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)

The immunoregulatory C-C chemokine, macrophage inflammatory protein-1 alpha (MIP-1 alpha) has suppressive activity on proliferation of stem cells and early subsets of myeloid progenitor cells. A receptor for C-C chemokines that binds MIP-1 alpha has been characterized, cloned, and shown to be related structurally to neuropeptide receptors that couple through G-proteins to phospholipase-C and adenyl cyclase. Yet, very little information on the intracellular mechanisms of action of MIP-1 alpha is available. We show here that the human factor-dependent cell line M07e is responsive to the cell cycle-suppressive effects of MIP-1 alpha, has specific membrane-binding sites for MIP-1 alpha, and that treatment of these cells with this chemokine increases the phosphatidylcholine (PC) and phosphocholine turnover rates in cells that are synergistically stimulated by the combination of granulocyte-macrophage colony-stimulating factor and steel factor but not these factors acting singly. Additional, MIP-1 alpha treatment induces a dose- and time-dependent increase in intracellular cAMP levels in M07e cells. Both exogenous PC and dibutyryl cAMP were found to suppress the proliferation of M07e colony-forming cells to a level similar to that of MIP-1 alpha, further implicating cAMP and PC metabolism in MIP-1 alpha-induced M07e suppression. RANTES, a related chemokine, with weak or incomplete binding to the cloned MIP-1 alpha receptor, did not suppress M07e colony-forming cells, nor did it increase intracellular cAMP levels, but it did enhance growth factor-induced PC turnover, further supporting the involvement of cAMP in MIP-1 alpha suppression while demonstrating that increased PC turnover alone is not sufficient for suppression. These findings support the idea that the human MIP-1 alpha receptor is coupled to phospholipid and cAMP metabolism in a manner similar to other 7-transmembrane, G-protein-linked receptors and suggest that a phosphatidylcholine hydrolytic cycle and an associated increase in cAMP are part of the mechanisms of action of MIP-1 alpha.
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PMID:Macrophage inflammatory protein-1 alpha enhances growth factor-stimulated phosphatidylcholine metabolism and increases cAMP levels in the human growth factor-dependent cell line M07e, events associated with growth suppression. 753 66

To address the role of dimerization in the function of the monocyte chemoattractant protein-1, MCP-1, we mutated residues that comprise the core of the dimerization interface and characterized the ability of these mutants to dimerize and to bind and activate the MCP-1 receptor, CCR2b. One mutant, P8A*, does not dimerize. However, it has wild type binding affinity, stimulates chemotaxis, inhibits adenylate cyclase, and stimulates calcium influx with wild type potency and efficacy. These data suggest that MCP-1 binds and activates its receptor as a monomer. In contrast, Y13A*, another monomeric mutant, has a 100-fold weaker binding affinity, is a much less potent inhibitor of adenylate cyclase and stimulator of calcium influx, and is unable to stimulate chemotaxis. Thus Tyr13 may make important contacts with the receptor that are required for high affinity binding and signal transduction. We also explored whether a mutant, [1+9-76]MCP-1 (MCP-1 lacking residues 2-8), antagonizes wild type MCP-1 by competitive inhibition, or by a dominant negative mechanism wherein heterodimers of MCP-1 and [1+9-76]MCP-1 bind to the receptor but are signaling incompetent. Consistent with the finding that MCP-1 can bind and activate the receptor as a monomer, we demonstrate that binding of MCP-1 in the presence of [1+9-76]MCP-1 over a range of concentrations of both ligands fits well to a simple model in which monomeric [1+9-76]MCP-1 functions as a competitive inhibitor of monomeric MCP-1. These results are crucial for elucidating the molecular details of receptor binding and activation, for interpreting mutagenesis data, for understanding how antagonistic chemokine variants function, and for the design of receptor antagonists.
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PMID:Monomeric monocyte chemoattractant protein-1 (MCP-1) binds and activates the MCP-1 receptor CCR2B. 983 83

