Adenylyl cyclase type II is stimulated by PKC via C-terminal phosphorylation.

Adenylyl cyclases of the type II family differ from other subforms in that they are conditionally stimulated via alpha(s)/betagamma subunits and regulated by PKC mediated phosphorylation. AC II, stably expressed in HEK 239 cells, was incubated with the PKC activator tetradecanoylphorbol acetate (TPA). Using cells metabolically labeled with [32P]phosphate, TPA caused concerted stimulation of basal and forskolin activated adenylyl cyclase together with incorporation of [32P]phosphate into AC II protein. Enhanced phosphorylation was also indicated by a monoclonal anti-phosphothreonine antibody. Assignment of TPA-induced [32P]phosphate-incorporation to specific sites was achieved by a combination of chemical and immunochemical methods. Three out of five [32P]labeled peptides that were generated by fragmentation with N-chlorosuccinimide were also recognized by the monoclonal antibody BBC-4 [S. Mollner, T. Pfeuffer, Eur. J. Biochem. 171 (1988) 265-271] directed against an epitope 8 kDa from the extreme C-terminus. These findings suggested Ser-871 (consensus sequence ARSLK) and Thr-1057 (CTCR) as acceptor candidates of phorbolester induced phosphoryl transfer.

Affinity labeling of forskolin-binding proteins. Comparison between glucose carrier and adenylate cyclase.

An [125I]iodoazidosalicylic acid derivative of forskolin was synthesized for identification of the diterpene's binding sites on the catalytic subunit of adenylate cyclase and on glucose transport proteins. The affinity label was selectively incorporated into proteins of Mr 40,000-60,000 in membranes from human erythrocytes and from various other tissues. The iodoazidosalicylic acid derivative also specifically labeled the catalytic moiety of adenylate cyclase from rabbit myocardial membranes. However, the structural requirements of the two forskolin-binding sites must be different, since the affinity of the photolabel for the glucose carriers is much higher than that for the cyclase catalyst. Furthermore, the label is readily competed with by D-glucose and cytochalasin B for its binding site on the glucose carrier but not on adenylate cyclase.

Differential effects of ceramides upon adenylyl cyclase subtypes.

Ceramides are reported to stimulate different effector systems, among them atypical protein kinases C (PKCs). When HEK 293 cells, stably expressing adenylyl cyclase type II (AC II), were treated with various ceramide derivatives, adenylyl cyclase activity was enhanced 8-15-fold. The stimulation by the most potent analog, C18/C24 ceramide, was comparable to that by the phorbolester TPA. The stimulatory effect of ceramide was not restricted to AC II, although the type I and type V enzymes were affected less dramatically. Unexpectedly, the dihydro derivatives of ceramides, generally serving as non-activating controls, exhibited only slightly lower stimulation than ceramides, whereas short-chain ceramides (e.g. C2) were without effect. The action of ceramides was at least partially inhibited by okadaic acid, suggesting involvement of a phosphatase. Furthermore, ceramides and TPA operated synergistically. While the PKC inhibitor staurosporine counteracted the action of phorbol-esters, it significantly (2.5x) enhanced the effect of ceramides.

Localisation of an ATP-binding site on adenylyl cyclase type I after chemical and enzymatic fragmentation.

Photolabeling of partially purified bovine brain adenylyl cyclase (AC I) with [gamma 32P]8-N3-ATP led to incorporation of 32P into the 115 kDa catalyst. Further treatment with N-chlorosuccinimide, which cleaves proteins at tryptophan residues, yielded a 14 kDa 32P-labeled fragment. The latter was immunoprecipitated by antibody BBC1, recognizing the extreme C-terminus of AC I, but not by antibody BBC2, recognizing a more remote epitope. Further fragmentation of photolabeled AC I by the proteases Glu-C and Asp-N yielded 32P-labeled peptides corresponding to 2.9 kDa and 5.6 kDa fragments, which were not recognized by any of these antibodies. This narrows the ATP binding site down to a 25 amino acid sequence containing a general motif G(X0-7)KG(X0-4)L/M(X5-7)S/T present in all eukaryotic adenylyl cyclases so far cloned, but also in a variety of bacterial adenylyl cyclases (Peterkofsky et al. (1993) Progr. Nucleic Acids Res. Mol. Biol. 44, 31-65).

Acylation of adenylyl cyclase catalyst is important for enzymic activity.

