Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Proteolytic removal and genetic deletion of the amino-terminal domain of G protein alpha subunit have shown that this region is necessary for interaction with beta gamma subunits. In the alpha subunits which undergo myristoylation, myristoylation of the amino-terminal glycine modulates the affinity of alpha subunit for the beta gamma complex. To determine the role of the same glycine in nonmyristoylated alpha subunits, we substituted it for alanine in Gs alpha and characterized the properties of the mutated chain G2A Gs alpha. The mutant could still bind guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) as revealed by its resistance to trypsin proteolysis and was able to interact with the membrane. However, G2A Gs alpha was a poor substrate for cholera toxin-catalyzed ADP-ribosylation either in the soluble form or when membrane-associated. Addition of beta gamma subunits increased the sedimentation rate of G2A Gs alpha in sucrose gradients. Binding experiments performed on cyc- membranes reconstituted by G2A Gs alpha showed that the GTP-induced shift of isoproterenol affinity for the beta-adrenergic receptors was reduced. On the same membranes, isoproterenol, GTP gamma S and NaF were 2-fold less effective for activating adenylylcyclase when compared to cyc- membranes reconstituted by Gs alpha. This differential stimulation of adenylylcyclase was not due to an affinity change for the effector but to a decrease in the maximal activation. Thus the G2A substitution affected beta gamma-dependent properties on reconstituted membranes such as receptor coupling and cholera toxin-catalyzed ADP-ribosylation and we propose that the decreased activation of adenylylcyclase might result from the same defect. Although not essential for association with beta gamma subunits, the amino-terminal glycine of nonmyristoylated Gs alpha might play a modulatory role in this interaction.
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PMID:Mutagenesis of the amino-terminal glycine to alanine in Gs alpha subunit alters beta gamma-dependent properties and decreases adenylylcyclase activation. 838 Jan 63

The addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly enhanced cAMP formation induced by carbacyclin, a stable prostacyclin analogue, in cultured mast cells (IC2 cells), but did not enhance basal or NaF plus AlCl3-induced cAMP formation. On the other hand, W-7, a calmodulin (CaM) inhibitor, almost completely suppressed the enhancing activity of TPA, suggesting the involvement of CaM in the enhancement by TPA of carbacyclin-induced cAMP formation. The enhancing activity of TPA disappeared in TPA-treated cells permeabilized with saponin in the presence of Ca2+, but reconstitution with CaM in the permeable cells resulted in remarkable restoration of the action of TPA. On the other hand, TPA treatment induced the phosphorylation and translocation of myristoylated alanine-rich C kinase substrate (MARCKS) from the membrane to the cytosol. Exogenously added protein kinase C (PKC) also phosphorylated MARCKS and induced its translocation in the cells permeabilized with saponin. Whereas the addition of CaM did not enhance the carbacyclin-stimulated GTPase activity and adenylate cyclase activity in the control permeable cells, in which MARCKS bound to the membrane, CaM markedly enhanced those activities in the PKC-treated permeable cells, which lost endogenous membrane-bound MARCKS. When MARCKS was added to the PKC-treated permeable cells, MARCKS bound to the membrane and inhibited the effects of CaM. These results suggest that activation of PKC enhances the prostacyclin-activated adenylate cyclase through a CaM/MARCKS system.
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PMID:Enhancement by protein kinase C of prostacyclin receptor-mediated activation of adenylate cyclase through a calmodulin/myristoylated alanine-rich C kinase substrate (MARCKS) system in IC2 mast cells. 838 May 84

