Thomas Pfeuffer

Inhibition of adenylyl cyclase isoforms V and VI by various Gβγ subunits

An intriguing development in the G‐protein signaling field has been the finding that not only the Gα subunit, but also Gβγ subunits, affect a number of downstream target molecules. One of the downstream targets of Gβγ is adenylyl cyclase, and it has been demonstrated that a number of isoforms of adenylyl cyclase can be either inhibited or stimulated by Gβγ subunits. Until now, adenylyl cyclase type I has been the only isoform reported to be inhibited by free Gβγ. Here we show by transient cotransfection into COS‐7 cells of either adenylyl cyclase V or VI, together with Gγ2 and various Gβ subunits, that these two adenylyl cyclase isozymes are markedly inhibited by Gβγ. In addition, we show that Gβ1 and Gβ5 subunits differ in their activity. Gβ1 transfected alone markedly inhibited adenylyl cylcase V and VI (probably by recruiting endogenous Gγ subunits). On the other hand, Gβ5 produced less inhibition of these isozymes, and its activity was enhanced by the addition of Gγ2. These results demonstrate that adenylyl cyclase types V and VI are inhibited by Gβγ dimers and that Gβ1 and Gβ5 subunits differ in their capacity to regulate these adenylyl cyclase isozymes.—Bayewitch, M. L., Avidor‐Reiss, T., Levy, R., Pfeuffer, T., Nevo, I., Simonds, W. F., Vogel, Z. Inhibition of adenylyl cyclase isoforms V and VI by various Gβγ subunits. FASEB J. 12, 1019–1025 (1998)

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 cyclases 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.

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.

Developmental changes in Gs and Golf 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.

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 α 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.

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 phorbolesters, it significantly (2.5×) 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 [γ 32P8‐N‐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.

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.

Interaction of adenylyl cyclase type 1 with βγ subunits of heterotrimeric G-proteins

All of the 10 known mammalian adenylyl cyclases (AC) (1) are stimulated by the α subunit of the activating G-protein (αs) but only a certain subset is regulated by G-protein βγ subunits. The isoforms 2, 4, and 7 (?) are conditionally activated by βγ subunits upon costimulation with αs while AC-1 activity is suppressed by βγ. A physical interaction between βγ and AC is implicated by the work of Taussig (2), Enomoto (3) as well by our own experiments (unpublished results).