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1.
Clin Immunol ; 265: 110305, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38972618

ABSTRACT

Auto-inflammatory skin diseases place considerable symptomatic and emotional burden on the affected and put pressure on healthcare expenditures. Although most apparent symptoms manifest on the skin, the systemic inflammation merits a deeper analysis beyond the surface. We set out to identify systemic commonalities, as well as differences in the metabolome and lipidome when comparing between diseases and healthy controls. Lipidomic and metabolomic LC-MS profiling was applied, using plasma samples collected from patients suffering from atopic dermatitis, plaque-type psoriasis or hidradenitis suppurativa or healthy controls. Plasma profiles revealed a notable shift in the non-enzymatic anti-oxidant defense in all three inflammatory disorders, placing cysteine metabolism at the center of potential dysregulation. Lipid network enrichment additionally indicated the disease-specific provision of lipid mediators associated with key roles in inflammation signaling. These findings will help to disentangle the systemic components of autoimmune dermatological diseases, paving the way to individualized therapy and improved prognosis.


Subject(s)
Dermatitis, Atopic , Hidradenitis Suppurativa , Lipidomics , Metabolomics , Psoriasis , Humans , Dermatitis, Atopic/immunology , Dermatitis, Atopic/blood , Dermatitis, Atopic/metabolism , Psoriasis/metabolism , Psoriasis/immunology , Psoriasis/blood , Hidradenitis Suppurativa/blood , Hidradenitis Suppurativa/metabolism , Hidradenitis Suppurativa/immunology , Lipidomics/methods , Female , Adult , Male , Metabolomics/methods , Middle Aged , Metabolome , Young Adult , Inflammation/metabolism , Inflammation/blood , Lipid Metabolism
2.
Prostaglandins Other Lipid Mediat ; 162: 106660, 2022 10.
Article in English | MEDLINE | ID: mdl-35714920

ABSTRACT

Prostanoids are potent lipid mediators involved in a wide variety of physiological functions like blood pressure regulation or inflammation as well as cardiovascular and malign diseases. Elucidation of their modes of action is mainly carried out in pre-clinical animal models by quantifying prostanoids in tissues of interest. Unfortunately, prostanoids are prone to post-mortem artifact formation and de novo synthesis can already be caused by external stimuli during the euthanasia of animals like prolonged hypercapnia or ischemia. Therefore, this study investigates the suitability and impact of fast cervical dislocation for the determination of prostanoids (6-keto-PGF1α, TXB2, PGF2α, PGD2, PGE2) in seven tissues of mice (spinal cord, brain, sciatic nerve, kidney, liver, lung, and spleen) to minimize time-dependent effects and approximate physiological concentrations. Tissues were dissected in a standardized sequence directly or after 10 min to investigate the influence of dissection delays. The enzyme inhibitor indomethacin (10 µM) in combination with low processing temperatures was employed to preserve prostanoid concentrations during sample preparation. Quantification of prostanoids was performed via LC-MS/MS. This study shows, that prostanoids are differentially susceptible to post-mortem artifact formation which is closely connected to their physiological function and metabolic stability in the respective tissues. Prostanoids in the brain, spinal cord, and kidney that are not involved in the regulatory response post-mortem, i.e. blood flow regulation (6-keto-PGF1α, PGE2, PGF2α) showed high reproducibility even after dissection delay and could be assessed after fast cervical dislocation if prerequisites like standardized pre-analytical workflows with immediate dissection and inhibition of residual enzymatic activity are in place. However, in tissues with high metabolic activity (liver, lung) more stable prostanoid metabolites should be used. Moreover, prostanoids in the spleen were strongly affected by dissection delays and presumably the method of euthanasia itself.


