Selective formation of Gsalpha-MHC I complexes after desensitization of human platelets with iloprost.

Prolonged treatment of human platelets with the adenylate cyclase-stimulating prostacyclin analog iloprost leads to reduction in cAMP formation. Previous studies have demonstrated that this may be ascribed to modification of both receptor and Gsalpha function rather than of the catalytic component of adenylate cyclase [Mollner, S., Deppisch, H. & Pfeuffer, T. (1992) Eur. J. Biochem. 210, 539-544]. Iloprost-induced desensitization was accompanied by the formation of a Gsalpha-containing 90-kDa product in membranes treated with the bifunctional cross-linker 1,6-bismaleimidohexane. The cAMP-inducing prostanoid PGD2, which does not promote desensitization, did not cause formation of the 90-kDa species either. The long-term effect of the common G-protein activator [AlF4]- on human platelet adenylate cyclase was shown in many respects to be comparable with that of iloprost. However, [AlF4]- treatment also failed to induce the 90-kDa species, showing that different mechanisms of desensitization were operating. Treatment of the cross-linked 90-kDa complex with PNGase F demonstrated the glycoprotein nature of the Gsalpha-associated component. The 90-kDa cross-linked product was purified by consecutive immunoaffinity chromatography and preparative PAGE to apparent homogeneity. Analysis of the purified protein by MS suggested that, besides Gsalpha, the heavy chain of MHC I (HLA-A2) was part of the complex. This was confirmed by coprecipitation of Gsalpha by the monoclonal anti-(MHC I) antibody W6/32.

Nonenzymatic palmitoylation at Cys 3 causes extra-activation of the alpha-subunit of the stimulatory GTP-binding protein Gs.

Treatment of crude stimulatory GTP-binding protein of adenylyl cyclase (Gs) from turkey erythrocyte membranes with hydroxylamine results in twofold enhancement of adenylyl cyclase activity following reconstitution with adenylyl cyclase type V expressed in Spodoptera frugiperda cells (Sf9) cells. Enhancement by hydroxylamine of immunoaffinity purified Gs was still 1.5-fold, while that of Gs purified according to the multiple-step procedure by Northup, J. K., Sternweis, P. C., Smigel, M. D., Schleifer, L. S., Ross, E. M. & Gilman, A. G. (Proc. Natl Acad. Sci. USA 78, 6516-6520, 1980) was close to unity. The alpha-subunit of the stimulatory GTP-binding protein expressed in Escherichia coli (r(alpha)s), likewise, failed to show an effect by hydroxylamine. Surprisingly, guanosine 5'-O-(3-thiotriphosphate) (GTP[S])-liganded r(alpha)s, treated with palmitoyl-CoA for 14 h at 4 degrees C resulted in sixfold enhancement of reconstitutive activity. In contrast, that of the GDP-liganded r(alpha)s(beta)gamma heterotrimer was not improved by palmitoylation and consecutive activation with GTP[S], although incorporation of [3H]palmitate into momomer and heterotrimer was identical. While adenylyl cyclase type-V activity reconstituted by r(alpha)s x GTP[S] was not influenced by betagamma-subunits, that activated by palmitoylated r(alpha)s x GTP[S] was considerably inhibited, suggesting a higher affinity of palmitoylated r(alpha)s for betagamma-subunits. On treatment of either form with the proteinase Lys-C, less than 25% of the label was found in a stable M(r) 38000 fragment with intact C terminus, but lacking the N-terminal portion. Absence of the latter did not affect activation by r(alpha)s, but caused a >90% loss of extra-activation by palmitoylated r(alpha)s. The results also indicate that nonenzymatic, much in the same way as physiological enzymatic, palmitoylation of alpha(s) occurs predominantly on Cys 3.

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.

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 calmodulin binding domain of nitric oxide synthase and adenylyl cyclase.

