A BSTR ACTReceptors activate adenylyl cyclases through the G␣ s subunit. Previous studies from our laboratory have shown in certain cell types that express adenylyl cyclase 6 (AC6), heterologous desensitization included reduction of the capability of adenylyl cyclases to be stimulated by G␣ This region contains a conserved motif present in most adenylyl cyclases; however, the PKA phosphorylation site is unique to members of the AC6 family. These observations suggest a mechanism of how isoform selective regulatory diversity can be obtained within conserved regions involved in signal communication.Transmembrane signaling through the receptor-G s -adenylyl cyclase complex has long been studied as a model for signal transduction through heterotrimeric G proteins. Receptor activation of G s results in the dissociation of G␣ s from G␤␥, and the activated G␣ s stimulates adenylyl cyclase (1). Nine G␣ s -regulated adenylyl cyclases have currently been cloned, and the different isoforms of adenylyl cyclases are differentially regulated (2-4). One aspect of this differential regulation is their susceptibility to participate in heterologous desensitization. Previous studies from our laboratory have shown that in addition to receptor desensitization (5), the glucagonsensitive adenylyl cyclase can undergo desensitization at the level of G s (6) and adenylyl cyclase (7). Desensitization at the level of adenylyl cyclase is a cAMP-dependent process and was observed in hepatocytes and S49 lymphoma cells where a decrease in G␣ s -mediated adenylyl cyclase activities was seen. Cloning of adenylyl cyclase cDNAs from hepatocytes and S49 lymphoma cells indicated that adenylyl cyclase 6 (AC6) was present in both cells (7). We reasoned that the common sensitivity in hepatocytes and S49 cells of adenylyl cyclase activity to protein kinase A (PKA) could be caused by AC6. Hence we studied the effects of PKA on G␣ s regulation of AC6. MATERIALS AND METHODS Materials. [␣-32 P]ATP was from New England Nuclear. Purified catalytic subunit of PKA was purchased from Promega. Tissue culture reagents and fetal calf serum were from GIBCO. WIPTIDE and reagents for peptide synthesis were from Bachem. Anti-FLAG M2 column was from Kodak. All other reagents used were the highest grade available.Expression of Adenylyl Cyclases. AC1, AC2, and AC6 were tagged with the FLAG epitope at the N terminus. The FLAG tagged AC1 (8) and AC6 were constructed by using a strategy similar to that used for AC2 (9). The epitope-tagged adenylyl cyclases were expressed in Hi-5 cells by infection with recombinant baculovirus containing the required adenylyl cyclase insert. Hi-5 cells were infected with a multiplicity of 5-10, and membranes were prepared from infected cells 48 hrs after infection as described (9).PKA Treatment. Hi-5 cell membranes containing the recombinant adenylyl cyclase were treated for 15 min in a solution of 25 mM Tris⅐HCl, 10 mM MgCl 2 , 0.8 mM ATP, and protease inhibitor mixture (10) with and without 50-75 nM PKA catalytic subunit. After treatment the...

Recombinant adenylyl cyclase isozyme Types I, II, VI, VII, and three splice variants of Type VIII were compared for their sensitivity to P-site-mediated inhibition by several adenine nucleoside derivatives and by the family of recently synthesized adenine nucleoside 3-polyphosphates (Dé saubry, L., Shoshani, I., and Johnson, R. A. (1996) J. Biol. Chem. 271, 14028 -14034). Inhibitory potencies were dependent on isozyme type, the mode of activation of the respective isozymes, and on P-site ligand. For the nucleoside derivatives potency typically followed the order 2,5-dideoxyadenosine (2,5-ddAdo) > ␤-adenosine > 9-(cyclopentyl)-adenine (9-CP-Ade) 9-(tetrahydrofuryl)-adenine (9-THF-Ade; SQ 22,536), with the exception of Type II adenylyl cyclase, which was essentially insensitive to inhibition by 9-CP-Ade. For the adenine nucleoside 3-polyphosphates inhibitory potency followed the order Ado < 2-dAdo < 2,5-ddAdo and 3-mono-< 3-di-< 3-triphosphate. Differences in potency of these ligands were noted between isozymes. The most potent ligand was 2,5-dd-3-ATP with IC 50 values of 40 -300 nM. The data demonstrate isozyme selectivity for some ligands, suggesting the possibility of isozyme-selective inhibitors to take advantage of differences in P-site domains among adenylyl cyclase isozymes. Differential expression of adenylyl cyclase isozymes may dictate the physiological sensitivity and hence importance of this regulatory mechanism in different cells or tissues.Adenylyl cyclase is potently and directly inhibited by analogs of adenosine via a domain referred to as the P-site from its requirement for an intact purine moiety (1-4). Domains for catalysis and inhibition have been distinguished by use of enzyme purification (5, 6), inhibition kinetics (7, 8), site-specific covalent ligands (9), and selective amino acid substitutions (10). These data suggest that the P-site is distinct from, yet homologous to and interacting with, the catalytic domain. The observation that purified native and recombinant Type I adenylyl cyclases are inhibited by P-site ligands, although exhibiting decreased sensitivity to inhibition (4 -6, 11), establishes the locus of the P-site on the enzyme per se and that inhibition is not via cell surface receptors or G-proteins.P-site-mediated inhibition has been characterized pharmacologically (1, 2, 4, 12-16). Inhibition requires an intact adenine moiety, and potency of inhibition is increased substantially for deoxyribose and especially 3Ј-phosphorylribose adenine nucleosides. Inhibitory potency follows the order: 3Ј-mono-Ͻ 3Ј-di Ͻ 3Ј-triphosphate and adenosine (Ado) Ͻ 2Ј-deoxy (d) 1 -Ado Ͻ 2Ј,5Ј-ddAdo, with 2Ј,5Ј-dd-3Ј-ATP being the most potent ligand and exhibiting an IC 50 ϳ40 nM (15). In addition, tolerance for large substitutions at the 3Ј-position and for other ribose modifications has been demonstrated (1, 2, 4).We reported previously that levels of 2Ј-d-3Ј-AMP and 3Ј-AMP varied considerably in different tissues and were dependent on the metabolic state of the animal (17). Moreover, sensitivity o...