Incubation of human thrombocytes in the presence of [3H]palmitic acid leads to incorporation of this fatty acid into the alpha subunit of Gs as described [Linder et al., Proc. Natl. Acad. Sci. USA 90 (1993) 3675-3679; Degtyarev et al., Biochemistry 32 (1993) 8057-8061] but also into the catalyst of adenylyl cyclase which has not been recognized before. Treatment of labeled membranes with hydroxylamine released the label from both components. Label incorporated into the catalyst could be identified as [3H]palmitate. At the same time chemical deacylation caused partial loss of adenylyl cyclase activity.

Isolation of homologous and heterologous complexes between catalytic and regulatory components of adenylate cyclase by forskolin-Sepharose.

Homologous and heterologous complexes between catalytic and GTP-binding components can be isolated by means of immobilized succinyldeacetylforskolin (forskolin-Sepharose). A heterologous complex is formed by reconstitution of forskolin-Sepharose bound catalytic function from rabbit myocardial membranes with the homogenous [3H]methyl-GTP-binding protein from duck erythrocyte membranes. Analysis of the reconstituted complex by sodium dodecyl sulfate polyacrylamide gelelectrophoresis reveals that only the Mr 42 000 component of the GTP-binding protein's Mr 42 000/Mr 35 000 heterodimer contributes to the formation of active adenylate cyclase.

Chemical and functional analysis of components of adenylyl cyclase from human platelets treated with phorbolesters.

Human platelets, prelabeled with [32P]phosphate were treated with tetradecanoylphorbol acetate (TPA) for 5 min at 37 degrees C. Phosphorylation of the components of adenylyl cyclase was determined in membranes using specific antibodies against G-proteins and the catalytic moiety. Less than 0.01 mol of [32P]phosphate/mol could be detected in immunoprecipitates using antibodies against sequences within the alpha-subunit of the GTP binding protein Gi. TPA, however, caused the incorporation of 0.67-1.1 mol of [32P]phosphate per mol of catalyst while 0.13-0.2 mol were found in the absence of TPA. Lack of modification of the alpha-subunit of Gi was also indicated by the results of reconstitution experiments with purified Gi alpha from bovine brain: adenylyl cyclase in membranes from untreated platelets was significantly more inhibited by added G1 alpha, than that from TPA treated cells. While beta, gamma-subunits were like-wise inhibitory no difference dependent on platelet-pretreatment could be observed.

Molecular cloning and expression of a novel type V adenylyl cyclase from rabbit myocardium.

A cDNA of a novel form of type V adenylyl cyclase has been cloned from rabbit myocardium using oligonucleotide probes derived from peptides that were produced by enzymatic cleavage of purified heart cyclase. A corresponding mRNA (6 kb) has been detected in rabbit myocardial tissue by Northern blot analysis. The cDNA encodes a protein of 1,264 amino acids exhibiting 12 putative membrane-spanning regions in its hydrophilicity profile. Sequence comparison to two other previously published type V adenylyl cyclase reveals amino-terminal domains of different length and low correlative homology, whereas the rest of the sequences is almost identical. The nonconserved amino-terminal region of the subtype consists of 214 amino acids and exceeds the length of the others by 40 and 80 residues, respectively. Its presence in membrane preparations from different tissues has been confirmed immunologically using an antibody directed against a synthetic peptide. The cloned adenylyl cyclase was functionally expressed in COS-1 cells to attain an enzymatic activity 3.5- to 14-fold above control in the presence of forskolin.

Interactions of pure beta gamma-subunits of G-proteins with purified beta 1-adrenoceptor.

The role of the beta gamma-subunits in the interaction of G-proteins was examined with beta 1-adrenoceptors purified from turkey erythrocytes and pure beta gamma-subunits prepared from turkey erythrocytes and bovine brain. On a non-denaturing polyacrylamide gel, the mobility of beta gamma-subunits was increased when incubated with beta 1-adrenoceptor and the beta 1-adrenergic agonist 1-(-)-isoproterenol, whereas on incubation with the antagonist 1-alprenolol the mobility was unchanged. Furthermore, the beta 1-adrenoceptor was retarded on a Sephadex G-50 column equilibrated with beta gamma-subunits and agonist. No retardation occurred in the presence of antagonist. These data suggest a direct interaction of activated beta 1-adrenoceptors with isolated beta gamma-subunits of G-proteins.

Trimeric G-proteins of the trans-Golgi network are involved in the formation of constitutive secretory vesicles and immature secretory granules.

