Islet-activating protein, pertussis toxin: a probe for functions of the inhibitory guanine nucleotide regulatory component of adenylate cyclase

doi:10.1016/0165-6147(84)90444-9

Trends in Pharmacological Sciences

Volume 5, 1984, Pages 277-279

https://doi.org/10.1016/0165-6147(84)90444-9 Get rights and content

Abstract

The guanine nucleotide binding regulatory proteins (N) play an important role in communication between membrane receptors and the adenylate cyclase catalytic unit. One (N_s) of the nucleotide regulatory proteins is involved in the activation, while the other (N_i) is involved in the inhibition of adenylate cyclase. Islet-activating protein (IAP), pertussis toxin, catalyses the transfer of the ADP-ribose moiety of NAD to the active subunit (M_r = 41 00) of N_u resulting in a complete loss of the N_i functions. IAP will be useful as a tool for further studies of the transmembrane signal transduction mechanism.

References (21)

T. Katada et al.
J. Biol. Chem.
(1981)
T. Katada et al.
J. Biol. Chem.
(1982)
F. Okajima et al.
Arch. Biochem. Biophys.
(1982)
T. Katada et al.
J. Biol. Chem.
(1979)
H. Kurose et al.
J. Biol. Chem.
(1983)
O. Hazeki et al.
J. Biol. Chem.
(1981)
T. Murayama et al.
J. Biol. Chem.
(1983)
T. Murayama et al.
Arch. Biochem. Biophys.
(1983)
T. Katada et al.
J. Biol. Chem.
(1982)
T. Katada et al.
Arch. Biochem. Biophys.
(1983)

There are more references available in the full text version of this article.

