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Results for “Receptors, Cyclic AMP”

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At least 19 recordsLinked to original sources
PubMed1978

A new extragenic suppressor of cya mutation. Mutant cyclic AMP receptor protein with an increased affinity for cyclic AMP.

A strain bearing an extragenic suppressor of cya mutation was isolated as a second-site revertant of an adenylate cyclase deficient strain. The mutant was unable to synthesize cAMP but showed normal fermentation profiles and growth properties on a variety of carbon sources. The site of reversion was mapped in, or near, the structural gene for the cAMP receptor protein. Structural alteration of the protein was directly demonstrated by the following biochemical observations: (i) A 10-fold decrease in the dissociation constant for cAMP, (ii) an acidic shift in the isoelectric point, and (iii) the altered binding properties to lambdah80dlac ps DNA.

Carbohydrate Metabolism↗
PubMed1979

Presence of free cyclic AMP receptor protein and regulation of its level by cyclic AMP in neuroblastoma-glioma hybrid cells.

Neuroblastoma-glioma hybrid cells of line 108CC-5 were found to contain high levels of soluble adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase activity and high levels of two specific cAMP receptor proteins, RI and RII. Treatment of the hybrid cells with dibutyryl cAMP increased the level of RI but did not significantly affect the level either of RII or of cAMP-dependent protein kinase activity. The effect of dibutyryl cAMP could be mimicked by prostaglandin E1 and 3-isobutyl-1-methylxanthine, both of which are known to raise cAMP levels in neuroblastoma-glioma hybrid cells. Both in control as well as in dibutyryl cAMP-treated cells, RII but not RI was associated with cAMP-dependent protein kinase. Several lines of evidence suggest that RI represents the free regulatory subunit of type I cAMP-dependent protein kinase. The presence of this regulatory subunit as free cAMP receptor protein in neuroblastoma-glioma hybrid cells may be of significance with respect to the regulation of growth and differentiation in tumor cells.

Animals↗
PubMed1977

Solubilization and photoaffinity labeling of renal membrane cyclic AMP receptors.

Renal cortical plasma membranes were solubilized with sodium deoxycholate. The membrane-bound cyclic AMP receptors retained biologic activity in the detergent-dispersed state exhibiting the properties of high affinity for cyclic AMP, saturability and specificity. Half-maximal binding of cycle [3H]-AMP to these receptors was found to occur at 0.06 muM and 1.5 pmol of cyclic [3H]AMP was bound per mg membrane protein at saturation (0.5 muM cyclic [3H]AMP). Sodium deoxycholate-solubilized membrane proteins were chromatographed on Biogel A-5m. Cyclic [3H]AMP receptors eluted in the internal volume at positions equivalent to molecular sizes of 50 000 and 20 000 daltons and in the void volume at molecular size greater than 450 000. After photoaffinity labeling the renal membrane receptors with cyclic [3H]AMP, we found peaks of tritium radioactivity which eluted at similar molecular size positions on this Bogel A-5m column. Further treatment of photoaffinity labeled membranes with sodium dodecyl sulfate, mercaptoethanol and urea, followed by polyacrylamide gel electrophoresis, showed bands of tritium-labeled receptor protein with relative mobilities corresponding to molecular sizes of 26 000 and 21 000 daltons. This study shows that porcine renal cortical membranes contain at least two molecular species of cyclic AMP receptors which may be associated with regulation of the membrane-bound cyclic AMP-dependent protein kinase.

Affinity Labels↗
PubMed1977

Effects of cyclic nucleotides on the conformational states of the alpha core of the cyclic AMP receptor protein.

The alpha core gragment produced by limited proteolysis contains the cyclic AMP binding domain and the two buried sulfhydryl groups of the cyclic AMP receptor protein. The buried sulfhydryl groups of the alpha core react with 5,5'-dithio-bis(2-nitrobenzoic acid) after denaturation by 3 M urea or digestion with subtilisin. The rate of sulfhydryl modification in the presence of 3 M urea or subtilisin is markedly decreased in the presence of cyclic nucleotides which are proposed to tighten the conformation of the alpha core. Incubation of the alpha core in 3 M urea or dithionitrobenzoic acid does not affect cyclic AMP binding while dithionitrobenzoic acid plus 3 M urea inhibits cyclic AMP binding suggesting a role for the buried sulfhydryls in cyclic AMP binding or their proximity to the cyclic AMP binding domain of the alpha core. The data are consistent with a ligand-induced conformational change in the alpha region of the native cyclic AMP receptor protein that is required for DNA binding.

