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cAMP receptor protein (CRP), allosterically activated by cAMP, regulates the expression of several genes in Escherichia coli. As binding of cAMP leads to undefined conformational changes in CRP, we performed a steady-state and time-resolved fluorescence study to show how the binding of the ligand influences the structure and dynamics of the protein. We used CRP mutants containing a single tryptophan residue at position 85 or 13, and fluorescently labeled with 1,5-I-AEDANS attached to Cys178. Binding of cAMP in the CRP-(cAMP)2 complex leads to changes in the Trp13 microenvironment, whereas its binding in the CRP-(cAMP)4 complex alters the surroundings of Trp85. Time-resolved anisotropy measurements indicated that cAMP binding in the CRP-(cAMP)2 complex led to a substantial increase in the rotational mobility of the Trp13 residue. Measurement of fluorescence energy transfer (FRET) between labeled Cys178 and Trp85 showed that the binding of cAMP in the CRP-(cAMP)2 complex caused a substantial increase in FRET efficiency. This indicates a decrease in the distance between the two domains of the protein from 26.6 A in apo-CRP to 18.7 A in the CRP-(cAMP)2 complex. The binding of cAMP in the CRP-(cAMP)4 complex resulted in only a very small increase in FRET efficiency. The average distance between the two domains in CRP-DNA complexes, possessing lac, gal or ICAP sequences, shows an increase, as evidenced by the increase in the average distance between Cys178 and Trp85 to approximately 20 A. The spectral changes observed provide new structural information about the cAMP-induced allosteric activation of the protein.

Three cAMP-binding proteins have been identified by photoaffinity labeling with 8-azido[32P]cAMP and purified from the mantle tissue of the sea mussel Mytilus galloprovincialis. Their molecular masses, determined by SDS/PAGE, were 54, 42 and 37 kDa. The purified 54-kDa protein, which had two cAMP-binding sites/monomer, was judged to be a regulatory (R) subunit of cAMP-dependent protein kinase since it re-associated with and inhibited purified catalytic (C) subunit of this enzyme from mussel, in the absence but not in the presence of cAMP. The molecular mass of the complex between Mytilus cAMP-binding protein and C subunit, estimated by analytical gel-filtration, was 220 kDa, a value which agrees with a R2C2 stoichiometry for the mussel cAMP-dependent protein kinase holoenzyme. On the basis of the elution pattern from DEAE-cellulose chromatography and its ability to be phosphorylated by purified C subunit of cAMP-dependent protein kinase, the 54-kDa protein could be classified as a type II regulatory subunit. Furthermore, no mobility shift on SDS/PAGE upon phosphorylation/dephosphorylation of Mytilus protein was observed, a similar behaviour to that shown by the mammalian RIIβ isoform. The 42-kDa and 37-kDa proteins, which were recognized by a specific antiserum against the 54-kDa protein and fail to be phosphorylated by Mytilus C subunit, are probably products generated by proteolysis of the 54-kDa protein, although they were shown even when inhibitors of the major types of proteases were used.

Two cAMP-binding proteins, cbpl and cbp2, were purified from the cytoplasm of the green alga Volvox carteri. Both proteins have a native molecular mass of 90 kDa as determined by gel filtration. cbp2 was purified to apparent electrophoretic homogeneity, having a subunit molecular mass of 42 kDa as determined by SDS/PAGE. The cbp1 preparation contains a 42-kDa and a 44-kDa band. The cAMP-binding activity is not associated with protein kinase activity. Tryptic peptides of cbp2 were sequenced by automated Edman degradation. Two pairs of peptides differ in one amino acid only, thus pointing to the presence of isoforms of cbp2. Both binding proteins differed from the cAMP-specific phosphodiesterases of V. carteri with respect to charge, molecular mass and binding affinity to N6-cAMP–agarose. Reverse-phase chromatography of the bound ligand revealed that the two binding proteins hydrolyse cAMP to 5′ AMP. The binding specificity of purified cbp1 and cbp2 was probed by a set of modified cAMP derivatives. Both proteins bind cAMP strictly specifically in the anti conformation; position 1 and 6 of the adenine moiety and at least one of the exocyclic O atoms of the ribose cyclic phosphate moiety are essential. 3-Isobutyl-1-methylxanthine is an effective inhibitor of binding but the natural methyl-xyanthines are not. At present it is not clear whether cbp1 and cbp2 are individual proteins or isoforms of one another.