Subject(s)
Prostaglandins , Tandem Mass Spectrometry , Animals , Chromatography, Liquid , Dinoprostone , Indomethacin/pharmacology , Mice , Prostaglandins/metabolism , Prostaglandins E , Prostaglandins F , Reproducibility of Results , Tandem Mass Spectrometry/methods
3.
Acta Neuropathol Commun ; 5(1): 42, 2017 06 02.
Article in English | MEDLINE | ID: mdl-28578681

ABSTRACT

Bioactive lipids contribute to the pathophysiology of multiple sclerosis. Here, we show that lysophosphatidic acids (LPAs) are dysregulated in multiple sclerosis (MS) and are functionally relevant in this disease. LPAs and autotaxin, the major enzyme producing extracellular LPAs, were analyzed in serum and cerebrospinal fluid in a cross-sectional population of MS patients and were compared with respective data from mice in the experimental autoimmune encephalomyelitis (EAE) model, spontaneous EAE in TCR1640 mice, and EAE in Lpar2 -/- mice. Serum LPAs were reduced in MS and EAE whereas spinal cord LPAs in TCR1640 mice increased during the 'symptom-free' intervals, i.e. on resolution of inflammation during recovery hence possibly pointing to positive effects of brain LPAs during remyelination as suggested in previous studies. Peripheral LPAs mildly re-raised during relapses but further dropped in refractory relapses. The peripheral loss led to a redistribution of immune cells from the spleen to the spinal cord, suggesting defects of lymphocyte homing. In support, LPAR2 positive T-cells were reduced in EAE and the disease was intensified in Lpar2 deficient mice. Further, treatment with an LPAR2 agonist reduced clinical signs of relapsing-remitting EAE suggesting that the LPAR2 agonist partially compensated the endogenous loss of LPAs and implicating LPA signaling as a novel treatment approach. Graphical summary of lysophosphatidic signaling in multiple sclerosis.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/metabolism , Lysophospholipids/metabolism , Multiple Sclerosis/metabolism , Adolescent , Adult , Animals , Biomarkers/metabolism , Cohort Studies , Cross-Sectional Studies , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Encephalomyelitis, Autoimmune, Experimental/pathology , Female , Humans , Immunologic Factors/pharmacology , Male , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Transgenic , Middle Aged , Multiple Sclerosis/drug therapy , Multiple Sclerosis/pathology , Myelin-Oligodendrocyte Glycoprotein , Peptide Fragments , Receptors, Lysophosphatidic Acid/agonists , Receptors, Lysophosphatidic Acid/genetics , Receptors, Lysophosphatidic Acid/metabolism , Young Adult
4.
Thromb Haemost ; 114(2): 297-312, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25904061

ABSTRACT

Endothelial chemokine CXC motif ligand 16 (CXCL16) expression is associated with atherosclerosis, while platelets, particularly those attaching to atherosclerotic plaque, contribute to all stages of atherosclerotic disease. This investigation was designed to examine the role of CXCL16 in capturing platelets from flowing blood. CXCL16 was expressed in human atherosclerotic plaques, and lesion severity in human carotid endarterectomy specimens was positively correlated with CXCL16 levels. CXCL16 expression in plaques was co-localised with platelets deposited to the endothelium. Immobilised CXCL16 promoted CXCR6-dependent platelet adhesion to the human vessel wall, endothelial cells and von Willebrand factor during physiologic flow. At low shear, immobilised CXCL16 captured platelets from flowing blood. It also induced irreversible platelet aggregation and a rise in intra-platelet calcium levels. These results demonstrate that endothelial CXCL16's action on platelets is not only limited to platelet activation, but that immobilised CXCL16 also acts as a potent novel platelet adhesion ligand, inducing platelet adhesion to the human vessel wall.


Subject(s)
Blood Platelets/pathology , Chemokines, CXC/metabolism , Endothelium, Vascular/metabolism , Plaque, Atherosclerotic/blood , Platelet Adhesiveness , Receptors, Scavenger/metabolism , Abciximab , Antibodies, Monoclonal/pharmacology , Blood Platelets/metabolism , Calcium/blood , Calcium Signaling , Carotid Artery Diseases/metabolism , Carotid Artery Diseases/pathology , Carotid Artery Diseases/surgery , Chemokine CXCL16 , Endarterectomy, Carotid , Hemorheology , Human Umbilical Vein Endothelial Cells , Humans , Immobilized Proteins/metabolism , Immunoglobulin Fab Fragments/pharmacology , In Vitro Techniques , Ligands , Plaque, Atherosclerotic/pathology , Platelet Aggregation , Platelet Glycoprotein GPIb-IX Complex/antagonists & inhibitors , Receptors, CXCR6 , Receptors, Chemokine/antagonists & inhibitors , Receptors, Chemokine/physiology , Receptors, Virus/antagonists & inhibitors , Receptors, Virus/physiology , von Willebrand Factor/metabolism
5.
Anal Bioanal Chem ; 406(28): 7103-16, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25192790