CCR-3 is a major receptor involved in regulating eosinophil trafficking. Initial analysis of chemokine receptors has demonstrated unique receptor events in different cell types, indicating the importance of investigating CCR-3 events in eosinophilic cell lines. We now report that the eosinophilic cell line, acute myelogenous leukemia (AML) 14.3D10, expresses eosinophil granule proteins and eotaxin, but has no detectable expression of eosinophil chemokine receptors. Treatment of the cell line with butyric acid and IL-5 results in a dose-dependent synergistic induction of CCR-3 and, to a lesser extent, CCR-1 and CCR-5. Interestingly, using a luciferase reporter construct under the control of the hCCR-3 promoter, the uninduced and induced cells display high, but comparable, levels of promoter activity. Differentiated AML cells developed enhanced functional activation, as indicated by adhesion to respiratory epithelial cells and chemokine-induced transepithelial migration. Chemokine signaling did not inhibit adenylate cyclase activity even though calcium transients were blocked by pertussis toxin. Additionally, chemokine-induced calcium transients were inhibited by pretreatment with PMA, but not forskolin. Eotaxin treatment of differentiated AML cells resulted in marked down-modulation of CCR-3 expression for at least 18 h. Receptor internalization was not dependent upon chronic ligand exposure and was not accompanied by receptor degradation. Thus, CCR-3 is a late differentiation marker on AML cells and uses a signal transduction pathway involving rapid and prolonged receptor internalization, calcium transients inhibitable by protein kinase C but not protein kinase A, and the paradoxical lack of inhibition of adenylate cyclase activity.
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PMID:Molecular analysis of CCR-3 events in eosinophilic cells. 1062 56

Monocyte chemoattracant-1 (MCP-1) stimulates leukocyte chemotaxis to inflammatory sites, such as rheumatoid arthritis, atherosclerosis, and asthma, by use of the MCP-1 receptor, CCR2, a member of the G-protein-coupled seven-transmembrane receptor superfamily. These studies identified a family of antagonists, spiropiperidines. One of the more potent compounds blocks MCP-1 binding to CCR2 with a K(d) of 60 nm, but it is unable to block binding to CXCR1, CCR1, or CCR3. These compounds were effective inhibitors of chemotaxis toward MCP-1 but were very poor inhibitors of CCR1-mediated chemotaxis. The compounds are effective blockers of MCP-1-driven inhibition of adenylate cyclase and MCP-1- and MCP-3-driven cytosolic calcium influx; the compounds are not agonists for these pathways. We showed that glutamate 291 (Glu(291)) of CCR2 is a critical residue for high affinity binding and that this residue contributes little to MCP-1 binding to CCR2. The basic nitrogen present in the spiropiperidine compounds may be the interaction partner for Glu(291), because the basicity of this nitrogen was essential for affinity; furthermore, a different class of antagonists, a class that does not have a basic nitrogen (2-carboxypyrroles), were not affected by mutations of Glu(291). In addition to the CCR2 receptor, spiropiperidine compounds have affinity for several biogenic amine receptors. Receptor models indicate that the acidic residue, Glu(291), from transmembrane-7 of CCR2 is in a position similar to the acidic residue contributed from transmembrane-3 of biogenic amine receptors, which may account for the shared affinity of spiropiperidines for these two receptor classes. The models suggest that the acid-base pair, Glu(291) to piperidine nitrogen, anchors the spiropiperidine compound within the transmembrane ovoid bundle. This binding site may overlap with the space required by MCP-1 during binding and signaling; thus the small molecule ligands act as antagonists. An acidic residue in transmembrane region 7 is found in most chemokine receptors and is rare in other serpentine receptors. The model of the binding site may suggest ways to make new small molecule chemokine receptor antagonists, and it may rationalize the design of more potent and selective antagonists.
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PMID:Identification of the binding site for a novel class of CCR2b chemokine receptor antagonists: binding to a common chemokine receptor motif within the helical bundle. 1077 Sep 25

Monocyte chemotactic protein-1 (MCP-1) binding to its receptor, CCR2B, plays an important role in a variety of diseases involving infection, inflammation, and/or injury. In our effort to understand the molecular basis of this interaction and its biological consequences, we recognized a conserved hexad of amino acids at the N-terminal extracellular domain of several chemokine receptors, including CCR2B. Human embryonic kidney 293 cells expressing Flag-tagged CCR2B containing site-directed mutations in this region, 21-26, including a consensus tyrosine sulfation site were used to determine MCP-1 binding and its biological consequences. The results showed that several of these amino acids are important for MCP-1 binding and consequent lamellipodium formation, chemotaxis, and signal transduction involving adenylate cyclase inhibition and Ca(2+) influx into cytoplasm. Mutations that prevented adenylate cyclase inhibition and Ca(2+) influx did not significantly inhibit lamellipodium formation and chemotaxis, suggesting that these signaling events are not involved in chemotaxis. CCR2B was found to be sulfated at Tyr(26); this sulfation was abolished by the substitution of Tyr with Ala and severely reduced by substitution of Asp(25), a part of the consensus sulfation site. The expressed CCR2B was found to be N:-glycosylated, as N:-glycosidase F treatment of the receptor or growth of the cells in tunicamycin reduced the receptor size to the same level, from 50 to 45 kDa. Thus, CCR2B is the first member of the CC chemokine receptor family shown to be a glycoprotein that is sulfated at the N-terminal Tyr. These post-translational modifications probably have significant biological functions.
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PMID:Monocyte chemotactic protein-1 receptor CCR2B is a glycoprotein that has tyrosine sulfation in a conserved extracellular N-terminal region. 1104 64