Binding Sites↗
PubMed1979

Cyclic AMP receptor triggers nuclear protein phosphorylation in a hormone-dependent mammary tumor cell-free system.

Adenosine 3',5'-monophosphate (cyclic AMP) receptor protein of 56,000 daltons increases markedly in mammary tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) after incubation of tumor slices with cyclic AMP, benzamide, and arginine. Incubation of cytosol from these tumor slices with nuclei from unincubated tumors results in nuclear uptake of the 56,000-dalton cyclic AMP receptor and in phosphorylation of the 76,000-dalton nuclear protein. Binding of the 56,000-dalton receptor and phosphorylation of the 76,000-dalton protein also occur in DMBA tumor nuclei when protein kinase type II of bovine heart is used. The results suggest that cyclic AMP receptor is involved in the nuclear events of a hormone-dependent mammary tumor.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1978

The dependence of Escherichia coli asparaginase II formation on cyclic AMP and cyclic AMP receptor protein.

The amount of asparaginase II in an Escherichia coli wild-type strain (cya+, crp+) markedly increased upon a shift from aerobic to anaerobic growth. However, no such increase occurred in a mutant (cya) lacking cyclic AMP synthesis unless supplemented with exogenous cyclic AMP. Since a mutant (crp) deficient in cyclic AMP receptor protein also did not support the anaerobic formation of this enzyme, it is concluded that the formation of E. coli asparaginase II depends on both cyclic AMP and cyclic AMP receptor protein.

Aerobiosis↗
PubMedDate not supplied

[Method of measuring binding constants and the number of specific cyclic AMP receptor sites in tissue extracts].

A method for evaluation of binding constants and of amount of cAMP binding sites in crude tissue extracts was developed. The method is based on equilibrium binding of 3H-cAMP by proteins with subsequent ultrafiltration. Hydrolysis of cAMP and its unspecific sorption by proteins were eliminated under the conditions selected. Rat spleen cytosole contained 3.57 +/- 0.34 pmol of cAMP binding sites per mg of protein with dissociation constant of protein-cAMP complex (1.68 +/- 0.28).10(-8) M. As shown by studies on kinetics, binding constants and specificity of binding, the method permitted to evaluate quantitatively cAMP-dependent protein kinases in crude tissue extracts and to estimate their affinity to cAMP.

Animals↗
PubMed1979

Interaction site of Escherichia coli cyclic AMP receptor protein on DNA of galactose operon promoters.

Cyclic AMP (cAMP) and its receptor protein (CRP) have a dual role in the regulation of the two promoters that control the galactose (gal) operon of Escherichia coli. One promoter, P1, requires cAMP-CRP for activity; the other, P2, is inhibited by these factors. We have examined the interactions site of cAMP-CRP on gal DNA by using two types of protection experiments, involving DNase digestion and methylation by dimethyl sulfate. Our results indicate that cAMP-CRP binds to gal DNA in a segment located between 50 and 24 base pairs preceding the P1 start point for transcription. Although the location of the cAMP-CRP interaction site is clearly different in gal and lac DNA, comparison of the DNA sequences suggests a similar recognition sequence. The location of the cAMP . CRP-binding site in gal further suggests that protein-protein interactions between RNA polymerase and cAMP . CRP play an important role in transcription initiation at the gal and possibly other cAMP-dependent promoters.

Base Sequence↗
PubMed2003

Cyclic AMP receptor protein-dependent activation of the Escherichia coli acsP2 promoter by a synergistic class III mechanism.

The cyclic AMP receptor protein (CRP) activates transcription of the Escherichia coli acs gene, which encodes an acetate-scavenging enzyme required for fitness during periods of carbon starvation. Two promoters direct transcription of acs, the distal acsP1 and the proximal acsP2. In this study, we demonstrated that acsP2 can function as the major promoter and showed by in vitro studies that CRP facilitates transcription by "focusing" RNA polymerase to acsP2. We proposed that CRP activates transcription from acsP2 by a synergistic class III mechanism. Consistent with this proposal, we showed that CRP binds two sites, CRP I and CRP II. Induction of acs expression absolutely required CRP I, while optimal expression required both CRP I and CRP II. The locations of these DNA sites for CRP (centered at positions -69.5 and -122.5, respectively) suggest that CRP interacts with RNA polymerase through class I interactions. In support of this hypothesis, we demonstrated that acs transcription requires the surfaces of CRP and the C-terminal domain of the alpha subunit of RNA polymerase holoenzyme (alpha-CTD), which is known to participate in class I interactions: activating region 1 of CRP and the 287, 265, and 261 determinants of the alpha-CTD. Other surface-exposed residues in the alpha-CTD contributed to acs transcription, suggesting that the alpha-CTD may interact with at least one protein other than CRP.