Cyclic AMP-dependent protein kinase II beta (PKAII beta) is the principal mediator of cAMP action in neurons. A Kinase Anchor Proteins (AKAPs) are enriched in forebrain neurons and have distinct high affinity binding domains for the regulatory subunit (RII beta) of PKAII beta and components of the dendritic cytoskeleton. The selective accumulation of AKAP.RII beta complexes near dendritic microtubules tethers PKAII beta in proximity with adenylate cyclase in the synaptic plasma membrane and cytoskeletal proteins that are substrates for the kinase, thereby creating intraneuronal target sites for signals carried by cAMP. We have characterized the targeting (anchoring) and tethering (RII beta binding) domains of a prototypic anchor protein AKAP75. Deletion of N-terminal residues 27-48 generated a truncated RII beta-binding protein that partitions equally between the cytosol and detergent-insoluble fractions of HEK293 cells. Further removal of a non-adjacent sequence (residues 77-91) produced a cytosolic protein with unimpaired RII beta binding activity. Thus, two noncontiguous domains mediate the intracellular localization of AKAP75. Boundaries for the RII beta tethering domain were mapped to residues 392-413 by scanning mutagenesis. Residues containing long aliphatic side chains are essential for the high affinity binding of RII beta by AKAP75. Contributions of hydrophobic amino acids to tethering activity also depend on the position of the residue in the sequence. Certain conservative mutations that should not alter significantly the overall hydrophobicity or helicity of the tethering region (e.g. replacement of Leu with Ala) diminish the RII beta binding activity of AKAP75.
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PMID:Characterization of distinct tethering and intracellular targeting domains in AKAP75, a protein that links cAMP-dependent protein kinase II beta to the cytoskeleton. 850 14

Despite their beneficial effects on cardiovascular derangements in patients with severe sepsis, high doses of sympathomimetics might contribute to an impaired neutrophil function. This study was conducted to examine whether various sympathomimetics [(-)-epinephrine (EPI), dopamine (DA) and dobutamine (DOB)] differ in their potency to suppress the formation of oxygen radicals by neutrophils and whether this potency correlates with their affinity to or intrinsic activity for beta-2 adrenoceptors (beta-2 AR). Oxygen radical production of human neutrophils was induced by N-formyl-methionyl-leucyl-phenyl-alanine and detected by chemiluminescence measurements. Dose-response curves for the inhibition of chemiluminescence by sympathomimetics were measured in the absence and presence of 0.1 microM CGP 20,712 A (1-[2(3-carbamoyl-4-hydroxy phenoxy)-ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl) phenoxy]-2-propanol methanesulfonate) and 0.1 microM ICI 118,551 (erythro-(+/-)-1-(7-methylindan-4-yloxy)-3 isopropylaminobutan-2-ol hydrochloride) to selectively antagonize beta-1 AR and beta-2 AR, respectively. Inhibition of chemiluminescence of neutrophils by EPI was approximately 100-fold more potent than that by DA and DOB. Only the inhibition curve by EPI exhibited two components, one at nanomolar and one at micromolar concentrations. The nanomolar component was sensitive against beta-2 AR blockade, whereas the micromolar one was insensitive against both beta AR antagonists. Dose-response curves for DA and DOB exhibited a simple hyperbolic shape at micromolar concentrations and were insensitive against both beta AR antagonists. Maximum inhibition by DA and DOB was equipotent to that by EPI. However, the EC50 for DA was much lower than its dissociation constants, KD, assayed in membrane preparations by radioligand binding, whereas the EC50 of DOB matched KD. This difference could not be explained by a different efficiency of signal transduction, which was determined in receptor-coupled adenylate cyclase activity and which only showed a slightly higher efficiency of DA (51%) than of DOB (34%). Therefore, sympathomimetics were also investigated in a cell-free system, in which chemiluminescence was generated by horseradish peroxidase with hydrogen peroxide as substrate. Surprisingly, all of the sympathomimetics suppressed chemiluminescence with micromolar concentrations. We conclude that sympathomimetics with high affinity and high intrinsic activity (EPI) inhibit neutrophil function via occupation of beta-2 AR, whereas sympathomimetics with low affinity (DA) or low intrinsic activity (DOB) may act by direct scavenging of oxygen radicals.
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PMID:Is inhibition of oxygen radical production of neutrophils by sympathomimetics mediated via beta-2 adrenoceptors? 881 92