ABSTRACT

Prostanoids, derivatives of arachidonic acid, are involved in inflammation and immune reactions. To understand the role of prostanoids produced by diverse immune cells, a highly sensitive quantitation method for prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), 6-keto prostaglandin F1α (6-keto PGF1α), prostaglandin F2α (PGF2α), and thromboxane B2 (TXB2) by means of nano-liquid chromatography-tandem mass spectrometry has been developed. It was validated according to the guidelines of the Food and Drug Administration (FDA) in terms of linearity, precision, accuracy, recovery, stability, and lower limit of quantitation (LLOQ). The LLOQ were 25 pg/mL in the injected solution (75 fg on column (o.c.)) for PGE2 and PGD2 and 37.5 pg/mL (112.5 fg on column) for 6-keto PGF1α, PGF2α, and TXB2, respectively. It was successfully applied to murine mast cells isolated from paws after zymosan injection and to CD4(+) and CD8(+) T lymphocytes from blood of sensitized versus non-sensitized mice in context of a delayed type hypersensitivity model. About 5,000 (T cells) to 40,000 (mast cells) cells were sufficient for quantitation. In the mast cells, the production of PGE2 increased at a significantly higher extent than the synthesis of the other prostanoids. The T lymphocytes did not show any difference in prostanoid production, no matter whether they were obtained from sensitized mice or non-sensitized mice.


Subject(s)
Chromatography, Liquid/methods , Mast Cells/metabolism , Prostaglandins/analysis , T-Lymphocytes/metabolism , Tandem Mass Spectrometry/methods , Animals , Cells, Cultured , Male , Mast Cells/cytology , Mice , Mice, Inbred C57BL , T-Lymphocytes/cytology
6.
Br J Pharmacol ; 151(4): 494-503, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17435797

ABSTRACT

BACKGROUND AND PURPOSE: Dipyrone is a potent analgesic drug that has been demonstrated to inhibit cyclooxygenase (COX). In contrast to classical COX-inhibitors, such as aspirin-like drugs, dipyrone has no anti-inflammatory effect and a low gastrointestinal toxicity, indicating a different mode of action. Here, we aimed to investigate the effects of dipyrone on COX. EXPERIMENTAL APPROACH: The four major metabolites of dipyrone, including the two pharmacologically active metabolites, 4-methyl-amino-antipyrine (MAA) and amino-antipyrine (AA), were used to characterise their binding to COX and haem as well as their effects on the biochemical properties of COX. Mass spectrometry, UV and visible photometry were used to study binding and prostaglandin production. Levels of anti-oxidant enzymes were assessed by Western blotting. KEY RESULTS: The pharmacologically active metabolites of dipyrone, MAA and AA, did not inhibit COX activity in vitro like classical COX inhibitors, but instead redirected the prostaglandin synthesis, ruling out inhibition of COX through binding to its active site. We found that MAA and AA formed stable complexes with haem and reacted with hydrogen peroxide in presence of haem, ferrous ions (Fe(2+)) or COX. Moreover, MAA reduced Fe(3+) to Fe(2+) and accordingly increased lipid peroxidation and the expression of anti-oxidant enzymes in cultured cells and in vivo. CONCLUSIONS AND IMPLICATIONS: Our data suggest that the pharmacologically active metabolites of dipyrone inhibit COX activity by sequestering radicals which initiate the catalytic activity of this enzyme or through the reduction of the oxidative states of the COX protein.