Full-length human cDNAs for all the different regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinases (PKA) were transcribed and translated in a cell-free in vitro system. The resulting proteins were characterized with respect to molecular size, isoelectric focusing, immunoreactivity, cAMP binding, and to what extent the RII protein subunits revealed mobility shifts upon phosphorylation by catalytic subunit of PKA. We were able to express cDNAs for all the human R (RI alpha, RI beta, RII alpha and RII beta) and C (C alpha, C beta and C gamma) subunits in a wheat-germ extract. [35S]Methionine-labelled in-vitro-translated products were analyzed by SDS/PAGE and revealed distinct protein bands with apparent molecular masses of 49 (RI alpha), 54-55 (RI beta), 51 (RII alpha) and 53 kDa (RII beta) for the R subunits. In vitro transcription/translation of the cDNAs for the C subunits of PKA gave proteins with molecular masses of approximately 40 kDa for all the different C subunits. Phosphorylation of RII alpha and RII beta by the C subunit of PKA, revealed a distinct mobility shift of the RII alpha subunit on one-dimensional SDS/PAGE (51-54 kDa), but not of RII beta (53 kDa). Further characterization of the R subunits by two-dimensional SDS/PAGE revealed that RI alpha was more acidic than RI beta, with pIs of 6.1-6.0 and 6.4-6.2, respectively. Furthermore, the RII alpha protein was more basic than RII beta, with pIs of approximately 5.4-5.3 and 5.3-5.1, respectively. All the in-vitro-translated R subunits could be photoaffinity labelled by the cAMP-analog 8-azido-[32P]cAMP and were also detected by immunoprecipitation with subunit-specific antibodies.

Drug-resistance selection in Dictyostelium discoideum transformants resulted in up to eight-times-higher ras protein levels. Over-production of the wild-type ras protein did not lead to an aberrant phenotype. Increased levels of the mutated [G12T]ras protein, however, were correlated with severe deficiencies in aggregation and development. This aberrant phenotype is associated with reduced cAMP binding, due to a lower number of cell-surface receptors. We show that both RNA and cAMP-receptor-protein levels are reduced. These results indicate that ras in Dictyostelium discoideum seems to be involved in regulating cAMP-receptor-gene expression.

We show that the yeast, Saccharomyces cerevisiae, contains two cAMP-binding proteins in addition to the well-characterized regulatory (R) subunit of cytoplasmic cAMP-dependent protein kinase (PKA). We provide evidence that they comprise a new type of cAMP receptor, membrane-anchored by covalently attached lipid structures. They are genetically not related to the cytoplasmic R subunit. The respective proteins can be detected in sral mutants, in which the gene for the R subunit of PKA has been disrupted and a monoclonal antibody raised against the cytoplasmic R subunit does not cross-react with the two membrane-bound cAMP-binding proteins. In addition, they differ from the cytoplasmic species also with respect to their location and the peptide maps of the photoaffinity-labeled proteins. Although they differ from one another in molecular mass and subcellular location, peptide maps of the cAMP-binding domains resemble each other and both proteins are membrane-anchored by lipid structures, one to the outer surface of the plasma membrane, the other to the outer surface of the inner mitochondrial membrane. Both anchors can be metabolically labeled by Etn, myo-Ins and fatty acids. In addition, the anchor structure of the cAMP receptor from plasma membranes can be radiolabeled by GlcN and Man. After cleavage of the anchor with glycosylphosphatidylinositol-specific phospholipase C from trypanosomes, the solubilized cAMP-binding protein from plasma membranes reacts with antibodies which specifically recognize the cross-reacting determinant from soluble trypanosomal coat protein, suggesting similarity of the anchors. Degradation studies also point to the glycosylphosphatidylinositol nature of the anchor from the plasma membrane, whereas the mitochondrial counterpart is less complex in that it lacks carbohydrates. The plasma membrane cAMP receptor is, in addition, modified by an N-glycosidically linked carbohydrate side chain, responsible mainly for its higher molecular mass.