Peptides corresponding to regions of the calmodulin-activated NO-synthase and of the calmodulin dependent adenylyl cyclase, which could represent the calmodulin binding domains of the two proteins, have been synthesized and tested for calmodulin binding. The chosen peptides were those in the sequence of the two proteins which most closely corresponded to the accepted general properties of the calmodulin binding domains, i.e., a hydrophobic sequence containing basic amino acids. In the case of the NO-synthase, the putative high-affinity calmodulin binding domain was identified by urea gel electrophoresis and fluorescence spectroscopy with dansylcalmodulin as peptide NO-30 (amino acids 725-754). The highest affinity calmodulin binding site of the calmodulin-dependent adenylyl cyclase was assigned to peptide AC-28 (amino acids 495-522) by titration with dansylcalmodulin and by the ability to inhibit the calmodulin-stimulated activity of purified calmodulin-stimulated adenylyl cyclase. The sequence 495-522 is located in the unit protruding into the cytosol from the sixth putative transmembrane domain of the molecule. It has the typical hydrophobic/basic composition of canonical calmodulin binding domains, and also contains, like most calmodulin binding domains, an aromatic amino acid in its N-terminal portion. It also contains two Cys residues in the central portion, which is an unusual feature of the calmodulin binding domain of this enzyme.

The alpha subunit of the stimulatory guanine-nucleotide-binding regulatory protein forms high-molecular-mass aggregates, concomitant with iloprost-induced desensitization of human platelet adenylyl cyclase.

Prolonged treatment of human platelets with the prostacyclin analog iloprost led to desensitization of the response to various prostaglandin derivatives. However, basal adenylyl cyclase activity and stimulation by agents acting directly via Gs, the stimulatory guanine-nucleotide-binding regulatory protein of adenylyl cyclase, were likewise decreased. Reconstitution of desensitized membranes with purified Gs from turkey erythrocytes indicated no alteration in the catalyst itself. However, the function of Gs (in cholate extracts) appeared to be severely impaired when reconstituted with adenylyl cyclase catalyst. Modification of Gs was also indicated by its altered sedimentation in sucrose density gradients. From Western blots, the alpha subunit of Gs, alpha s, from control platelets sedimented as a 5.6S species, while that from desensitized cells appeared at higher S values (in a polydisperse distribution). Activation by guanosine 5'-[gamma-thio]triphosphate of Gs from control platelets shifted alpha s to 3.5-3.7S, while activation of Gs from desensitized platelets induced such shift only for a minor portion of alpha s. This small fraction alone appeared to be susceptible to ADP-ribosylation by cholera toxin/[32P]NAD. Furthermore, an antibody directed against the C-terminal hexadecapeptide of alpha s precipitated much less alpha s from cholate extracts derived from desensitized platelets. Modification of alpha s during desensitization was also suggested from cross-linking experiments using the homobifunctional agent bismaleimidohexane: alpha s from desensitized platelets formed a single product of 80 kDa, while that from untreated platelets yielded a doublet (100 kDa and 110 kDa).

Chronic lithium regulates the expression of adenylate cyclase and Gi-protein alpha subunit in rat cerebral cortex.

A possible role for adenylate cyclase and guanine nucleotide-binding proteins (G proteins) in contributing to the chronic actions of lithium on brain function was investigated in rat cerebral cortex. It was found that chronic treatment of rats with lithium (with therapeutically relevant serum levels of approximately 1 mM) increased levels of mRNA and protein for the calmodulin-sensitive (type 1) and calmodulin-insensitive (type 2) forms of adenylate cyclase and decreased levels of mRNA and protein for the inhibitory G-protein subunits Gi alpha 1 and Gi alpha 2. Chronic lithium did not alter levels of other G-protein subunits, including Go alpha, Gs alpha, and G beta. Lithium regulation of adenylate cyclase and Gi alpha was not seen in response to short-term lithium treatment (with final serum levels of approximately 1 mM) or in response to chronic treatment at a lower dose of lithium (with serum levels of approximately 0.5 mM). The results suggest that up-regulation of adenylate cyclase and down-regulation of Gi alpha could represent part of the molecular mechanism by which lithium alters brain function and exerts its clinical actions in the treatment of affective disorders.

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.

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.

cDNA sequence of cyclophilin from Dictyostelium discoideum.