Subject(s)
Cyclooxygenase Inhibitors/pharmacology , Dipyrone/pharmacology , Ampyrone/analogs & derivatives , Ampyrone/metabolism , Ampyrone/pharmacology , Animals , Dipyrone/metabolism , Heme/metabolism , Hydrogen Peroxide/metabolism , Iron/metabolism , Male , Rats , Rats, Sprague-Dawley
7.
J Biol Chem ; 276(50): 47583-9, 2001 Dec 14.
Article in English | MEDLINE | ID: mdl-11590159

ABSTRACT

Using the yeast two-hybrid assay and the second of the two large cytosolic domains of type V adenylyl cyclase (ACV) as bait, we identified a small region (amino acids 1028-1231) in the protein associated with Myc (PAM) as an interaction site for ACV. This small region of PAM as well as purified full-length PAM inhibited the activity of ACV. Additionally, full-length PAM was a very potent inhibitor of ACI and AC activities in S49 cyc(-) cells and HeLa cells with IC(50) values in the pm and low nm range. Moreover, the regulator of chromatin condensation 1-like domain of PAM (amino acids 446-1062) was sufficient and as potent as full-length PAM at inhibiting the activity of ACV. Interestingly, full-length PAM did not inhibit ACII activity that was stimulated by either forskolin of Galpha(s). When endogenous levels of PAM in HeLa cells were decreased using antisense oligodeoxynucleotides, the basal cAMP content was elevated, and the dose-response curve for vasoactive intestinal peptide-elicited cAMP accumulation in HeLa cells was shifted to the left. Therefore, we conclude that PAM is a very potent, novel inhibitor of specific isoforms of AC. Furthermore, the regulator of chromatin condensation 1-like domain of PAM is sufficient to exert the effects of the full-length protein on AC and decreases in endogenous PAM levels in HeLa cells can modulate both basal and agonist stimulated cAMP accumulation.


Subject(s)
Adaptor Proteins, Signal Transducing , Adenylyl Cyclase Inhibitors , Carrier Proteins/physiology , Enzyme Inhibitors/pharmacology , Mixed Function Oxygenases , Adenylyl Cyclases/chemistry , Carrier Proteins/genetics , Carrier Proteins/pharmacology , Cloning, Molecular , Cyclic AMP/metabolism , Cytoplasm/metabolism , Dose-Response Relationship, Drug , HeLa Cells , Humans , Inhibitory Concentration 50 , Microscopy, Fluorescence , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/pharmacology , Protein Binding , Protein Isoforms , Protein Structure, Tertiary , Recombinant Proteins/metabolism , Two-Hybrid System Techniques , Ubiquitin-Protein Ligases , Vasoactive Intestinal Peptide/metabolism
8.
Gene ; 269(1-2): 13-25, 2001 May 16.
Article in English | MEDLINE | ID: mdl-11376933

ABSTRACT

Signal transduction through the cell membrane requires the participation of one or more plasma membrane proteins. For many transmembrane signaling events adenylyl cyclases (ACs) are the final effector enzymes which integrate and interpret divergent signals from different pathways. The enzymatic activity of adenylyl cyclases is stimulated or inhibited in response to the activation of a large number of receptors in virtually all cells of the human body. To date, ten different mammalian isoforms of adenylyl cyclase (AC) have been cloned and characterized. Each isoform has its own distinct tissue distribution and regulatory properties, providing possibilities for different cells to respond diversely to similar stimuli. The product of the enzymatic reaction catalyzed by ACs, cyclic AMP (cAMP) has been shown to play a crucial role for a variety of fundamental physiological cell functions ranging from cell growth and differentiation, to transcriptional regulation and apoptosis. In the past, investigations into the regulatory mechanisms of ACs were limited by difficulties associated with their purification and the availability of the proteins in any significant amount. Moreover, nearly every cell expresses several AC isoforms. Therefore, it was difficult to perform biochemical characterization of the different AC isoforms and nearly impossible to assess the physiological roles of the individual isoforms in intact cells, tissue or organisms. Recently, however, different molecular biological approaches have permitted several breakthroughs in the study of ACs. Recombinant technologies have allowed biochemical analysis of adenylyl cyclases in-vitro and the development of transgenic animals as well as knock-out mice have yielded new insights in the physiological role of some AC isoforms. In this review, we will focus mainly on the most novel approaches and concepts, which have delineated the mechanisms regulating AC and unravelled novel functions for this enzyme.


