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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