strongly with Gi-alpha, detects as little as 5 ng of purified Gi-alpha and readily detects Gi-alpha in brain membranes, but in neutrophil membranes, the antiserum detects an approximately 40-kDa band that corresponds to less than 10% of the expected amount of pertussis toxin substrate(s). The results show that human neutrophil membranes contain relatively large amounts of pertussis toxin substrate(s), but that the predominant pertussis toxin substrate is immunochemically distinct from previously identified substrates, transducin, Gi, and Go.     

Identification of a 43-kilodalton human T lymphocyte membrane protein as a receptor for pertussis toxin

Pertussis toxin (PTx), an exotoxin of Bordetella pertussis has been used as a molecular probe to study stimulus-response coupling in a wide variety of cells. We have previously shown that PTx activates the same signal transduction pathways as Ag or mAb directed against the CD3-T cell Ag receptor complex in human T cells. Because the EC50 for mitogenic stimulation by PTx was 1.7 nM, we suspected that the toxin was specifically interacting with a membrane protein or receptor. We have used both chemical cross-linking and Western blotting techniques to demonstrate that PTx shows specific binding to a 43 kDa-membrane protein on cells that respond to PTx by rapid second messenger production. The PTx receptor can be detected in both the E6-1 Jurkat cell line and a CD3-TCR-negative Jurkat line, demonstrating that it is not coordinately expressed with the Ag receptor complex. The 43 kDa-protein is also found in the HPB-ALL human T cell line and PBL, but not in a murine T cell hybridoma or human neutrophils, both of which are unresponsive to PTx activation. These data suggest that the biochemical basis for the mitogenic activity of PTx may lie in its binding to a specific membrane receptor that is capable of transmitting an activation signal.     

Effects of pertussis toxin on vasodilation and cyclic GMP in bovine mesenteric arteries and demonstration of a 40 kD soluble protein ribosylation substrate for pertussis toxin

The inhibitory nucleotide-regulatory protein (Gl) has been shown to lose its adenylate cyclase inhibitory effect upon treatment with pertussis toxin. To find out whether a pertussis sensitive mechanism is involved in the regulation of the cGMP-system, bovine mesenteric arteries were incubated in buffer containing pertussis toxin, and the relaxation and intracellular cGMP accumulation induced by different groups of vasodilating agents were studied. The present results show a pertussis toxin induced decrease in relaxation as well as a decrease in the cGMP-elevation induced by the endothelium dependent vasodilators acetylcholine and calcium ionophore A 23187. Arteries treated with atrial natriuretic peptide showed no alterations in relaxation or cGMP content after incubation with pertussis toxin. A 40 kD soluble ribosylation substrate for pertussis toxin was identified in bovine mesenteric artery. These results suggest that a pertussis toxin sensitive mechanism is involved in the vasodilating mechanism of acetylcholine and calcium ionophore A 23187, while no evidence for such a mechanism could be found regarding the vasodilatory action of atrial natriuretic peptide.     

Mastoparan, a wasp venom, stimulates insulin release by pancreatic islets through pertussis toxin sensitive GTP-binding protein

A wasp venom, mastoparan, rapidly stimulated insulin release by rat pancreatic islets in a dose-related manner. The amount of insulin released in response to 58 microM mastoparan far exceeded that induced by 27.8 mM glucose. Mastoparan stimulated insulin release to similar degrees at ambient glucose concentrations of 1.7 mM and 5.6 mM. The islets obtained from pertussis toxin-treated rats showed unequivocally less response to mastoparan. Pretreatment of islets with bromophenacyl bromide, a phospholipase A2 inhibitor, abolished their responsiveness to mastoparan. Pretreatment of islets with nifedipine, a Ca2+ channel blocker, was without effect. Mastoparan is a unique stimulator of insulin release by the pancreatic islets, which acts through GTP-binding protein(s) and phospholipase A2.     

Functional modification by cholera-toxin-catalyzed ADP-ribosylation of a guanine-nucleotide-binding regulatory protein serving as the substrate of pertussis toxin.

