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)

Octopamine receptors from American cockroach, Periplaneta americana (Pa oa1), and fruit fly, Drosophila melanogaster (OAMB), were cloned and permanently expressed in HEK-293 cells, and found to activate adenylate cyclase activity and increase [Ca2+]i levels through G-protein coupled receptor signaling pathways. Sequencing information (GenBank accession number AY333178) and functional data of Pa oa1 were recently published. Saturation binding analysis with 3H-yohimbine was performed with Pa oa(1) and OAMB expressed in COS-7 cells. The K(d) values were determined to be 28.4 and 43.0 nM, respectively. B(max) was determined to be 11.8 and 8.04 pmol receptor/mg protein, respectively. Competitive binding data using cell membranes expressing either OAMB or Pa oa1 demonstrated significantly decreased binding activity in binding assays performed in the presence of plant essential oils, eugenol, cinnamic alcohol, and trans-anethole. Eugenol decreased cAMP level in HEK-293 cells expressing Pa oa1, but trans-anethole increased cAMP in HEK-293 cells expressing OAMB. All three chemicals increased [Ca2+]i level in both cell models. Toxicity data against fruit flies and American cockroaches demonstrated species differences in response to treatment with tested plant essential oils. The toxicity of tested chemicals against wild type and octopamine mutant (iav) fly strains suggested that an octopamine receptor mediates the toxicity of cinnamic alcohol, eugenol, trans-antehole, and 2-phenethyl propionate against fruit flies. Collectively, the data suggest a correlation between cellular changes induced by tested plant essential oils and their toxicity against fruit fly and American cockroach.
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PMID:Molecular and pharmacological analysis of an octopamine receptor from American cockroach and fruit fly in response to plant essential oils. 1598 84

Various cellular signaling pathways induced by nociceptin activation of ORL1 (opioid receptor-like 1 receptor) develop homologous desensitization. Multiple lines of evidence suggest that agonist-induced phosphorylation of serine (Ser)/threonine (Thr) residues at intracellular carboxyl tail leads to homologous desensitization of G protein-coupled receptors. In the present study, we investigated the functional role played by C-terminal Ser/Thr residues in agonist-induced desensitization and phosphorylation of ORL1. In HEK 293 cells expressing wild-type ORL1 and ORL1(CDelta21), which lacks C-terminal 21 amino acids, nociceptin inhibition of adenylate cyclase activity exhibited homologous desensitization after 1 h pretreatment of nociceptin. In contrast, ORL1(CDelta34), which differs with ORL1(CDelta21) by lacking C-terminal Ser(334), Ser(335) and Ser(343) residues, failed to develop agonist-induced desensitization. Point mutant (S343A) ORL1 underwent homologous desensitization after nociceptin pretreatment. Substituting Ser(334) or Ser(335) with alanine greatly impaired nociceptin-induced ORL1 desensitization. In HEK 293 cells expressing double mutant (S334A/S335A) ORL1, nociceptin pretreatment failed to significantly affect the efficacy and potency by which nociceptin inhibits forskolin-stimulated cAMP formation. Mutation of Ser(334) and Ser(335) also greatly reduced nociceptin-induced ORL1 phosphorylation. These results suggest that two C-terminal serine residues, Ser(334) and Ser(335), are required for homologous desensitization and agonist-induced phosphorylation of ORL1.
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PMID:Two C-terminal amino acids, Ser(334) and Ser(335), are required for homologous desensitization and agonist-induced phosphorylation of opioid receptor-like 1 receptors. 1601 91