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Strong G-Protein-Mediated Inhibition of Sodium Channels
2018, Cell Reports
Citation Excerpt :
GPCRs can be coupled to a range of different G-protein complexes, the primary families being Gi/o, Gq, Gs, and G12 (Neves et al., 2002). Pertussis toxin (PTx) is a specific inhibitor of Gi/o signaling (Ui, 1984). Preincubation with PTX (200 ng/mL) for either 16–24 hr (n = 8) or 48–72 hr (n = 6) did not alter the cinacalcet-induced inhibition of VGSC currents, indicating that the pathway was mediated by G-proteins other than Gi/o (Figures 4F and 4G).
Voltage-gated sodium channels (VGSCs) are strategically positioned to mediate neuronal plasticity because of their influence on action potential waveform. VGSC function may be strongly inhibited by local anesthetic and antiepileptic drugs and modestly modulated via second messenger pathways. Here, we report that the allosteric modulators of the calcium-sensing receptor (CaSR) cinacalcet, calindol, calhex, and NPS 2143 completely inhibit VGSC current in the vast majority of cultured mouse neocortical neurons. This form of VGSC current block persisted in CaSR-deficient neurons, indicating a CaSR-independent mechanism. Cinacalcet-mediated blockade of VGSCs was prevented by the guanosine diphosphate (GDP) analog GDPβs, indicating that G-proteins mediated this effect. Cinacalcet inhibited VGSCs by increasing channel inactivation, and block was reversed by prolonged hyperpolarization. Strong cinacalcet inhibition of VGSC currents was also present in acutely isolated mouse cortical neurons. These data identify a dynamic signaling pathway by which G-proteins regulate VGSC current to indirectly modulate central neuronal excitability.
Rapid membrane responses to dihydrotestosterone are sex dependent in growth plate chondrocytes
2012, Journal of Steroid Biochemistry and Molecular Biology
Citation Excerpt :
Chondrocytes from the resting zone of the growth plate were treated with testosterone and DHT, and rapid effects on phospholipase A2 (PLA2) and PKC signaling pathways determined. 5α-Dihydrotestosterone (DHT); testosterone; G-protein inhibitor pertussis toxin (PTX, Gαi inhibitor) [35], cholera toxin (CTX, Gαs activator) [36], and GDPβs (general G-protein inhibitor) [37,38]; and PLA2 inhibitors AACOCF3 [39] and quinacrine [40] were obtained from Sigma Chemical Co. (St. Louis, MO). Thapsigargin, which inhibits a Ca-ATPase pump in the endoplasmic reticulum [41]; verapamil hydrochloride and nifedipine, which are inhibitors of specific L type Ca2+ channels on the cell membrane [42,43]; the transcription inhibitor actinomycin D [44]; the translation inhibitor cycloheximide [45,46]; U73122 (phospholipase C inhibitor) [47] and cyproterone acetate (CPA), which is an inhibitor of the classical androgen receptor (AR) [48] were purchased from Sigma Chemical Co. (St. Louis, MO).
Sex steroids are important regulators for longitudinal growth, bone mass accrual, and sexual dimorphism of the skeleton. 17β-Estradiol regulates proliferation and differentiation of female chondrocytes via a membrane-associated signaling pathway in addition to its estrogen receptor (ER) mediated effects. In contrast, testosterone does not elicit a similar membrane response, either in male or female cells. Whereas female rat growth plate chondrocytes convert testosterone to 17β-estradiol, male chondrocytes produce 5α-dihydrotestosterone (DHT). Previously DHT was found to mediate sex-specific effects of testosterone in male cells, but it is not known if a membrane-signaling pathway is involved. In this study, we hypothesized that DHT can induce sex-specific rapid membrane effects similar to other steroid hormones. Confluent cultures of chondrocytes isolated from resting zones of growth plates of both male and female rats were treated with 10⁻¹⁰–10⁻⁷ M testosterone or DHT for 3, 9, 90 and 270 min and protein kinase C (PKC) and phospholipase A2 (PLA2) activities were measured. To examine potential signaling pathways involved in PKC activation, male chondrocytes were treated with 10⁻⁷ M DHT for 9 min in the presence or absence of the phospholipase C (PLC) inhibitor U73122, the secretory PLA2 inhibitor quinacrine or the cytosolic PLA2 inhibitor AACOCF3; the Gαi inhibitor pertussis toxin (PTX) or Gαs activator cholera toxin (CTX), and the general G-protein inhibitor GDPβS; thapsigargin, an inhibitor of a Ca-ATPase pump in the endoplasmic reticulum; verapamil and nifedipine, inhibitors of specific L type Ca2+ channels on the cell membrane; and cyproterone acetate (CPA), which is an inhibitor of the classical androgen receptor (AR); as well as the transcription inhibitor actinomycin D, or the translation inhibitor cycloheximide. DHT induced a dose-dependent increase in PKC and PLA2 activity in male cells with the highest increase at 10⁻⁷ M DHT (p < 0.05), whereas testosterone had no effect. PKC activity was augmented at 9 and 90 min, and then decreased to baseline at 270 min. Neither testosterone nor DHT affected PKC in female cells. U73122, quinacrine, and AACOCF3 inhibited DHT-induced activation of PKC. DHT treatment for 9 min had no effect in [³H]-thymidine incorporation in quiescent confluent cultures but caused a dose dependent increase in alkaline phosphatase specific activity. Inhibition of PLC reduced the response of to DHT in a dose dependent manner, indicating that PLC is involved. In conclusion, our study indicates that DHT, but not testosterone, has sex-specific rapid membrane effects in male growth plate chondrocytes involving PLC and PLA2-mediated PKC signaling pathways. Together with previous observations showing that male cells convert testosterone to DHT, these results suggest that DHT might act in the membrane through an autocrine/paracrine mechanism.