A cDNA encoding a protein homologous to cyclophilins from other species has been isolated from a Dictyostelium discoideum cDNA library. From the deduced amino acid sequence a protein with a molecular mass of 19 kD and 64% identity with human cyclophilin is predicted. Southern blot analysis indicates that there is one cyclophilin gene in the D. discoideum genome. The mRNA is present in all developmental stages.

Monoclonal antibodies against various forms of the adenylyl cyclase catalytic subunit and associated proteins.

Monoclonal antibodies against partially purified adenylyl cyclase from bovine brain cortex were raised in mice. Three types of antibody were obtained. Type 1 was specific for the calmodulin-sensitive enzyme. Type II also recognized this enzyme, but recognized the calmodulin-insensitive enzymes from a variety of species and tissues as well. Type I antibodies precipitated their antigens in both the native and denatured forms, while type II strongly favored the denatured forms. Type III antibodies precipitated adenylyl cyclase activity, but as shown by Western blot analysis, were directed against 38-kDa and 45-kDa glycoproteins. The 38-kDa protein was identified as synaptophysin.

Diabetes-induced alterations in the expression, functioning and phosphorylation state of the inhibitory guanine nucleotide regulatory protein Gi-2 in hepatocytes.

Levels of the G-protein alpha-subunits alpha-Gi-2, alpha-Gi-3 and the 42 kDa, form of alpha-Gs were markedly decreased in hepatocyte membranes from streptozotocin-diabetic animals as compared with normals. In contrast, no detectable changes in alpha-Gi subunits were seen in liver plasma membranes of streptozotocin-diabetic animals, although levels of the 45 kDa form of Gs were increased. G-protein beta subunits in plasma membranes were unaffected by diabetes induction. Analysis of whole-liver RNA indicated that the induction of diabetes had little effect on transcript levels of Gi-3, caused an increase in Gs transcripts and decreased transcript number for Gi-2, albeit to a much lesser extent than was observed upon analysis of hepatocyte RNA. In both hepatocyte and liver plasma membranes, immunoblot analysis showed that levels of the catalytic unit of adenylate cyclase were increased upon induction of diabetes. Under basal conditions, alpha-Gi-2 from hepatocytes of diabetic animals was found to be both phosphorylated to a greater extent than alpha-Gi-2 isolated from hepatocytes of normal animals, and furthermore was resistant to any further phosphorylation upon challenge of hepatocytes with angiotensin, vasopressin or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. Treatment of isolated plasma membranes from normal, but not diabetic, animals with purified protein kinase C caused the phosphorylation of alpha-Gi-2. Treatment of membranes from diabetic animals with alkaline phosphatase caused the dephosphorylation of alpha-Gi-2 and rendered it susceptible to subsequent phosphorylation with protein kinase C. Low concentrations of the non-hydrolysable GTP analogue guanylyl 5'-imidodiphosphate inhibited adenylate cyclase activity in both hepatocyte and liver plasma membranes from normal, but not diabetic, animals.

Olfactory adenylyl cyclase. Identification and purification of a novel enzyme form.

Rat olfactory adenylyl cyclase has been identified by means of a monoclonal antibody BBC-2, which reacts with both Ca2+/calmodulin-sensitive and -insensitive forms of adenylyl cyclase (Mollner, S., and Pfeuffer, T. (1988) Eur. J. Biochem. 171, 265-271). The antibody recognized a 180-kDa polypeptide in olfactory cilia but not in decilitated olfactory epithelial membranes. A protein of the same mobility was observed when olfactory adenylyl cyclase was purified by forskolin-agarose affinity chromatography followed by radioiodination. Its identity was further established by cross-linking to [32P]ADP-ribosylated Gs alpha (GTP-binding protein), to yield a single radiolabeled product of Mr approximately 220. Olfactory adenylyl cyclase has a approximately 3-fold higher turnover number, as assessed from stoichiometric binding of [35S]guanosine 5'-(3-O-thio)triphosphate. Therefore, the considerably higher specific adenylyl cyclase activity in olfactory cilia must be due to a approximately 100-fold higher molar concentration of enzyme in this tissue.