N-arachidonoylethanolamine (anandamide, AEA), is a full agonist at both cannabinoid CB(1) receptors and "transient receptor potential vanilloid" type 1 (TRPV1) channels, and N-palmitoylethanolamine (PEA) potentiates these effects. In neurons of the rat dorsal root ganglia (DRG), TRPV1 is activated and/or sensitised by AEA as well as upon activation of protein kinases C (PKC) and A (PKA). We investigated here the effect on AEA levels of PKC and PKA activators in DRG neurons. AEA levels were significantly enhanced by both phorbol-miristoyl-acetate (PMA), a typical PKC activator, and forskolin (FSK), an adenylate cyclase stimulant, as well as by thrombin, which also activates PKC by stimulating protease-activated receptors (PARs). The levels of the other endocannabinoid and TRPV1-inactive compound, 2-arachidonoylglycerol (2-AG), were enhanced only by thrombin and to a lesser extent than AEA, whereas PEA was not affected by any of the treatments. Importantly, FSK- and PMA-induced elevation of AEA levels was not sensitive to intracellular Ca2+ chelation with BAPTA-acetoxymethyl (AM) ester. In human embryonic kidney (HEK-293) cells, which constitutively express PARs, thrombin, PMA and FSK elevated AEA levels, and the effects of the two former compounds were counteracted by the PKC inhibitor, RO318220, whereas the effect of FSK was reduced by the PKA inhibitor RpcAMPs. In conclusion, we report that AEA levels are stimulated by both PKC, either directly or after thrombin receptor activation, and PKA, possibly in a way independent from intracellular calcium. Since AEA activates TRPV1, these findings may suggest the existence of an amplificatory cascades on this receptor in sensory neurons.
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PMID:Functional lipidomics. Calcium-independent activation of endocannabinoid/endovanilloid lipid signalling in sensory neurons by protein kinases C and A and thrombin. 1832 52

TRPV1 is a nociceptive, Ca2+-selective ion channel involved in the development of several painful conditions. Sensitization of TRPV1 responses by cAMP-dependent PKA crucially contributes to the development of inflammatory hyperalgesia. However, the pathways involved in potentiation of TRPV1 responses by cAMP-dependent PKA remain largely unknown. Using HEK cells stably expressing TRPV1 and the mu opioid receptor, we demonstrated that treatment with the adenylate cyclase activator forskolin significantly increased the multimeric TRPV1 species. Pretreatment with the mu opioid receptor agonist morphine reversed this increased TRPV1 multimerization. FRET analysis revealed that treatment with forskolin did not cause multimerization of pre-existing TRPV1 monomers on the plasma membrane and that intracellular pools of TRPV1 exist mostly as monomers in this model. This suggests that increased TRPV1 multimerization occurred from an intracellular store of inactive TRPV1 monomers. Treatment with forskolin also caused an increase in TRPV1 expression on the plasma membrane not resulting from increased TRPV1 expression, and this rapid TRPV1 translocation was inhibited by treatment with morphine. Thus, potentiation of TRPV1 responses by cAMP-dependent PKA involves plasma membrane insertion of functional TRPV1 multimers formed from an intracellular store of inactive TRPV1 monomers. This potentiation occurs rapidly and can be dynamically modulated by activation of the mu opioid receptor under conditions where cAMP levels are raised, such as with inflammation. Increased translocation and multimerization of TRPV1 channels provide a cellular mechanism for fine-tuning of nociceptive responses that allow for rapid modulation of TRPV1 responses independent of transcriptional changes.
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PMID:Rapid, opioid-sensitive mechanisms involved in transient receptor potential vanilloid 1 sensitization. 1848 91

G protein-coupled receptors (GPCRs) play an essential role in the regulation of cardiovascular function. Therapeutic modulation of GPCRs has proven to be beneficial in the treatment of human heart disease. Myocardial "orphan" GPCRs, for which the natural ligand is unknown, represent potential novel therapeutic targets for the treatment of heart disease. Here, we describe the expression pattern, signaling pathways, and possible physiological role of the orphan GPR22. GPR22 mRNA analysis revealed a highly restricted expression pattern, with remarkably abundant and selective expression in the brain and heart of humans and rodents. In the heart, GPR22 mRNA was determined to be expressed in all chambers and was comparable with transcript levels of the beta(1)-adrenergic receptor as assessed by Taqman PCR. GPR22 protein expression in cardiac myocytes and coronary arteries was demonstrated in the rat heart by immunohistochemistry. When transfected into HEK-293 cells, GPR22 coupled constitutively to G(i)/G(o), resulting in the inhibition of adenyl cyclase. No constitutive coupling to G(s) or G(q) was observed. Myocardial mRNA expression of GPR22 was dramatically reduced following aortic banding in mice, suggesting a possible role in response to the stress associated with increased afterload. The absence of detectable GPR22 mRNA expression in the hearts of GPR22(-/-) mice had no apparent effect on normal heart structure or function; however, these mice displayed increased susceptibility to functional decompensation following aortic banding. Thus, we described, for the first time, the expression pattern and signaling for GPR22 and identified a protective role for GPR22 in response to hemodynamic stress resulting from increased afterload.
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PMID:Myocardial expression, signaling, and function of GPR22: a protective role for an orphan G protein-coupled receptor. 1858 85