Acetate-CoA Ligase↗
PubMed1977

Identification, characterization, and quantitative measurement of cyclic AMP receptor proteins in cytosol of various tissues using a photoaffinity ligand.

Two protein bands, present in cytosol fractions from each of seven rat tissues examined, specifically incorporated 32P-labeled 8-azidoadenosine 3':5'-monophosphate (8-N3-[32P]cAMP), a photoaffinity label for cAMP-binding sites. These proteins had apparent molecular weights of 47,000 and 54,000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. These two proteins were characterized in three of the tissues, namely, heart, uterus, and liver, by the total amount of 8-N3-[32P]cAMP incorporation, by the dissociation constant (Kd) for 8-N3-[32P]cAMP, and by the nucleotide specific inhibition of 8-N3-[32P]cAMP incorporation. Several lines of evidence were obtained that the protein with an apparent molecular weight of 47,000 represents the regulatory subunit of a type I cAMP-dependent protein kinase, while the protein with an apparent molecular weight of 54,000 represents the regulatory subunit of a type II cAMP-dependent protein kinase. Almost all of the cAMP receptor protein found in the cytosol of these tissues, as measured by 8-N3-[32P]cAMP incorporation, was associated with these two protein kinases, in agreement with the idea that most effects of cAMP are mediated through protein kinases. The photoaffinity labeling with 8-N3-[32P]cAMP can be used to estimate quantitatively the amounts of regulatory subunit of type I and type II cAMP-dependent protein kinases in various tissues.

Affinity Labels↗
PubMed1975

Beta-adrenergic receptors, cyclic AMP, and ion transport in the avian erythrocyte.

(1) Interaction of beta-adrenergic catecholamines with specific receptors on the cell membrane causes activation of adenylate cyclase, generation of intracellular cyclic 3',5'-AMP, and enhanced sodium and potassium transport. The effects of catecholamines on each of these functions are characteristic of beta1-type receptor interactions and are specific for 1(--)isomers. The order of effectiveness of agonists is isoproterenol greater than norepinephrine approximately equal to epinephrine. (2) exogenous cyclic 3',5'-AMP enhances sodium or potassium transport in a fashion identical to that caused by catecholamines. (3) the receptor contains an ethanolamine site that recognizes the stereospecific function of the agonist. Interaction at this site can be determined by measuring specific binding of a high specific activity-radioiodinated beta-blocker analogue (125-iodohydroxybenzylpindolol). Catecholamines bind at this site with affinities that parallel their biologic effectiveness as beta-adrenergic agonist. Beta-Blockers interact at this site with affinities that parallel their potencies as specific inhibitors of biologic function. (4) the adenylate cyclase complex contains a receptor site for guanyl nucleotides that is distinct from the catalytic site for ATP. Interaction at this site can be determined by measuring binding of radioactive guanylylimidodiphosphate (GMPPNP). GMPPNP markedly enhances the adenylate cyclase response to beta-adrenergic catecholamines with a 10-fold increase in apparent affinity of isoproterenol for activation of adenylate cyclase. Concomitantly there is a shift in Km and Vmax for ATP at the catalytic site. (5) Activation of adenylate cyclase by agonist or GMPPNP alone is reversible (GMPPNP remains bound even though the system returns to basal activity). The addition of isoproterenol plus GMPPNP, however, results in a state of enzyme activity which is apparently irreversible even though, under these conditions, the beta-adrenergic catecholamine freely dissociates from its receptor site. Under these conditions also the activated enzyme is not inhibited by the specific beta-blocker propranolol. (6) There is no evidence under adenylate cyclase assay conditions for a direct interaction of guanyl nucleotides with the beta-adrenergic receptor site nor for direct interaction of the agonist with the guanyl nucleotide site.

Animals↗