Subject(s)
Adenylyl Cyclases/physiology , Signal Transduction/physiology , Adenylyl Cyclases/chemistry , Adenylyl Cyclases/genetics , Adenylyl Cyclases/metabolism , Animals , Chromosome Mapping , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Protein alpha Subunits, Gs/metabolism , Humans , Mice , Mice, Knockout , Mice, Transgenic , Structure-Activity Relationship
9.
J Biol Chem ; 276(25): 22742-7, 2001 Jun 22.
Article in English | MEDLINE | ID: mdl-11279012

ABSTRACT

The Drosophila Sprouty (SPRY) protein has been shown to inhibit the actions of epidermal growth factor and fibroblast growth factor. However, the role of mammalian SPRY proteins has not been clearly elucidated. We postulated that human Sprouty2 (hSPRY2) is an inhibitor of cellular migration and proliferation. Indeed, using stably transfected HeLa cells, which expressed hemagglutinin (HA)-tagged hSPRY2 or hSPRY2 tagged at the C terminus with red fluorescent protein, we demonstrated that hSPRY2 inhibits the migration of cells in response to serum, epidermal growth factor, fibroblast growth factor, and platelet-derived growth factor. Additionally, hSPRY2 also inhibited the growth of HeLa cells in response to serum. Previously, two C-terminal domains on hSPRY2, which are necessary for its colocalization with microtubules (residues 123-177) or translocation to membrane ruffles (residues 178-194), have been identified (Lim, J., Wong, E. S., Ong, S. H., Yusoff, P., Low, B. C., and Guy, G. R. (2000) J. Biol. Chem. 275, 32837-32845). Therefore, using TAT-tagged hSPRY2 and its mutants, we determined the role of these two C-terminal domains in the inhibition of cell migration and proliferation. Our data show that the deletion of either of these two regions in hSPRY2 abrogates its ability to modulate cell migration in response to different growth factors and proliferation in response to serum. Therefore, we conclude that hSPRY2 inhibits the actions of a number of growth factors, and its C terminus, which is homologous among various SPRY isoforms, is important in mediating its biological activity.


Subject(s)
Cell Division/physiology , Cell Movement/physiology , Drosophila Proteins , Insect Proteins/physiology , Membrane Proteins , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Cloning, Molecular , DNA Primers , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique , Green Fluorescent Proteins , HeLa Cells , Humans , Insect Proteins/chemistry , Insect Proteins/genetics , Luminescent Proteins/metabolism , Recombinant Fusion Proteins/metabolism
10.
J Biol Chem ; 275(34): 25915-9, 2000 Aug 25.
Article in English | MEDLINE | ID: mdl-10852906

ABSTRACT

Adenylyl cyclase, the enzyme that converts ATP to cAMP, is regulated by its stimulatory and inhibitory GTP-binding proteins, G(s) and G(i), respectively. Recently, we demonstrated that besides catalyzing the synthesis of cAMP, type V adenylyl cyclase (ACV) can act as a GTPase-activating protein for Galpha(s) and also enhance the ability of activated receptors to stimulate GTP-GDP exchange on heterotrimeric G(s) (Scholich, K., Mullenix, J. B., Wittpoth, C., Poppleton, H. M., Pierre, S. C., Lindorfer, M. A., Garrison, J. C., and Patel, T. B. (1999) Science 283, 1328-1331). This latter action of ACV would facilitate the rapid onset of signaling via G(s). Because the C1 region of ACV interacts with the inhibitory GTP-binding protein Galpha(i), we investigated whether the receptor-mediated activation of heterotrimeric G(i) was also regulated by ACV and its subdomains. Our data show that ACV and its C1 domain increased the ability of a muscarinic receptor mimetic peptide (MIII-4) to enhance activation of heterotrimeric G(i) such that the amount of peptide required to stimulate G(i) in steady-state GTPase activity assays was 3-4 orders of magnitude less than without the C1 domain. Additionally, the MIII-4-mediated binding of guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) to G(i) was also markedly increased in the presence of ACV or its C1 domain. In contrast, the C2 domain of ACV was not able to alter either the GTPase activity or the GTPgammaS binding to G(i) in the presence of MIII-4. Furthermore, in adenylyl cyclase assays employing S49 cyc(-) cell membranes, the C1 (but not the C2) domain of ACV enhanced the ability of peptide MIII-4 as well as endogenous somatostatin receptors to activate endogenous G(i) and to inhibit adenylyl cyclase activity. These data demonstrate that adenylyl cyclase and its C1 domain facilitate receptor-mediated activation of G(i).