SPC3 is a multibranched peptide containing eight identical GPGRAF motifs which are derived from the human immunodeficiency virus (HIV)-1 gp120 V3 loop consensus sequence. This molecule was reported to prevent the infection of CD4+ cells by various HIV-1 and HIV-2 strains. However, the molecular mode of action of SPC3 remains unclear. Here, we investigated the possibility that SPC3 could interact with alpha/beta-chemokine receptors following observations that, first, the V3 loop is likely to be involved in alpha/beta-chemokine receptor-dependent HIV entry and, second, natural ligands of these receptors are potent inhibitors of cell infection. To address this point, we examined the effects of SPC3 on Xenopus oocytes either uninjected or expressing exogenous human CXCR4 alpha-chemokine receptors. Extracellular applications of micromolar concentrations of SPC3 onto Xenopus oocytes trigger potent inward chloride currents which can be inhibited by increasing extracellular Ca2+ concentration. This effect can be blocked by chloride channel antagonists and is highly specific to SPC3 as it is not triggered by structural analogs of SPC3. The SPC3-induced chloride conductance in oocytes is alpha/beta-chemokine receptor dependent because: (i) SPC3 alters the sensitivity of this channel to external applications of human recombinant MIP-1alpha, a natural ligand of human CCR5 receptor, and (ii) the amplitude of the inward current could be increased by the expression of exogenous human CXCR4 chemokine receptor. The effect of SPC3 appears to rely on the activation of a phospholipase A2 signaling pathway, but is not affected by changes in cytosolic Ca2+ concentration, or by alterations in Gi/Go protein, adenylate cyclase, phospholipase C or protein kinase C activity. Altogether, the data indicate that SPC3 is capable of activating a surface alpha/beta-chemokine-like receptor-mediated signaling pathway in competent cells, thereby triggering, either directly or indirectly, a Ca2+-inactivated chloride conductance.
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PMID:Ion channel activation by SPC3, a peptide derived from the HIV-1 gp120 V3 loop. 1115 2

Extracellular superoxide dismutase (EC-SOD) is synthesized in mesenchymally derived cells and prevents the oxygen radical-induced injury. We studied whether kidney mesangial cells (MCs) produce EC-SOD and how its production is associated with chemokine secretion. Under unstimulated condition, MCs produced EC-SOD, and its production was correlated positively with cyclic adenosine monophosphate (cAMP), but negatively with interleukin (IL)-6 or IL-8 production. By prednisolone or phorbol myristate acetate treatment, EC-SOD levels were correlated negatively with levels of IL-6 and IL-8. The presence of adenylate cyclase inhibitor 2',3'-dideoxyadenosine lost the prednisolone effect. The stimulation of EC-SOD production might be one of the important effects of prednisolone via cAMP pathway in MCs.
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PMID:Extracellular superoxide dismutase and glomerular mesangial cells: its production and regulation. 1202 21

