Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein.

Abstract

GTP and isoproterenol activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in washed membranes prepared from C6 glioma cells was enhanced by incubation with islet-activating protein, one of the pertussis toxins, ifthe incubation mixture was supplemented with NAD and ATP. The action of the protein was observed immediately after its addition and increased progressively in magnitude as the protein concentration or the incubation time increased. There was simultaneous incorporation of radioactivity from the ADP-ribose moiety of variously labeled NAD into the membrane protein with a molecular weight of 41,000. We conclude that islet-activating protein enhances receptor-mediated GTP-induced activation of membrane adenylate cyclase as a result of ADP-ribosylation of a membrane protein, probably one of the components of the receptor-adenylate cyclase system. Islet-activating protein (lap) has recently been isolated from the culture medium of Bordetella pertussis (1, 2) as one of the pertussis toxins (3, 4). Injection of lap into animals in vivo (5) or addition of it to intact cell preparations in vitro (6-8) markedly modifies cellular cAMP responses to a variety of receptor agonists; receptor-mediated stimulation of cAMP accumulation in cells is potentiated, whereas receptor-mediated inhibition is abolished. This action of lap has been observed with rat pancreatic islet cells (5-7), rat cardiac cells (8), C6 glioma cells (9), 3T3 fibroblasts, and NG 108-15 hybrid cells (unpublished data). The lap-induced modification of these cAMP responses seems to be due to a change in generation of cAMP via adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1], rather than to its breakdown by phosphodiesterase, since it was observed in the presence ofan inhibitor ofthe diesterase. Indeed, ,-adrenergic receptor-mediated increases of the membrane cyclase activity were enhanced and a-adrenergic receptor-mediated decreases were attenuated by previous treatment with lap of C6 glioma cells (9) and pancreatic islet cells (10), respectively. Thus, a mechanism whereby adenylate cyclase is linked to membrane receptors would be affected by lap.Exposure of intact cell preparations to lap has thus far been the sole means to achieve lap-induced modification of membrane adenylate cyclase responses, because addition of the pertussis toxin to cell-free preparations such as diluted homogenate or washed membranes was without effect. In this paper, we show that direct addition of lap to the membrane preparation from rat C6 glioma cells immediately enhanced GTP-dependent adenylate cyclase activity only in the presence of NAD and ATP. The enhancement was associated with covalent incorporation ofradioactivity from the ADP-ribose moiety of NAD into a membrane protein with a Mr of 41,000.

Inhibition of formation of cyclic AMP and cyclic GMP by vasopressin in smooth-muscle cells is insensitive to pertussis toxin

“…The functional integrity of G1 in these cells was further strengthened by the observation that thrombin-mediated inhibition of cyclic AMP accumulation was inhibited by pertussis-toxin pretreatment of the cells (Table 4). DISCUSSION Pretreatment of different cell types with pertussis toxin produces inhibition of agonist-induced responses [13][14][15][16][17][18][19][20][21][22] The data in the present paper demonstrate that this is indeed the case. This is supported by the following findings.…”

“…Thus Ca2", chemotaxis, superoxide production, cyclic AMP and cyclic GMP phosphodiesterases, liberation of arachidonic acid from phospholipids and degradation of inositol phospholipids [13][14][15][16][17][18][19][20][21][22]. Rat aortic smoothmuscle cells (A-10; American Type Culture Collection CRL 1476) display vasopressin receptors of V1 subtype [23], ,-adrenergic receptors [24] and ANF receptors [25].…”

Inactivation of Gi proteins induces an antidepressant-like effect in the mouse forced-swimming test

“…The administration of pertussis toxin (PTX), a bacterial toxin produced by Bordetella pertussis that ADP-ribosylates and inactivates the α subunit of Gi-protein family (Katada and Ui, 1982), enhanced the mobility time of mice and, therefore, counteracted the depressant-like condition induced in the test. These data evidence the important role played by Gi proteins in the signal transduction mechanism responsible for the modulation of depressive states.…”

Differential prevention of morphine amnesia by antisense oligodeoxynucleotides directed against various Gi‐protein α subunits

“…administration of morphine produced an impairment of memory functions of intensity comparable to that exerted by the amnesic drugs scopolamine. The morphine-induced amnesia was completely prevented by pretreatment with pertussis toxin (PTX), a bacterial toxin produced by Bordetella pertussis that ADP-ribosylates and inactivates the a subunit of Gi proteins (Katada & Ui, 1982), indicating the important role played by the Gi proteins in the signal transduction mechanism activated by morphine. The Gi protein subfamily is composed of di erent members, Gi 1 , Gi 2 and Gi 3 (Simon et al, 1991).…”