The chemoattractant cAMP induces the activation of adenylate cyclase in Dictyostelium discoideum. Upon prolonged incubation with cAMP, cells become desensitized via two distinct processes: a decrease in the number of available cAMP-binding sites (down regulation) and modification of the receptor (presumably via phosphorylation) correlated with adaptation. These processes occur simultaneously, but differ in the cAMP dose dependency and reversibility. In this study we investigated the mechanism of adaptation; cells were incubated with a cAMP analog to induce desensitization mediated by adaptation. The cells were then washed, lysed and the interaction between cAMP, receptor, guanine-nucleotide-binding regulatory proteins (G proteins) and GTP was investigated. (1) cAMP receptors that are phosphorylated in vivo remain phosphorylated for at least 45 min after lysis. (2) Desensitization did not alter basal cAMP binding to the receptor nor the inhibitory effect of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) on this binding. (3) The stimulatory effect of cAMP on GTP[S] binding was also unchanged, while basal GTP[S] binding and the kinetics of binding were only slightly different. (4) Basal high-affinity GTPase activity was not altered but cAMP stimulation was reduced from 43 +/- 7% in control lysates to 14 +/- 4% in lysates from desensitized cells. (5) cAMP stimulation of GTPase was decreased by pretreatment of cells with pertussis toxin from 43 +/- 7% to 17 +/- 8% but this was not further altered in lysates from desensitized pertussis-toxin-treated cells. These observations indicate that during desensitization the phosphorylated receptor can still interact with G proteins. Furthermore, desensitization did not affect cAMP stimulation of GTP[S] binding but strongly reduced cAMP stimulation of GTPase, suggesting that a G protein becomes activated. This G protein is pertussis toxin sensitive and may be the inhibitor G protein (G(i)). This would imply that adenylate cyclase desensitizes because G(i) becomes activated.

Polysomal RNAs were isolated from control neuroblastoma cells and those treated with 1,N6-dibutyrl-adenosine 3',5'-phosphate (Bt2cAMP) and translated in wheat germ lysates. Comparison of proteins synthesized in vitro on two-dimensional gel electrophoretograms showed that there was a specific induction in the synthesis of a protein, Mr 48000, by the polysomal RNAs from Bt2cAMP-treated cells. This protein was identified as the R1 cAMP-binding protein by its coelectrophoresis with unlabelled binding protein and by its specific retention on 8-(6-aminohexylamino)-adenosine 3',5'-phosphate linked to Sepharose. Quantification of the proteins synthesized in vitro with subsaturating inputs of polysomal RNAs showed that there was a 1.4--1.7-fold increase in the synthesis of the R1 cAMP-binding protein by polysomal RNAs isolated from Bt2cAMP-treated cells. There was a similar increase when purified polyadenylated mRNA populations were compared. showing there was no change in the ratio of adenylated to nonadenylated mRNAs in the induced mRNA population. There was no corresponding increase in the synthesis of the R2 cAMP-binding protein although the relative synthesis of several other proteins was also increased and the synthesis of actin and the alpha and beta-tubulin subunits was decreased. The increased levels of the R1 cAMP-binding protein found in Bt2cAMP-treated neuroblastoma cells are therefore partly caused by a specific accumulation of its mRNA on polysomes. The mRNA content of the cytoplasmic messenger ribonucleoprotein (mRNP) population of control cells was insufficient to account for this increase by a translocation of R1 mRNA from the mRNP to the polysome fraction in Bt2cAMP-treated cells. The increase in polysomal R1 mRNA is therefore caused by its increased transcription of post-transcriptional processing or its decreased rate of degradation in Bt2cAMP-treated cells. Although the R1 and R2 binding proteins have identical molecular weights and similar pI values, the specific induction of the mRNA for R1 cAMP-binding protein and the differential distribution of the R1 and R2 mRNAs between the polysomal and messenger ribonucleoprotein compartments show that these two cAMP-binding proteins are encoded by different mRNA populations.

The effects of numerous cAMP analogs present in the [3H]cAMP binding reaction on subsequent dissociation of [3H]cAMP from the regulatory subunit of CAMP-dependent protein kinase I and I1 were analyzed. Certain analogs with modification at either C-8 or C-2 showed relative selectivity for one (site 1) of two intrachain cAMP binding sites of both isozymes. Modification at C-6 caused selectivity for the second site (site 2). The combination of a site-I-directed and site-Zdirected analog inhibited [3H]cAMP binding much more than did either analog alone. In general, there was a correlation between the site 1 selectivity and the ability of the analog to stimulate the binding of [3H]clMP, which selects site 2. The site-1-directed analogs stimulated the initial rate of [3H]cIMP binding. The stimulatory effect was enhanced in the presence of a polycationic protein such as histone and was inhibited by high ionic strength. The type I and II isozymes exhibited large differences in analog specificity for this effect. For type I, of the analogs tested the most efficacious for stimulating [3H]cIMP binding were those containing a nitrogen atom attached to C-8, 8-aminobutylamino-CAMP being the most effective. Type I1 responded best to analogs con- taining a sulfur atom attached to C-8, 8-SH-CAMP being the most effective of those tested. The stimulatory effect was accentuated in the presence of MgATP when using type I, but this nucleotide had no effect when using type II. It is proposed that in intact tissues cAMP binding to site 1 of either isozyme stimulates the binding to site 2.