The genes for either the TSH receptor (TSH-R) or the stimulatory guanine nucleotide-binding protein subunit (Gs alpha) can undergo somatic mutations in thyroid cells, leading to constitutive activation of adenylyl cyclase and the formation of clonal hyperfunctioning thyroid adenomas. Autonomously functioning thyroid adenomas are thought not to be common precursors of thyroid cancer. If this is the case, mutations of the TSH-R or Gs alpha would not be expected to be highly prevalent in thyroid carcinomas. In this paper we report the results of a screen for structural defects in exon 10 of the TSH-R (which includes the whole serpentine structure, but not the extracellular domain) and of Gs alpha in 30 thyroid carcinomas. Five of these were from patients with functioning metastasis, as we hypothesized that if mutations of these genes were to play a role in the progression to malignancy, they would be more likely to manifest in thyroid cancers that retain unusual differentiated function (i.e. capable of synthesizing enough thyroid hormone to render patients euthyroid or hyperthyroid after total thyroidectomy). None of the 30 tumors had activating point mutations of Gs alpha. Only 2 of 30 had somatic mutations of the TSH-R (codon 632: ACC to GCC, Thr to Ala; and ACC to ATC, Thr to Ile, respectively), the latter in a patient with a thyroid hormone-producting follicular carcinoma. These results suggest that events leading to constitutive activation of the adenylate cyclase signal transduction cascade are not a frequent event in the progression toward differentiated thyroid carcinomas.
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PMID:Structural studies of the thyrotropin receptor and Gs alpha in human thyroid cancers: low prevalence of mutations predicts infrequent involvement in malignant transformation. 892 35

We observed four families with loss of function mutations of the TSH receptor gene. One patient had a homozygous Pro162 Ala substitution. The three other were compound heterozygotes: 1) Gln324-->Stop and Asp410 Asn2), Cys41 Ser and Phe525 Leu, 3) Cys390 Trp and Trp546-->Stop. In all patients, the plasma TSH concentration was increased, whereas T3 and T4 concentrations were normal. The TSH levels were normal in the heterozygous parents. These results confirmed the recessive character of TSH receptor defects. Expression of the various mutated receptors in transfected COS-7 cells demonstrated the impairment of their function. We studied the expression of the receptors on the cell surface by immunofluorescence, their ability to bind hormone, and their capacity to activate adenylate cyclase. Some mutations allowed us to identify sites that are especially important for receptor function. The substitution Cys390 Trp abolished high affinity hormone binding. Receptor mutated at Asp410 Asn bound the hormone normally, but failed to activate adenylate cyclase. This result underscores the role of this acidic extracellular residue, close to the first transmembrane segment, in signal transmission. The Phe525 Leu substitution also markedly impaired adenylate cyclase activation, underlining the importance of the second intracellular loop in receptor signaling.
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PMID:Four families with loss of function mutations of the thyrotropin receptor. 895 20

Structural determinants within the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor that mediate G-protein activation of adenylate cyclase and phospholipase C are unknown. We investigated the role of the N-terminal region of the third intracellular loop of the opossum PTH/PTHrP receptor in coupling to two signal transduction pathways. We mutated residues in this region by tandem-alanine scanning and expressed these mutant receptors in COS-7 cells and/or Xenopus oocytes. All mutant receptors retained high affinity PTH binding in COS-7 cells, indistinguishable from wild-type receptors. Receptors with tandem-alanine substitutions in two N-terminal segments (377RVL379 and 381TKLR384) demonstrated impaired adenylate cyclase and phospholipase C activation. Receptor mutants with single-alanine substitutions scanning these two segments showed three different signaling defects in COS-7 cells. 1) Two mutant receptors (V378A and L379A) had reduced inositol phosphate (IP), but normal cAMP responses to PTH. 2) Mutant receptor T381A showed reduced cAMP, but wild-type IP responses to PTH. 3) Mutant receptor K382A demonstrated both markedly reduced cAMP and IP production due to PTH. In oocytes, mutants T381A and K382A showed decreased PTH-stimulated cAMP accumulation and intracellular Ca2+ mobilization. Thus, the N-terminal region of the third intracellular loop of this receptor plays a critical role in coupling to both Gs- and Gq-mediated second-messenger generation.
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PMID:The N-terminal region of the third intracellular loop of the parathyroid hormone (PTH)/PTH-related peptide receptor is critical for coupling to cAMP and inositol phosphate/Ca2+ signal transduction pathways. 896 99