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Citation Excerpt :
Notably, the primary amino acid sequence of pierisin is highly similar to the bacterial protein toxin MTX (mosquitocidal toxin) from Bacillus sphaericus SSII-1, which is known to be a protein-specific mono-ADP-ribosyltransferase (Thanabalu et al., 1993; Schirmer et al., 2002; Carpusca et al., 2006). Many bacterial ADP-ribosyltransferases specifically modify nucleotide-binding proteins, including heterotrimeric G proteins (e.g., cholera toxin (Cassel and Selinger, 1977) or pertussis toxin (Ui, 1984)), elongation factor 2 (e.g., diphtheria toxin (Collier, 1990) or Pseudomonas exotoxin A (Iglewski et al., 1977)) and low molecular mass GTPases (e.g., Clostridium botulinum C3 toxin (Aktories et al., 1987), Pseudomonas exoenzyme S (Barbieri and Sun, 2004) or Photorhabdus luminescens toxin complex PTC5 (Lang et al., 2010)). Other bacterial protein toxins ADP-ribosylate the ATP-binding protein actin e.g. the family of binary ADP-ribosylating toxins including C. botulinum C2 toxin (Aktories et al., 1986) or P. luminescens toxin complex PTC3 (Lang et al., 2010).
Pierisin-like proteins comprise a growing family of ADP-ribosyltransferases expressed in various species of white butterflies. The prototype pierisin-1 from the cabbage butterfly, Pieris rapae, was identified as a potent apoptosis-inducing agent, acting on various types of carcinoma cell lines by mono-ADP-ribosylation of DNA. The characterization of pierisin-like proteins is hampered by its potent toxicity, which prevents its expression as a recombinant protein in Escherichia coli. Here we characterized a new member of the pierisin protein family named pierisin-1b, which was cloned from P. rapae. Pierisin-1b consists of 849 amino acids residues and shares 63%–91% identity with already described pierisins. For expression of pierisin-1b a novel in vitro translation system was utilized. Obtained protein exhibits specific ADP-ribosyltransferase activity on deoxyguanosine residues of DNA leading to induction of apoptosis and cell death.
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2007, Journal of Surgical Research
Citation Excerpt :
Dulbecco’s minimal essential medium (DMEM) and Dulbecco’s PBS (dPBS) were purchased from Cellgro. Pertussis toxin catalyzes the ADP-ribosylation of the α-subunit of Gi and Go specifically, preventing coupling with receptors [12], and was used at a standard inhibitory concentration. Pertussis toxin does not affect Gαq, even when modified at its C-terminus to mimic Gi [13].
Urokinase plasminogen activator (uPA) is involved in vessel remodeling and mediates smooth muscle cell migration. Migration in response to uPA is dependent on both the growth factor binding domain at the aminoterminal end and the kringle (K) domain of the molecule. uPA is readily degraded in vivo into these constitutive domains. The aim of this study was to examine cell signaling during the migration of smooth muscle cell in response to the kringle domain of urokinase.
Murine arterial smooth muscle cells were cultured in vitro. Migration assays were performed in the presence of K with and without the plasmin inhibitors (aprotinin and ϵ-aminocaproic acid), the Gαi inhibitor Pertussis toxin, the MMP inhibitor (GM6001), the PI3-K inhibitors, Wortmannin and LY294002, and the MAPK inhibitors PD98089 (MEK1 inhibitor) and SB203580 (p38^MAPK inhibitor). Western blotting was performed for ERK 1/2 and p38^MAPK phosphorylation after stimulation with K in the presence and absence of the inhibitors. Statistics were analyzed by one-way ANOVA (n = 6).
The kringle domain (K) induced a plasmin-independent, MMP-dependent increase in cell migration (2-fold, P < 0.05) compared to control. This migratory response to K was Gαi mediated and dependent on both ERK 1/2 and p38^MAPK activation. K induced time-dependent increases in the phosphorylation of ERK 1/2 (3-fold, P < 0.05) and p38^MAPK (3-fold, P < 0.05). Activation of p38^MAPK and ERK 1/2 was completely inhibited by the PI3-K inhibitors. We explored a potential role for the epidermal growth factor receptor (EGFR). K induced EGFR phosphorylation and the presence of AG1478, the EGFR inhibitor, inhibited both cell migration and akt activation in response to K.
Kringle domain of uPA induces smooth muscle cell migration through a G-protein-coupled PI3-K-dependent process involving both ERK 1/2 and p38^MAPK and is mediated in part through EGFR. Defining the differences in response to key molecular domains of uPA is vital to understand its role in vessel remodeling.