Dimethylbenzanthracene-induced rat carcinomas possess activities binding cyclic adenosine 3':5'-monophosphate (cAMP) and estrogen. When dimethylbenzanthracene-induced tumors regress after ovariectomy of the host, a change in the specific binding of cAMP and estrogen occurs in the tumors. Six days after ovariectomy, cAMP binding increases 5-fold in the nuclei and 2-fold in the cytosol of tumors, while nuclear and cytoplasmic estrogen binding decreases by 80% and 50%, respectively. These changes in activities binding cAMP and estrogen are detectable within 1 day after ovariectomy and the changes are reversed when resumption of tumor growth is induced by the injection of 17beta-estradiol. When dimethylbenzanthracene-induced tumors fail to regress after ovariectomy, the change in activities binding cAMP and estrogen does not occur. Significant increases in the cAMP level as well as in adenylate cyclase and cAMP-phosphodiesterase activities are also found in the regressing tumors. Concomitant with the increase of cAMP-binding activity is an increase in histone kinase activity in the regressing tumor. These data suggest the involvement of cAMP in the growth control of a hormone-dependent mammary rumor.

cAMP binding to the 'stable' cAMP-binding sites in the regulatory subunit of the cAMP-dependent protein kinase type I was investigated using a set of 18 selected derivatives. All the tested analogues were competitive with [3H]cAMP and inhibitor constants from 12 nM to 20 microM with the free regulatory subunit were determined. The cAMP molecule seemed to be bound by these specific hydrogen bonds to the 5' and 3' oxygen, the 2' hydroxyl, and an ion pair interaction between the negative charge in equatorial position and a positively charged amino acid side chain. The adenine base is rather unspecifically bound with no hydrogen bonds involved. This binding specificity of the 'stable' site is similar to the requirement for dissociation as determined by the activation of the kinase by a respective analogue. This indicates that occupation of the 'stable' sites leads to activation of the protein kinase. The presence of the catalytic subunit reduced the affinity of most analogues. The binding of one derivative with the negative charge fixed in the axial position is not influenced by the addition of the catalytic subunit and ATP. A plausible model for a conformational change during the activation process in the 'stable' site is discussed.

The high-affinity cAMP-binding site of form-II yeast glyceraldehyde-3-phosphate dehydrogenase has a marked specificity for adenosine derivatives, such ligands including N6-substituted adenosine derivatives active as cytokinins in plant systems and adenine nucleotides. Of a wide range of nucleotides and nucleosides examined only adenosine derivatives bind to the cAMP binding site. A variety of antimitotic compounds (including colchicine, colcemid and phenylcarbamate derivatives), adrenergic receptor antagonists (alprenolol and propranolol) and non-steroidal anti-inflammatory agents (notably indomethacin and flufenamic acid) displace cAMP from glyceraldehyde-3-phosphate dehydrogenase. Colchicine, colcemid, N6-furfuryladenosine, indomethacin, flufenamic acid and propranolol inhibit cAMP binding to the enzyme in an apparently competitive fashion. Given the evolutionary conservatism and abundance of glyceraldehyde-3-phosphate dehydrogenase, the affinity of the cAMP-binding site of this enzyme for a variety of structurally-disparate pharmacologically-active compounds compromises simple one-site interpretations of physiological responses to these agents.

The biological effects of 5′-amido analogues of adenosine 3′:5′-monophosphate (cAMP) and monophosphothioate, with and without N-substituents, were investigated. The unsubstituted amino analogues stimulated the cAMP-dependent protein kinase from bovine muscle while N-substitution prevented this effect. Some of the N-alkyl derivatives, however, inhibited the protein kinase activity. Only adenosine 5′-amido 3′:5′-monophosphate was found to compete for the binding of [3H]cAMP to cAMP binding protein from bovine muscle. Phosphodiesterases from bovine heart and rabbit brain hydrolyzed 5′-amido-adenosine 3′: 5′-monophosphate, but only the former hydrolyzed the corresponding phosphothioate. The heart diesterase was inhibited competitively by N-substituted derivatives of 5′-amido-adenosine 3′: 5′-monophosphate and 5′-amido-adenosine 3′: 5′-monophosphothioate, but 5′-N-n-octyl-amino-adenosine 3′:5′-monophosphate did not inhibit any of the brain diesterases tested. Results suggest that steric and electronic requirements of the cyclophosphate ring determine the catalytic effects of the cAMP on protein kinase, while alteration in other regions of the molecule exercise an appreciably smaller effect and largely affect binding parameters. Phosphodiesterases show similar requirements for binding and reactivity.