Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.6.1.1 (adenylate cyclase)
 19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The relationship between receptor number and agonist-induced intracellular responses has been well studied in receptors coupled to adenylate cyclase; however, for receptors coupled to phospholipase C (PLC), very little is known about the effect of receptor number on receptor-mediated processes. To explore this issue, we investigated the effect of the number of receptors for gastrin-releasing peptide (GRP) on ligand affinity and on the ability to activate intracellular messengers [PLC, tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK)] and cause receptor modulation (internalization, desensitization, down-regulation) and ligand degradation. Three BALB 3T3 cell lines were made that stably expressed the gastrin-releasing peptide receptor (GRP-R) with receptor numbers varying by 280-fold (GRP-R-Low, GRP-R-Med, and GRP-R-Hi). Each cell line had the same affinity for agonist. The efficacy for bombesin to increase [3H]inositol phosphates but not tyrosine phosphorylation of p125FAK correlated well with receptor number. In contrast, the EC50 value for [3H]inositol phosphate generation for bombesin was the same in each cell line. Receptor number did not alter internalization. In the absence of protease inhibitors, there was an inverse correlation between receptor number and receptor down-regulation and desensitization. However, with protease inhibitors present, GRP-R-Med and GRP-R-Hi down-regulated significantly less than the GRP-R-Low. Similarly, GRP-R-Low desensitized significantly more than GRP-R-Med or GRP-R-Hi. GRP-R-Hi caused significantly greater ligand degradation than GRP-R-Low, and protease inhibitors completely inhibited degradation by GRP-R-Low and inhibited degradation by 70% for GRP-R-Hi. In conclusion, we show that for the PLC-coupled GRP-R, receptor number had little or no effect on binding affinity, potency for activating PLC, tyrosine phosphorylation of p125FAK, or extent of receptor internalization. In contrast, receptor number had an effect on ligand degradation, down-regulation, desensitization, and efficacy of PLC activation without altering the efficacy of tyrosine phosphorylation of p125FAK. These results demonstrate that the effect of receptor number differs for the different functions mediated by the GRP receptor and differs from that reported for adenylate cyclase-coupled receptors such as receptors mediating the action of adrenergic agents, secretin, and opioids.
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PMID:Effect of gastrin-releasing peptide receptor number on receptor affinity, coupling, degradation, and modulation. 914 10

Myosin light chain (MLC) phosphorylation catalyzed by the Ca(2+)- calmodulin-dependent MLC kinase (MLCK) is critical to thrombin-mediated endothelial cell gap formation and barrier dysfunction. We have tested the hypothesis that the Ca2+ ionophore ionomycin stimulates MLCK-dependent endothelial cell contraction and permeability. Ionomycin significantly increased albumin clearance and decreased electrical resistance across confluent bovine pulmonary microvascular and macrovascular endothelial cell monolayers in a concentration-dependent manner that was temporally similar to that produced by thrombin. In contrast, however, ionomycin produced a significant Ca(2+)-dependent reduction in the levels of phosphorylated MLC with evidence of serine/threonine phosphatase activation. Potential MLCK-independent mechanisms of endothelial cell permeability were examined with little evidence to support a role for stimulated nitric oxide synthase or phospholipase A2 activities. Importantly, ionomycin produced 1) reductions in the activities of the barrier protective adenylate cyclase and the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A, 2) dramatic dose- and time-dependent inhibition of endothelial cell tyrosine kinase activities, and 3) marked decreases in the phosphotyrosine content of the p125 focal adhesion kinase. These data indicate that ionomycin produces endothelial cell barrier dysfunction by mechanisms that are independent of MLCK activation and may involve reductions in endothelial cell tethering forces via inhibition of protein kinase A and tyrosine kinase activities, especially the p125 focal adhesion kinase.
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PMID:Mechanisms of ionomycin-induced endothelial cell barrier dysfunction. 925 54

Bombesin (Bn) receptor subtype 3 (BRS-3) is an orphan receptor that is a predicted member of the heptahelical G-protein receptor family and so named because it shares a 50% amino acid homology with receptors for the mammalian bombesin-like peptides neuromedin B (NMB) and gastrin-releasing peptide. In a recent targeted disruption study, in which BRS-3-deficient mice were generated, the mice developed obesity, diabetes, and hypertension. To date, BRS-3's natural ligand remains unknown, its pharmacology unclear, and cellular basis of action undetermined. Furthermore, there are few tissues or cell lines found that express sufficient levels of BRS-3 protein for study. To define the intracellular signaling properties of BRS-3, we examined the ability of [D-Phe6,beta-Ala11,Phe13, Nle14]Bn-(6-14), a newly discovered peptide with high affinity for BRS-3, and various Bn receptor agonists and antagonists to alter cellular function in hBRS-3-transfected BALB 3T3 cells and hBRS-3-transfected NCI-H1299 non-small cell lung cancer cells, which natively express very low levels of hBRS-3. This ligand stimulated a 4-9-fold increase in [3H]inositol phosphate formation in both cell lines under conditions where it caused no stimulation in untransfected cells and also stimulated an increase in [3H]IP1, [3H]IP2, and 3H]IP3. The elevation of [3H]IP was concentration-dependent, with an EC50 of 20-35 nM in both cell lines. [D-Phe6,beta-Ala11,Phe13,Nle14]Bn-(6-14) stimulated a 2-3-fold increase in [Ca2+]i, a 3-fold increase in tyrosine phosphorylation of p125(FAK) with an EC50 of 0.2-0.7 nM, but failed to either stimulate increases in cyclic AMP or inhibit forskolin-stimulated increases. None of nine naturally occurring Bn peptides or three synthetic Bn analogues reported to activate hBRS-3 did so with high affinity. No high affinity Bn receptor antagonists had high affinity for the hBRS-3 receptor, although two low affinity antagonists for gastrin-releasing peptide and NMB receptors, [D-Arg1,D-Trp7,9, Leu11]substance P and [D-Pro4,D-Trp7,9,10]substance P-(4-11), inhibited hBRS-3 receptor activation. The NMB receptor-specific antagonist D-Nal,Cys,Tyr,D-Trp,Lys,Val, Cys,Nal-NH2 inhibited hBRS-3 receptor activation in a competitive fashion (Ki = 0.5 microM). Stimulation of p125(FAK) tyrosine phosphorylation by hBRS-3 activation was not inhibited by the protein kinase C inhibitor, GF109203X, or thapsigargin, alone or in combination. These results show that hBRS-3 receptor activation increases phospholipase C activity, which causes generation of inositol phosphates and changes in [Ca2+]i and is also coupled to tyrosine kinase activation, but is not coupled to adenylate cyclase activation or inhibition. hBRS-3 receptor activation results in tyrosine phosphorylation of p125(FAK), and it is not dependent on activation of either limb of the phospholipase C cascade. Although the natural ligand is not a known bombesin-related peptide, the availability of [D-Phe6,beta-Ala11, Phe13,Nle14]Bn-(6-14), which functions as a high affinity agonist in conjunction with hBRS-3-transfected cell lines and the recognition of three classes of receptor antagonists including one with affinity of 0.5 microM, should provide important tools to assist in the identification of its natural ligand, the development of more potent selective receptor antagonists and agonists, and further exploration of the signaling properties of the hBRS-3 receptor.
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PMID:Ability of various bombesin receptor agonists and antagonists to alter intracellular signaling of the human orphan receptor BRS-3. 959 99