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Brefeldin A, a fungal metabolite that inhibits membrane transport, induces the mono(ADP-ribosyl)ation of two cytosolic proteins of 38 and 50 kDa as judged by SDS/PAGE. The 38-kDa substrate has been previously identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We report that the 50-kDa BFA-induced ADP-ribosylated substrate (BARS-50) has native forms of 170 and 130 kDa, as determined by gel filtration of rat brain cytosol, indicating that BARS-50 might exist as a multimeric complex. BARS-50 can bind GTP, as indicated by blot-overlay studies with [alpha-32P]GTP and by photoaffinity labeling with guanosine 5'-[gamma-32P] [beta,gamma-(4-azidoanilido)]triphosphate. Moreover, ADP-ribosylation of BARS-50 was completely inhibited by the beta gamma subunit complex of G proteins, while the ADP-ribosylation of GAPDH was unmodified, indicating that this effect was due to an interaction of the beta gamma complex with BARS-50, rather than with the ADP-ribosylating enzyme. Two-dimensional gel electrophoresis and immunoblot analysis shows that BARS-50 is a group of closely related proteins that appear to be different from all the known GTP-binding proteins.

Heterotrimeric GTP-binding regulatory proteins (G proteins) have been identified as part of signal transduction systems in a wide variety of organisms. In this paper, we establish the presence of a G protein associated with the plasma membranes of the apical bud of etiolated peas. The GTPase activity is induced by low fluences of blue light administered to plasma membrane-enriched fractions. The activity is not responsive to red-light irradiation and is specific for GTP. The threshold for the excitation of the GTPase activity in vitro is less than 10(-1) mumol.m-2 of blue light, consistent with participation in the blue low-fluence system identified in the same tissue. A 40-kDa polypeptide is recognized by polyclonal antisera directed against the alpha subunit of the G protein transducin. The polypeptide also serves as a substrate for ADP-ribosylation by cholera and pertussis toxins. The ability of the 40-kDa polypeptide to serve as substrate for the toxin-mediated ribosylation is mediated by blue-light irradiation, implying that the 40-kDa polypeptide is the alpha subunit of a blue-light-stimulated G protein. The 40-kDa polypeptide binds a nonhydrolyzable photoaffinity-labeling analog of GTP only after irradiation with blue light. The protein we have described may function as an alpha subunit of a G protein active in the process of light-mediated development in higher plants.

Fluoride activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] is significantly enhanced (2 to 5 times) by a protein factor isolated from rat brain. The fluoride-dependent adenylate cyclase stimulator (FCS) is nondialyzable, trypsin-labile, and stable at 90 degrees C for 10 min. FCS stimulates adenylate cyclase activity only in the presence of NaF (2-25 mM) and this effect is independent of added GTP, 5'-guanylylimidodiphosphate, or calcium. FCS has been purified roughly 3000-fold from a 12,000 X g supernatant fraction of rat brain homogenate. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and sucrose density gradient sedimentation suggest that FCS is a monomer with an apparent Mr of 59,000. Isoelectric focusing indicates FCS has a pI of 8.9. FCS from rat brain stimulates fluoride-activated adenylate cyclase from a variety of cell types, and FCS can also be isolated from rat liver. The effects of FCS are not reversed by washing membranes when the membranes and FCS are preincubated with NaF. The Km of adenylate cyclase for ATP and the fluoride concentration causing half-maximal activation are unchanged by FCS; however, FCS increases the Vmax by 2.5-fold. FCS may act to increase the catalytic efficiency of fluoride-activated complexes of the GTP-binding unit with adenylate cyclase or to enhance the formation of additional active complexes.

Previous studies have noted profound similarities between the regulation of light-activated 3',5'-cyclic nucleotide phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) in retinal rods and hormone-activated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in a variety of tissues. We report here the functional exchange of components isolated from the photoreceptor system, which displayed predicted functional characteristics when incubated with recipient adenylate cyclase systems from rat cerebral cortical and hypothalamic synaptic membranes and frog erythrocyte ghosts. We demonstrate functional exchange of photoreceptor components at each of three loci: the hormone receptor, the GTP-binding protein (GBP), and the catalytic moiety of adenylate cyclase. Illuminated (but not unilluminated) rhodopsin was fund to mimic the hormone-receptor complex, causing GTP-dependent activation of adenylate cyclase. The photoreceptor GBP complexed with guanosine 5'-[beta, gamma)imidotriphosphate (p[NH]ppG) produced a marked activation of recipient adenylate cyclase systems. Much smaller activation was observed when GBP was not complexed with p[NH]ppG. A heat-stable photoreceptor phosphodiesterase inhibitor reduced both basal and Mn2+-activated adenylate cyclase activities and this inhibition was reversed by photoreceptor GBP.p[NH]ppG. These data demonstrate a remarkable functional compatibility between subunits of both systems and furthermore imply that specialized peptide domains responsible for protein-protein interactions are highly conserved.

GTP-binding proteins have been demonstrated to stimulate and inhibit rat brain adenylate cyclase without the prior addition of hormone. Exposure of rat cerebral cortex membranes to hydrolysis-resistant GTP analogs results in inhibition (or stimulation) of adenylate cyclase, which persists subsequent to buffer washing. The hydrolysis-resistant GTP photoaffinity probe P3-(4-azidoanilido)-P1-5' GTP (AAGTP) can promote a similar persistent inhibition of adenylate cyclase, and, after removal of unbound AAGTP and subsequent UV photolysis, AAGTP is covalently linked to the 40-kDa inhibitory GTP binding protein, GNi (inhibitory guanine nucleotide binding regulatory subunit of adenylate cyclase). Under conditions where the persistent inhibition of adenylate cyclase is overcome by subsequent incubation with 5'-guanylyl imidodiphosphate or NaF, AAGTP bound to the 40-kDa GNi protein is diminished while that bound to the 42-kDa stimulatory GTP-binding protein (GNs) is increased. Additionally, we have identified a 32-kDa protein that binds AAGTP with an affinity similar to that of GNs. This protein does not appear to be a byproduct of proteolysis as demonstrated by Staphylococcus aureus V8 protease digestion experiments, and it is not a substrate for ADP-ribosylation by bacterial toxins. The sum of the AAGTP bound by the GNi and GNs proteins is constant, and the transfer of nonphotoactivated AAGTP to GNs from GNi is stable to buffer washing. Furthermore, this alteration in the AAGTP-labeling pattern corresponds to the shift in adenylate cyclase from inhibition to stimulation. These data raise the possibility that hydrolysis-resistant GTP analogs might be exchanged directly between the GNi and GNs and that there exists some interaction between those proteins in the regulation of adenylate cyclase activity.