Regulation of basal activities of adenylyl cyclase (AC) 2 and 6, expressed in Sf9 cells by infection with recombinant baculovirus, was studied. An antipeptide antibody that recognizes AC2 and AC6 with equal sensitivity was used to establish that equivalent levels were expressed.

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Receptor activation of heterotrimeric G proteins dissociates G␣ from the G␤␥ complex, allowing both to regulate effectors. Little is known about the effectorinteraction regions of G␤␥. We had used molecular modeling to dock a peptide encoding the region of residues 956-982 of adenylyl cyclase (AC) 2 onto G␤ to identify residues on G␤ that may interact with effectors. Based on predictions from the model, we synthesized peptides encoding sequences of residues 86-105 (G␤86-105) and 115-135 (G␤115-135) from G␤. The G␤86-105 peptide inhibited G␤␥ stimulation of AC2 and blocked G␤␥ inhibition of AC1 and by itself inhibited calmodulin-stimulated AC1, thus displaying partial agonist activity. Substitution of Met-101 with Asn in this peptide resulted in the loss of both the inhibitory and partial agonist activities. Most activities of the G␤115-135 peptide were similar to those of G␤86-105 but G␤115-135 was less efficacious in blocking G␤␥ inhibition of AC1. Substitution of Tyr-124 with Val in the G␤115-135 peptide diminished all of its activities. These results identify the region encoded by amino acids 84-143 of G␤ as a surface that is involved in transmitting signals to effectors.Heterotrimeric G proteins serve as signal transducers for a wide variety of receptors. Both G␣ and G␤␥ subunits can communicate receptor signals (1-5). Regions of G␤␥ complex involved in communicating the signal to effectors have not been well characterized. We had identified the region of residues 956-982 of adenylyl cyclase (AC) 2 as being involved in receiving signals from G␤␥ (6). By using the yeast twohybrid system, the AC2 region of residues 956-982 has been subsequently shown to interact with G␤ but not G␥ subunits (7). In recent studies we found that the peptide encoding residues 956-982 of AC2 can be crosslinked to G␤ when it is part of the free G␤␥ complex but not when it is part of the heterotrimer, indicating that the putative binding surface on G␤ for the AC2 peptide is occluded by interactions with G␣. On the basis of constraints deduced from the crosslinking studies and other biophysical criteria, we docked the AC2 peptide containing residues 956-982 onto the crystal structure of G␤ by using molecular modeling techniques (8). From this docking model, we have identified the regions of G␤ that are predicted to interact with the AC2 peptide. Herein we have tested whether peptides encoding the effector-interaction surface of G␤ predicted from the modeling (8) can modulate G␤␥ regulation of AC1 and AC2. MATERIALS AND METHODSMaterials. Reagents for peptide synthesis were from Bachem. [␣-32 P]ATP was from New England Nuclear. Tissue culture reagents and fetal calf serum was from GIBCO. All other chemicals used were the highest grade available.Peptide Synthesis. Peptides were synthesized on an Applied Biosystems peptide synthesizer (model 431A) and purified by HPLC on acetonitrile gradients. Purified peptides were lyophilized and stored at Ϫ20ЊC. When required peptides were dissolved in water to a final concentration of 1-3 mM....

1. Serotonin (5-HT), small cardioactive peptide B (SCPB) and FMRFamide have well-established facilitatory and inhibitory effects on sensory neurons and their connections with motor neurons mediating withdrawal reflexes in Aplysia. Little is known, however, about their effects on interneurons contributing to those reflexes. As a first step, we examined the effects of these three transmitters on the identified inhibitory interneuron RP14 in isolated pleural-pedal ganglia. 2. Bath application of 5-HT hyperpolarized RP14, inhibited its spontaneous activity and decreased its excitability. In addition, 5-HT decreased the amplitude of inhibitory postsynaptic potentials produced by RP14 in tail sensory and motor neurons. 3. In contrast, bath application of SCPB increased spontaneous activity in RP14. Subsequent application of 5-HT to the bath, which still contained SCPB, inhibited RP14. Therefore, the effects of SCPB were essentially opposite to those of 5-HT on this inhibitory interneuron. 4. FMRFamide had little effect on RP14. It did not produce an obvious change in its resting membrane potential and produced only a transient increase in its spontaneous activity. 5. These results suggest that various neuromodulators have differential effects on elements of the neuronal circuit underlying the tail-withdrawal reflex of Aplysia. Differential modulation may determine the overall behavioral manifestations associated with sensitization.

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