There is evidence that the conserved glutamine at residue 54 in the beta-subunit of human LH and and CG (hCG) is important for biological activity. Mutation to Arg in LH has been reported to impair receptor binding, leading to a documented case of hypogonadism, whereas in hCG the mutation has been shown to result in defective subunit association. Functional distinctions between LH and hCG have been described, but the significance of peptide-chain differences between the two has not been investigated systematically. We therefore compared the role of Gln-54 and its neighboring residues in both hormones, through replacement by amino acids with contrasting properties using site-directed mutagenesis. The mutant subunits were coexpressed with alpha-subunit in mammalian (Chinese hamster ovary) cells and the secreted hormones assayed for heterodimer formation, receptor binding, and steroidogenesis in murine Leydig cell tumor (MA-10) cells. Basic (Arg, Lys) substitution for Gln-54 in either hormone markedly impaired subunit association (<20% of wild-type) and the heterodimers that were formed were inactive (<5% of wild-type) in both assays. Arg-substituted hCG was also inactive in an adenylate cyclase assay using HEK-293 cells expressing rat LH/hCG receptor. After acidic (Glu) or neutral (Ala) substitution, heterodimer formation was less impaired (50-60% of wild-type), but effects on receptor interaction differed between the two hormones. The LH mutants still lacked binding activity, whereas the hCG products were fully active. The importance of residue 54 for receptor interaction appears to be sharply localized because mutation at adjacent positions (Pro-53 and Val-55) did not impair the activity of either hormone. Diminished heterodimer formation by Ile-53 mutation in LH (but not hCG), together with the similar effects of basic mutations at 54, imply long-distance effects as these residues are remote from alpha in the crystal structure. Our findings indicate that position 54 in LH and hCG is a determinant for both subunit association and receptor interaction. The differing responses between LH and hCG to certain mutations suggest that structural characteristics of the peptide chains may confer functional differences despite their close sequence homology.
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PMID:A functional determinant in human luteinizing hormone and chorionic gonadotropin: differential effect of mutations about beta-GLN-54. 907 24

The binding to [125I]PACAP27 and adenylate cyclase activity have been investigated using rat brain membranes with substituted analogues of PACAP and VIP, including their hybrid peptides. Binding of [125I]PACAP27 was rapid, specific and reversible. Scatchard analysis revealed a single class of binding site, with a Kd = 457 +/- 117 pM, and a Bmax = 2.63 +/- 0.24 pmol.mg protein-1. Hybrids of PACAP, in which specific residues were substituted with the corresponding residues of VIP, and vice versa, as well as related analogues, were then tested for binding and adenylate cyclase activity. The results showed that N-terminal residues were important for recognition. In particular, multiple substituted analogues of PACAP by VIP, and vice versa, demonstrated that positions 4, 5 and 9 play a dominant role in the recognition of PACAP Type I receptor in rat brain membranes and account for the differences observed between PACAP and VIP. Substitutions in the C-terminal region at positions 24, 25 and 26 are not crucial for recognition specificity. PACAP-analogues provide evidence that positions 1 and 6 are essential for receptor recognition. The flexibility at position 21 also appears to play a role as substitution with Ala or Phe is tolerated, while Pro shows a significant loss both in binding affinity and adenylate cyclase activity.
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PMID:Recognition of pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide (PACAP/VIP) hybrids and related peptides by rat brain membranes. 934 25

The structure of human parathyroid hormone (PTH) related protein (residues 1-34) containing an Ala substituted for an Ile in position 15 was studied by two-dimensional proton nuclear magnetic resonance spectroscopy. This mutant retains quite high levels of adenylate cyclase activity based on slightly reduced PTH receptor binding capacity. Three segments of helix were revealed extending from His5 to Lys11, Lys13 to Arg19, and from Phe22 to Thr33/Ala34, with a decided kink between the first two helices around Gly12. N- and C-terminal helices were stabilized by charged and hydrophobic side chain interactions between His5 and Glu30, Asp17 and both His9 and His25, and between Leu8 and Ala29, resulting in a globular molecule occupying a single conformation. While the structure of the entire mid-molecule region differed greatly from the structure of the native peptide, the structure of both N- and C-terminal regions remains essentially unaltered. The residues responsible for initiating signal transduction in the mutant are located in the vicinity of the residues responsible for receptor binding. The C-terminal amphipathic helix forming the receptor binding site exhibits reduced binding as a result of the closely applied N-terminal signal transduction-activating region. Although not contributing directly to receptor binding, the N-terminal region can sterically affect hormone binding through modifications to certain N-terminal side chains.
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PMID:Solution structure of parathyroid hormone related protein (residues 1-34) containing an Ala substituted for an Ile in position 15 (PTHrP[Ala15]-(1-34)). 936 20


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