Non-hydrolysable analogues of GTP, such as GTP gamma S and GMP-PNP, have previously been shown to inhibit the formation of constitutive secretory vesicles (CSVs) and immature secretory granules (ISGs) from the trans-Golgi network (TGN). Using a cell-free system, we show here that the formation of these vesicles is also inhibited by [A1F4]-, a compound known to act on trimeric G-proteins. Addition of highly purified G-protein beta gamma subunits stimulated, in a differential manner, the cell-free formation of both CSVs and ISGs. ADP-ribosylation experiments revealed the presence of a pertussis toxin-sensitive G-protein alpha subunit in the TGN. We conclude that trimeric G-proteins regulate the formation of secretory vesicles from the TGN.

The hormonal regulation of adenylate cyclase.

The regulation of adenylate cyclase by hormones and by GTP regulatory proteins was investigated in native membrane systems and in systems reconstituted from purified components. These studies can be summarized as follows. The stimulatory beta 1-adrenoceptor catalyses the activation of a complex between the GTP stimulatory protein GS and the catalytic unit C. The agonist-receptor complex can activate a few cyclase units in native membrane systems as well as in reconstituted systems. GS from turkey erythrocytes is functionally different from rabbit liver GS, the latter being more amenable to activation by guanyl nucleotides in the absence of hormone. The coupling between the beta 1-adrenoceptor GS and C is efficient when compared with the coupling obtained in native membrane systems. GTP/GDP exchange at the alpha S subunit requires the presence of the beta gamma subunits. A mechanism for the inhibition of adenylate cyclase by the inhibitory GTP regulatory protein Gi is suggested.

Developmental changes in Gs and G(olf) proteins and adenylyl cyclases in mouse brain membranes.

Guanine nucleotide-binding (G) proteins, Gs and G(olf) mediate the increase in cAMP formation through the activation of adenylyl cyclases. The developmental profiles of Gs, G(olf) and adenylyl were determined in mouse striatum and whole brain using immunobloting with specific antisera. Gs and the 115 kDa and 150 kDa adenylyl cyclases were present at the earliest age tested, embryonic day (E) 14.5 G(olf) and the 160 kDa adenylyl cyclase emerged in parallel, postnatally; during this period the increase in the relative abundance of the 150 kDa was observed. Gpp[NH]p activated Gs/G(olf) in a dose dependent manner, with a smaller response observed in embryos compared to adults. Mn2+ and forskolin activated the adenylyl cyclases and this activation increased during development. At E 14.5, maximal activation with Mn2+ and forskolin elicited a similar increase in cAMP levels, but from postnatal day 1, a nearly two fold higher response was obtained with forskolin compared to Mn2+; at the same time the 160 kDa adenylyl cyclase was detected. These data suggest that the appearance of certain forms of stimulatory G proteins was developmentally correlated with the expression of specific adenylyl cyclases.

The expression of a novel Ca2+/CaM stimulated adenylyl cyclase activity in the neuroblastoma cell line Lan-1 is regulated by cell density.

In the human neuroblastoma cell line Lan-1, the mRNA encoding the Ca2+/calmodulin (CaM) sensitive adenylyl cyclase type-1 (AC-1) was detected by reverse transcription-polymerase chain reaction (RT-PCR) as well as by Northern blotting. However, neither Ca2+/CaM stimulated AC activity was found nor could AC-1 type protein be detected by a specific antibody (anti-1Cl). In contrast, when cells were grown to high cell density, Ca2+/CaM stimulated AC-activity could be indeed found in membranes. The large increase in activity was paralleled by the appearance of a 110 kDa protein detected by the monoclonal AC antibody BBC-2. At the same time a 150 kDa adenylyl cyclase species present in growing cells was absent. The 110 kDa protein co-migrated with bovine AC-1 and was slightly larger than the human AC-1. Unexpectedly, however, the antibody anti-1CI was not able to precipitate the newly induced Lan-1 AC. In addition, no increase in type-1 AC mRNA could be detected either by PCR or by Northern blotting. Treatment of Lan-1 cells with 10 microM retinoic acid for 7 days caused growth arrest and morphological differentiation of the cells, yet the induction of the Ca2+/CaM-stimulated AC activity was much lower than in the dense grown control cultures. It is concluded that the Ca2+/CaM-activated AC of M(r) 110 kDa in Lan-1 cells is not related to the previously known Ca2+/CaM stimulated AC isoforms, and might thus represent a novel AC.