The subunit structure of islet-activating protein (IAP), pertussis toxin, has been analyzed to study a possibility that this protein is one of the A-B toxins [Gill, D. M. (1978) in Bacterial Toxins and Cell Membranes (Jeljaszewicz, J., & Wadstrom, T., Eds.) pp 291-332, Academic Press, New York]. Heating IAP with 1% sodium dodecyl sulfate caused its dissociation into five dissimilar subunits named S-1 (with a molecular weight of 28 000), S-2 (23 000), S-3 (22 000), S-4 (11 700), and S-5 (9300), as revealed by polyacrylamide gel electrophoresis; their molar ratio in the native IAP was 1:1:1:2:1. The molecular weight of IAP estimated by equilibrium ultracentrifugation was 117 000 which was not at variance with the value obtained by summing up molecular weights of the constituent subunits. The preparative separation of these IAP subunits was next undertaken; exposure of IAP to 5 M ice-cold urea for 4 days followed by column chromatography with carboxymethyl-Sepharose caused sharp separation of S-1 and S-5, leaving the other subunits as two dimers. These dimers were then dissociated into their constituent subunits, i.e., S-2 and S-4 for one dimer and S-3 and S-4 for the other, after 16-h exposure to 8 M urea; these subunits were obtained individually upon further chromatography on a diethylaminoethyl-Sepharose column. Subunits other than S-1 were adsorbed as a pentamer by a column using haptoglobin as an affinity adsorbent. The same pentamer was obtained by adding S-5 to the mixture of two dimers. Neither this pentamer nor other oligomers (or protomers) exhibited biological activity in vivo. Recombination of S-1 with the pentamer at the 1:1 molar ratio yielded a hexamer which was identical with the native IAP in electrophoretic mobility and biological activity to enhance glucose-induced insulin secretion when injected into rats. In the broken-cell preparation, S-1 was biologically as effective as the native IAP; both catalyzed ADP-ribosylation of a protein in membrane preparations from rat C6 glioma cells. In conclusion, IAP is an oligomeric protein consisting of an A (active) protomer (the biggest subunit) and a B (binding) oligomer which is produced by connecting two dimers by the smallest subunit in a noncovalent manner. Rationale for this terminology is discussed based on the A-B model.

Leucine-rich repeat kinase 2 (LRRK2), a product of a causative gene for the autosomal-dominant form of familial Parkinson's disease (PARK8), harbors a Ras-like small GTP binding protein-like (ROC) domain besides the kinase domain, although the relationship between these two functional domains remains elusive. Here we show by thin-layer chromatographic analysis that LRRK2 stably binds GTP but lacks a GTPase activity in HEK293 and Neuro-2a cells. A ROC domain mutation that converts LRRK2 to a guanine nucleotide-free form (T1348N) abolishes the kinase activity of LRRK2 as well as its phosphate incorporation upon metabolic labeling. The phosphorylation of LRRK2 was inhibited by potential inhibitors for cyclic AMP-dependent protein kinase. These data suggest that binding of GTP to the ROC domain regulates the kinase activity of LRRK2 as well as its phosphorylation by other kinase(s).

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.

Human platelet membrane proteins were phosphorylated by exogenous, partially purified Ca2 +-activated phospholipid-dependent protein kinase (protein kinase C). The phosphorylation of one of the major substrates for protein kinase C (Mr = 41000) was specifically suppressed by the fl subunit of the inhibitory guaninenucleotide-binding regulatory component (Gi, Ni) of adenylate cyclase. The free a subunit of Gi ( M , = 41 000) also served as an excellent substrate for the kinase (> 0.5 mol phosphate incorporated per mol of subunit), but the Gi oligomer (a . fl. y) did not. Treatment of cyc-S49 lymphoma cells, which are deficient in GJNS (the stimulatory component) but contain functional Gi/Ni, with the phorbol ester, 12-0-tetradecanoylphorbol 13-acetate, a potent activator of protein kinase C, did not alter stimulation of adenylate cyclase catalytic activity by forskolin, whereas the Gi/Ni-mediated inhibition of the cyclase by the hormone, somatostatin, was impaired in these membranes. The results suggest that the a subunit of the inhibitory guanine-nucleotide-binding regulatory component of adenylate cyclase may be a physiological substrate for protein kinase C and that the function of the component in transducing inhibitory hormonal signals to adenylate cyclase is altered by its phosphorylation.