Two different adenylyl cyclases in brain distinguished by monoclonal antibodies.

Four monoclonal antibodies, raised against the 115-kDa adenylyl cyclase from bovine brain [Pfeuffer, E. et al. (1985) EMBO J. 4, 3675-3679] have been selected and designated BBC-1 to BBC-4. BBC-1 and BBC-3 are highly specific for the 115-kDa enzyme from bovine brain. The two other antibodies, BBC-2 and BBC-4, recognize an additional 150-kDa adenylyl cyclase in bovine brain, but also in brain tissue from other species. In membranes from lung and myocardium (bovine and rabbit) only the 150-kDa species is detected by the crossreacting antibodies BBC-2 and BBC-4. The two adenylyl cyclases from brain can be separated by calmodulin-Sepharose: only the enzyme of 115 kDa but not that of 150 kDa was retained by the affinity resin and could be stimulated by Ca2+/calmodulin. The data obtained with these antibodies of defined specificity provide for the first time direct evidence for the presence of two distinct adenylyl cyclase species in brain tissue.

Adenylate cyclase from bovine brain cortex: purification and characterization of the catalytic unit.

The non-stimulated (basal) adenylate cyclase from bovine brain cortical membranes was purified 10 000-fold to apparent homogeneity by Lubrol PX extraction and two cycles of affinity chromatography on forskolin-agarose. The final product appears as one major band (mol. wt. 115 000) on SDS-polyacrylamide gels. Further identification was achieved by affinity cross-linking using Gs (stimulatory GTP-binding protein) that was [32P]ADP-ribosylated by cholera-toxin/[32P]NAD: cross-linking with disuccinimidyl suberate gave products with mol. wts. of 160 000, approximately 270 000 and higher. The distribution of these products was dependent on the concentration of cross-linker, suggesting aggregation of two or more adenylate cyclase complexes. In contrast, photo-affinity cross-linking with 4-azidobenzoyl-[32P]Gs yielded a single product with a mol. wt. of 160 000. Purified adenylate cyclase was completely unresponsive towards stimulators (GTP-analogs, NaF) acting via Gs suggesting that this component was removed during purification. On the other hand, stimulation by forskolin and by added activated Gs was preserved but to a smaller degree as compared with the crude enzyme. In contrast, the stimulation of Ca2+/calmodulin was only marginal. Purified adenylate cyclase reversibly bound to wheat germ agglutinin-Sepharose. This suggests that bovine brain adenylate cyclase is a glycoprotein.

Murein hydrolase (N-acetyl-muramyl-L-alanine amidase) in human serum.

An enzyme was identified in human serum which unlike lysozyme cleaved the amide bond between N-acetyl-muramic acid and L-alanine of the peptide side chain of the rigid layer (murein) of Escherichia coli. The N-acetyl-muramyl-L-alanine amidase released all of the peptide side chains including those to which the lipoprotein is bound. A portion of the peptide side chains of the Micrococcus lysodeikticus murein was also hydrolysed from the polysaccharide chains. E. coli, M. lysodeikticus, Bacillus subtilis and Staphylococcus aureus were not killed by the amidase. Treatment of E. coli with EDTA or osmotic shock rendered the cells sensitive to the amidase and they were killed. Possible biological functions of the amidase are discussed. The enzyme was separated from lysozyme in human serum. Gel permeation chromatography indicated a molecular weight of the active enzyme of 82,000 while gel electrophoresis in the presence of sodium dodecyl sulfate revealed a molecular weight of 75,000. Thus, the enzyme probably consists of a single polypeptide chain. Incubation with neuraminidase rendered the amidase more basic suggesting the release of sialic acid residues. The modified glycoprotein disclosed an increased activity to murein. Enzyme activity was inhibited by p-chloromercuribenzene sulfonate and ethyleneglycol-bis(2-aminomethyl) tetraacetate (EGTA) at 1 and 0.2 mM concentration, respectively, whereas EDTA up to 5 mM was without effect. The amidase was also inactivated by agents that reduce disulfide bridges.