The alpha subunits of Gi (Gi alpha) and Gs (guanine-nucleotide-binding proteins involved in adenylate cyclase inhibition and stimulation, respectively) was ADP-ribosylated by cholera toxin in differentiated HL-60 cell membranes upon stimulation of chemotactic receptors by fMLF (fM, N-formylmethionine). The ADP-ribosylation site of Gi alpha modified by cholera toxin appeared to be different from that modified by pertussis toxin [Iiri, T., Tohkin, M., Morishima, N., Ohoka, Y., Ui, M. & Katada, T. (1989) J. Biol. Chem. 264, 21,394-21,400]. This allowed us to investigate how the two types of ADP-ribosylation influence the function of the signal-coupling protein. The major findings observed in HL-60 cell membranes, where the same Gi alpha molecule was ADP-ribosylated by treatment of the membranes with either toxin, are summarized as follows. (a) More fMLF bound with a high affinity to cholera-toxin-treated membranes than to the control membranes. The high-affinity binding was, however, not observed in pertussis-toxin-treated membranes. (b) Although fMLF stimulated guanine nucleotide binding and GTPase activity in control membranes, stimulation was almost completely abolished in pertussis-toxin-treated membranes. In contrast, fMLF-dependent stimulation of GTPase activity, but not that of guanine nucleotide binding was attenuated in cholera-toxin-treated membranes. (c) Gi alpha, once modified by cholera toxin, still served as a substrate of pertussis-toxin-catalyzed ADP-ribosylation; however, the ADP-ribosylation rate of modified Gi was much lower than that of intact Gi. These results suggested that Gi ADP-ribosylated by cholera toxin was effectively capable of coupling with fMLF receptors, resulting in formation of high-affinity fMLF receptors, and that hydrolysis of GTP bound to the alpha subunit was selectively impaired by its ADP-ribosylation by cholera toxin. Thus, unlike the ADP-ribosylation of Gi by pertussis toxin, cholera-toxin-induced modification would be of great advantage to the interaction of Gi with receptors and effectors that are regulated by the signal-coupling protein. This type of modification might also be a candidate for unidentified G proteins which were less sensitive to pertussis toxin and appeared to be involved in some signal-transduction systems.     

Purification and characterization of a new GTP-binding protein of Mr 24,000 in bovine brain membranes

A GTP-binding protein with an Mr of 24,000 was purified from a cholate extract of bovine brain membranes in addition to the previously reported alpha beta gamma-trimeric GTP-binding proteins (G proteins). Partial amino acid sequence analysis of the purified 24-kDa protein revealed that it was not identical to any of the low Mr GTP-binding proteins already reported, but similar to the rac-gene products serving as the substrate of an ADP-ribosyltransferase (C3) purified from the culture medium of Clostridium botulinum type C. However, the 24-kDa protein was not ADP-ribosylated by the botulinum C3 enzyme. The 24-kDa protein was purified as a nucleotide-free form and characterized by the following unique properties distinct from those of alpha beta gamma-trimeric G proteins. (1) Mg2+ was essentially required for nucleotide binding to the 24-kDa protein; there was a progressive increase in its binding affinity for nucleotides as the concentration of the divalent cation was increased. (2) Nucleotides previously bound to the 24-kDa protein were rapidly dissociated from the protein in Mg(2+)-free medium, in accord with the fact that the protein was indeed purified as a nucleotide-free form with Mg(2+)-free solutions. (3) The 24-kDa protein apparently exhibited much lower GTPase activity than do alpha beta gamma-trimeric G proteins because the product GDP was released from the 24-kDa protein in exchange for the substrate GTP only at a very low rate. Based on these findings, a possible role of the 24-kDa protein in cellular signalling is discussed in comparison with well characterized alpha beta gamma-trimeric G proteins.     

Dopamine receptors on photoreceptor membranes couple to a GTP-binding protein which is sensitive to both pertussis and cholera toxin

Dopamine receptors with a pharmacological profile similar to D2 receptors are coenriched with rhodopsin in preparations of bovine retinal membranes. A high density of these receptors are present on photoreceptor membranes. The affinity of the agonist apomorphine for these receptors is decreased by the guanine nucleotides GTP and GppNHp. Treatment of photoreceptor membranes with pertussis or cholera toxin also decreased the affinity of apomorphine and eliminated the effect of GTP.     

Effects of phosphorylation of inhibitory GTP-binding protein by cyclic AMP-dependent protein kinase on its ADP-ribosylation by pertussis toxin, islet-activating protein

Pretreatment of rat cardiac myocytes with the beta-adrenergic agonist, db-cAMP or forskolin decreased ADP-ribosylation of 40-41 kDa protein by islet-activating protein (IAP) in cell membranes. Addition of activated cyclic AMP-dependent protein kinase (protein kinase A) catalytic subunit and MgCl2 also decreased ADP-ribosylation of 40-41 kDa protein by IAP in cell membranes. The alpha- and beta-subunits of partially purified inhibitory GTP-binding protein (Gi) were both phosphorylated by protein kinase A. The amounts of phosphate incorporated into the subunits of Gi were 0.34 and 0.18 mol/mol protein. These show that phosphorylation of Gi by protein kinase A results in a decrease in its ADP-ribosylation by IAP.