Dopamine (DA) is a physiologically important biogenic amine in insect peripheral and nervous tissues.We recently cloned two DA receptors (BmDopR1 and BmDopR2) from the silkworm Bombyx mori and identified them as D1-like receptors, which activate adenylate cyclase to increase intracellular cAMP levels. In this study, these two receptors were stably expressed in HEK-293 cells, and the dose-responsiveness to DA and their pharmacological properties were examined using cAMP assays. BmDopR1 showed a dose-dependent increase in cAMP levels at DA concentrations up to 10(-7) M with EC(50) of 3.30 nM, while BmDopR2 required 10(-6) M DA for activation. In BmDopR1-expressing cells, DA at 10(-6)-10(-4) M induced 30-50% lower cAMP production than 10(-7) MDA. BmDopR2-expressing cells showed a standard sigmoidal dose-response, with maximum cAMP levels attained with 10(-5)-10(-4) M DA and EC(50) of 1.30 microM. Both receptors had similar agonist profiles, and the typical vertebrate D1-like receptor agonist SKF-38393 was ineffective. Experiments with antagonists revealed that BmDopR1 exhibits D1-like features. However, the pharmacology of BmDopR2 was distinct from D1-like receptors; the typical vertebrate D1-like receptor antagonist SCH-23390 was less potent than the nonselective antagonist flupenthixol and the D2-like receptor antagonist chlorpromazine. The rank order of activities of several antagonists for BmDopR1 and BmDopR2 was more similar to that of Drosophila melanogaster DA receptors than Apis mellifera DA receptors. These data suggest that DA receptors could be potential targets for specific insecticides or insectistatics.
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PMID:Comparative pharmacology of two D1-like dopamine receptors cloned from the silkworm Bombyx mori. 1950 4

The Slack gene encodes a Na(+)-activated K(+) channel and is expressed in many different types of neurons. Like the prokaryotic Ca(2+)-gated K(+) channel MthK, Slack contains two 'regulator of K(+) conductance' (RCK) domains within its carboxy terminal, domains likely involved in Na(+) binding and channel gating. It also contains multiple consensus protein kinase C (PKC) and protein kinase A (PKA) phosphorylation sites and although regulated by protein kinase C (PKC) phosphorylation, modulation by PKA has not been determined. To test if PKA directly regulates Slack, nystatin-perforated patch whole-cell currents were recorded from a human embryonic kidney (HEK-293) cell line stably expressing Slack. Bath application of forskolin, an adenylate cyclase activator, caused a rapid and complete inhibition of Slack currents however, the inactive homolog of forskolin, 1,9-dideoxyforskolin caused a similar effect. In contrast, bath application of 8-bromo-cAMP did not affect the amplitude nor the activation kinetics of Slack currents. In excised inside-out patch recordings, direct application of the PKA catalytic subunit to patches did not affect the open probability of Slack channels nor was open probability affected by direct application of protein phosphatase 2B. Preincubation of cells with the protein kinase A inhibitor KT5720 also did not change current density. Finally, mutating the consensus phosphorylation site located between RCK domain 1 and domain 2 from serine to glutamate did not affect current activation kinetics. We conclude that unlike PKC, phosphorylation by PKA does not acutely modulate the function and gating activation kinetics of Slack channels.
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PMID:cAMP-dependent kinase does not modulate the Slack sodium-activated potassium channel. 1954 Feb 51