Somatostatin, misoprostol and galanin inhibit gastrin- and PACAP-stimulated secretion of histamine and pancreastatin from ECL cells by blocking specific Ca<sup>2+</sup> channels
2005, Regulatory Peptides
The oxyntic mucosa is rich in ECL cells. They secrete histamine and chromogranin A-derived peptides, such as pancreastatin, in response to gastrin and pituitary adenylate cyclase-activating peptide (PACAP). Secretion is initiated by Ca²⁺ entry. While gastrin stimulates secretion by opening L-type and N-type Ca²⁺ channels, PACAP stimulates secretion by activating L-type and receptor-operated Ca²⁺ channels. Somatostatin, galanin and prostaglandin E₂ (PGE₂) inhibit gastrin- and PACAP-stimulated secretion from the ECL cells. In the present study, somatostatin and the PGE₂ congener misoprostol inhibited gastrin- and PACAP-stimulated secretion 100%, while galanin inhibited at most 60–65%. Bay K 8644, a specific activator of L-type Ca²⁺ channels, stimulated ECL-cell secretion, an effect that was inhibited equally effectively by somatostatin, misoprostol and galanin (75–80% inhibition). Pretreatment with pertussis toxin, that inactivates inhibitory G-proteins, prevented all three agents from inhibiting stimulated secretion (regardless of the stimulus). Pretreatment with nifedipine (10 μM), an L-type Ca²⁺ channel blocker, reduced PACAP-evoked pancreastatin secretion by 50–60%, gastrin-evoked secretion by ∼ 80% and abolished the response to Bay K 8644. The nifedipine-resistant response to PACAP was abolished by somatostatin and misoprostol but not by galanin. Gastrin and PACAP raised the intracellular Ca²⁺ concentration in a biphasic manner, believed to reflect mobilization of internal Ca²⁺ followed by Ca²⁺ entry. Somatostatin and misoprostol blocked Ca²⁺ entry (and histamine and pancreastatin secretion) but not mobilization of internal Ca²⁺.
The present observations on isolated ECL cells suggest that Ca²⁺ entry rather than mobilization of internal Ca²⁺ triggers exocytosis, that gastrin and PACAP activate different (but over-lapping) Ca²⁺ channels, that somatostatin, misoprostol and galanin interact with inhibitory G-proteins to block Ca²⁺ entry via L-type Ca²⁺ channels, and that somatostatin and misoprostol (but not galanin) in addition block N-type and/or receptor-operated Ca²⁺ channels.
Natriuretic peptide receptor-C signaling and regulation
2005, Peptides
The natriuretic peptides (NP) are a family of three polypeptide hormones termed atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). ANP regulates a variety of physiological parameters by interacting with its receptors present on the plasma membrane. These are of three subtypes NPR-A, NPR-B, and NPR-C. NPR-A and NPR-B are guanylyl cyclase receptors, whereas NPR-C is non-guanylyl cyclase receptor and is coupled to adenylyl cyclase inhibition or phospholipase C activation through inhibitory guanine nucleotide regulatory protein (Gi). ANP, BNP, CNP, as well as C-ANP_4–23, a ring deleted peptide that specifically interacts with NPR-C receptor inhibit adenylyl cyclase activity through Gi protein. Unlike other G-protein-coupled receptors, NPR-C receptors have a single transmembrane domain and a short cytoplasmic domain of 37 amino acids, which has a structural specificity like those of other single transmembrane domain receptors. A 37 amino acid cytoplasmic peptide is sufficient to inhibit adenylyl cyclase activity with an apparent Ki similar to that of ANP_99–126 or C-ANP_4–23. In addition, C-ANP_4–23 also stimulates phosphatidyl inositol (PI) turnover in vascular smooth muscle cells (VSMC) which is attenuated by dbcAMP and cAMP-stimulatory agonists, suggesting that NPR-C receptor-mediated inhibition of adenylyl cyclase and resultant decreased levels of cAMP may be responsible for NPR-C-mediated stimulation of PI turnover. Furthermore, the activation of NPR-C receptor by C-ANP_4–23 and CNP inhibits the mitogen-activated protein kinase activity stimulated by endothelin-3, platelet-derived growth factor, phorbol-12 myristate 13-acetate, suggesting that NPR-C receptor might also be coupled to other signal transduction system or that there may be an interaction of the NPR-C receptor and some other signaling pathways. In this review article, NPR-C receptor coupling to different signaling pathways and their regulation will be discussed.

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¹: Dr. Michio Ui graduated from the Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo in 1955 Since 1958 he has been in the Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University as an Instructor until 1965, as an Associate Professor until 1973 and then as a Professor and the Chairman of the department. His principal research interests are metabolic physiology and biochemical pharmacology, currently focusing on the mechanisms of transmembrane signal transduction.

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Trends in Pharmacological Sciences

Islet-activating protein, pertussis toxin: a probe for functions of the inhibitory guanine nucleotide regulatory component of adenylate cyclase

Abstract

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

Arch. Biochem. Biophys.