Monocyte chemotactic protein-1 (MCP-1) binds its G-protein-coupled seven transmembrane (TM) receptor, CCR2B, and causes infiltration of monocytes/macrophages into areas of injury, infection or inflammation. To identify functionally important amino acid residues in CCR2B, we made specific mutations of nine residues selected on the basis of conservation in chemokine receptors and located TM1 (Tyr(49)), TM2 (Leu(95)), TM3 (Thr(117) and Tyr(120)), and TM7 (Ala(286), Thr(290), Glu(291), and His(297)) and in the extracellular loop 3 (Glu(278)). MCP-1 binding was drastically affected only by mutations in TM7. Reversing the charge at Glu(291) (E291K) and at His(297) (H297D) prevented MCP binding although substitution with Ala at either site had little effect, suggesting that Glu(291) and His(297) probably stabilize TM7 by their ionic interaction. E291A elicited normal Ca(2+) influx. H297A, Y49F in TM1 and L95A in TM2 that showed normal MCP-1 binding did not elicit Ca(2+) influx and elicited no adenylate cyclase inhibition at any MCP-1 concentration. MCP-1 treatment of HEK293 cells caused lamellipodia formation only when they expressed CCR2B. The mutants that showed no Ca(2+) influx and adenylate cyclase inhibition by MCP-1 treatment showed lamellipodia formation and chemotaxis. Our results show that induction of lamellipodia formation, but not Ca(2+) influx and adenylate cyclase inhibition, is necessary for chemotaxis.
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PMID:Site-directed mutagenesis of CCR2 identified amino acid residues in transmembrane helices 1, 2, and 7 important for MCP-1 binding and biological functions. 1562 46

The chemokine receptor CCR2 binds four pro-inflammatory monocyte chemoattractant proteins, designated MCP1/CCL2, MCP2/CCL8, MCP3/CCL7 and MCP4/CCL13. This study demonstrates the important biology of this receptor during the response to the chemokine milieu. Competitive chemotaxis and calcium flux assays were performed utilising mixtures of chemokines to assess a hierarchal arrangement of chemokine prepotency; these demonstrated that the MCP2-CCR2 interaction is able to supersede signals generated by RANTES, another pro-inflammatory chemokine, or the homeostatic chemokine SDF1. These observations were validated using three physiologically relevant monocytic cell lines. Having identified the importance of CCR2, experiments were then performed to examine the signal transduction processes coupled to this receptor. G protein coupling was initially examined; Cholera toxin reduced the chemotactic response to MCP2 (p<0.001), whilst the response to the other MCP chemokines remained normal. The response to MCP2 was uniquely inhibited by elevated concentrations of cAMP and, unlike MCP1, 3 and 4 (p<0.05), MCP2 failed to inhibit adenylate cyclase. Expression of dominant negative H-ras demonstrated that each MCP chemokine required active ras in order to elicit ERK activation and a chemotactic response. Unlike MCP1, MCP2 failed to induce nuclear translocation of activated ERK1 or subsequent induction of c-Myc expression. Akt activation also showed ligand-specific differences, with MCP2 producing a delayed response compared to the other MCP chemokines. Together these data highlight the importance of CCR2 and suggest that it is a powerful tool for fine tuning the immune response.
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PMID:Chemokine-mediated inflammation: Identification of a possible regulatory role for CCR2. 1708 10

IL (interleukin)-6 exerts pro- as well as anti-inflammatory activities. Beside many other activities, IL-6 is the major inducer of acute phase proteins in the liver, acts as a differentiation factor for blood cells, as migration factor for T-cells and is a potent inducer of the chemokine MCP-1 (monocyte chemoattractant protein-1). Recent studies have focused on the negative regulation of IL-6 signal transduction through the IL-6-induced feedback inhibitors SOCS (suppressor of cytokine signalling) 1 and SOCS3 or the protein tyrosine phosphatases SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) and TcPTP (T-cell protein tyrosine phosphatase). Studies on the cross-talk between pro-inflammatory mediators (IL-1, tumour necrosis factor, lipopolysaccharide) and IL-6 elucidated further regulatory mechanisms. Less is known about the regulation of IL-6 signal transduction by hormone/cytokine signalling through G-protein-coupled receptors. This is particularly surprising since many of these hormones (such as prostaglandins and chemokines) play an important role in inflammatory processes. In the present study, we have investigated the inhibitory activity of PGE(1) (prostaglandin E(1)) on IL-6-induced MCP-1 expression and have elucidated the underlying molecular mechanism. Surprisingly, PGE(1) does not affect IL-6-induced STAT (signal transducer and activator of transcription) 3 activation, but does affect ERK (extracellular-signal-regulated kinase) 1/2 activation which is crucial for IL-6-dependent expression of MCP-1. In summary, we have discovered a specific cross-talk between the adenylate cyclase cascade and the IL-6-induced MAPK (mitogen-activated protein kinase) cascade and have investigated its impact on IL-6-dependent gene expression.
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PMID:Prostaglandin E1 inhibits IL-6-induced MCP-1 expression by interfering specifically in IL-6-dependent ERK1/2, but not STAT3, activation. 1827 57


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