Membrane rafts and caveolae are specialized microdomains of the cell membrane that form physical platforms for compartmentalization of signalling molecules. Here, we intended to gain insight into the consequences of caveolar localization in G protein-coupled receptor function. We analysed beta(2)-adrenoceptor signalling in purified CRLDF (caveolin-rich low density fractions) of beta(2)-adrenoceptor-overexpressing HEK-293 cells. beta(2)-adrenoceptor and Gs immunoreactivities and forskolin-stimulated adenylate cyclase activity were all detected in CRLDF obtained by the conventional raft purification method that uses Triton X-100 solubilization. However, Triton X-100 caused a complete loss of the functional coupling between beta(2)-adrenoceptor, Gs and adenylate cyclase. Therefore, we developed an optimized purification method based on n-octyl-beta-d-glucopyranoside solubilization, where the functional properties of beta(2)-adrenoceptor, Gs and adenylate cyclase were preserved in the CRLDF. Using this method, we showed that isoproterenol-stimulated adenylate cyclase activity was similar in CRLDF and bulk membrane preparations of HEK-293 cells that overexpress beta(2)-adrenoceptor or beta(2)-adrenoceptor-Gs fusion. Accordingly, treatment of cells with methyl-beta-cyclodextrin, a caveola-disrupting agent, did not affect beta(2)-adrenoceptor-induced cAMP response. Likewise, these responses were insensitive to caveolin 1 and 2 overexpression. On the other hand, methyl-beta-cyclodextrin treatment did decrease beta(2)-adrenoceptor-induced ERK phosphorylation. However, the latter effect of methyl-beta-cyclodextrin could be attributed to a non-specific effect rather than its ability to disrupt membrane microdomains. We showed that localization in the raft microdomains did not affect the signalling efficiency of beta(2)-adrenoceptor-Gs-adenylate cyclase pathway, and that methyl-beta-cyclodextrin may inhibit signalling by directly affecting the signalling system independently of its caveola-disrupting property.
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PMID:beta2-Adrenoceptor, Gs and adenylate cyclase coupling in purified detergent-resistant, low density membrane fractions. 2004 6

The transient receptor potential channel melastatin member 8 (TRPM8) is expressed in sensory neurons, where it constitutes the main receptor of environmental innocuous cold (10-25 degrees C). Among several types of G protein-coupled receptors expressed in sensory neurons, G(i)-coupled alpha 2A-adrenoreceptor (alpha 2A-AR), is known to be involved in thermoregulation; however, the underlying molecular mechanisms remain poorly understood. Here we demonstrated that stimulation of alpha 2A-AR inhibited TRPM8 in sensory neurons from rat dorsal root ganglia (DRG). In addition, using specific pharmacological and molecular tools combined with patch-clamp current recordings, we found that in heterologously expressed HEK-293 (human embryonic kidney) cells, TRPM8 channel is inhibited by the G(i) protein/adenylate cyclase (AC)/cAMP/protein kinase A (PKA) signaling cascade. We further identified the TRPM8 S9 and T17 as two key PKA phosphorylation sites regulating TRPM8 channel activity. We therefore propose that inhibition of TRPM8 through the alpha 2A-AR signaling cascade could constitute a new mechanism of modulation of thermosensation in both physiological and pathological conditions.
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PMID:The transient receptor potential channel TRPM8 is inhibited via the alpha 2A adrenoreceptor signaling pathway. 2011 Mar 57

Nicotinic acid (niacin) has been widely used as a favorable lipid-lowering drug for several decades, and the orphan G protein-coupled receptor GPR109A has been identified to be a receptor for niacin. Mechanistic investigations have shown that as a G(i)-coupled receptor, GPR109A inhibits adenylate cyclase activity upon niacin activation, thereby inhibiting free fatty acid liberation. However, the underlying molecular mechanisms that regulate signaling and internalization of GPR109A remain largely unknown. To further characterize GPR109A internalization, we made a construct to express GPR109A fused with enhanced green fluorescent protein (EGFP) at its carboxyl-terminal end. In stable GPR109A-EGFP-expressing HEK-293 cells, GPR109A-EGFP was mainly localized at the plasma membrane and was rapidly internalized in a dose- and time-dependent manner upon agonist stimulation. GPR109A internalization was completely blocked by hypertonic sucrose, indicating that GPR109A internalizes via the clathrin-coated pit pathway. Further investigation demonstrated that internalized GPR109A was recycled to the cell surface after the removal of agonist, and recycling of the internalized receptors was not blocked by treatment with acidotropic agents, NH(4)Cl and monensin. Pertussis toxin pretreatment not only inhibited forskolin-induced cAMP accumulation and intracellular Ca(2+) mobilization; it also significantly attenuated agonist-promoted GPR109A internalization. Moreover, RNA interference experiments showed that knockdown of GRK2 (G protein-coupled receptor kinase 2) and arrestin3 expression significantly impaired receptor internalization. Taken together, these results indicate that the agonist-induced internalization of GPR109A receptors is regulated by GRK2 and arrestin3 in a pertussis toxin-sensitive manner and that internalized receptor recycling is independent of endosomal acidification.
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PMID:Internalization of the human nicotinic acid receptor GPR109A is regulated by G(i), GRK2, and arrestin3. 2046 Mar 84


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