Subject(s)
Adenylyl Cyclases/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Signal Transduction , Adenylyl Cyclase Inhibitors , Animals , Colforsin/pharmacology , GTP Phosphohydrolases/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Mice , Molecular Mimicry , Receptor, Muscarinic M4 , Receptors, Muscarinic/metabolism , Somatostatin/pharmacology , Tumor Cells, Cultured
11.
Biochem Biophys Res Commun ; 270(1): 131-6, 2000 Apr 02.
Article in English | MEDLINE | ID: mdl-10733916

ABSTRACT

Several studies have examined the role of palmitoylation of G protein alpha subunits by nonenzymatic in vitro acylation using palmitoyl-CoA. Here, we investigated nonenzymatic palmitoylation of purified G(salpha) in vitro. GDP-bound G(salpha) was stoichiometrically autoacylated on cysteine residue(s) with micromolar concentrations of palmitoyl-CoA. The acylation led to a complete loss of steady-state GTPase activity and GTPgammaS binding to G(salpha). Mutation of Cys 3 to Ala in G(salpha) did not prevent either palmitoylation or its consequent functional alterations. However, stoichiometric palmitoylation of His(6)-G(salpha) did not alter its GTPase activity or GTPgammaS binding. Isoelectric focusing of tryptic peptides from autoacylated wild type, His(6)-tagged, and C3A mutant of G(salpha) showed that Cys 160 is the site of in vitro palmitoylation. Therefore, we conclude that in vitro palmitoylation of G(salpha) occurs on Cys 160 and this modification decreases the ability of the protein to exchange GTP for GDP; N-terminus elongation of G(salpha) prevents this latter effect without altering palmitoylation.


Subject(s)
Cysteine/metabolism , GTP-Binding Protein alpha Subunits, Gs/metabolism , Palmitic Acid/metabolism , Acylation , Cell-Free System , Cysteine/genetics , GTP-Binding Protein alpha Subunits, Gs/genetics , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Guanosine Diphosphate/metabolism , Mutation , Palmitoyl Coenzyme A/metabolism , Protein Conformation , Protein Processing, Post-Translational
12.
Proc Natl Acad Sci U S A ; 96(17): 9551-6, 1999 Aug 17.
Article in English | MEDLINE | ID: mdl-10449730

ABSTRACT

The two large cytoplasmic domains (C1 and C2) of adenylyl cyclases (AC), when expressed separately and mixed together, reconstitute enzyme activity that can be regulated by various modulators. Therefore, we have used the C1 or its C1a subdomain and C2 regions from type I AC (ACI) and type V AC (ACV) to identify the region on ACI that interacts with beta gamma subunits of heterotrimeric G proteins. In addition, we also used a chimeric C1 domain (VC1aIC1b) in which the C1a region was derived from ACV and the C1b region was from ACI. By mixing the C1 or C1a or VC1aIC1b domains with C2 regions of ACI or ACV, we have shown that the C1a region (amino acids 236-471) of ACI is sufficient to observe beta gamma-mediated inhibition of enzyme activity, which is stimulated by either constitutively active G(salpha) (G(salpha)*) or Ca(2+)/calmodulin (CaM). Although the C1b region and C2 domain of ACI were by themselves not sufficient for inhibition of activity by beta gamma subunits, the presence of both of these regions formed another beta gamma interaction site that was sufficient to observe G(salpha)*- or Ca(2+)/CaM-stimulated activity. Inhibition of AC activity attributable to interaction of beta gamma subunits at either of the two sites was blocked by a peptide (QEHA) that has previously been shown to inhibit the effects of beta gamma on various effectors. Moreover, the C1 region of ACI was sufficient to observe G(ialpha1)-elicited inhibition of Ca(2+)/CaM-stimulated activity. Although the C1a region of ACV was sufficient for inhibition of activity by G(ialpha1), the presence of C1b region from either ACI or ACV increased sensitivity to inhibition by the inhibitory G protein. Thus, the inhibitory influences of G(ialpha1) are mediated on the C1 regions of both ACI and ACV. The effects of beta gamma on ACI can be mediated by interactions with the C1a region and a beta gamma interacting site formed by the C1b and C2 domains of this enzyme.


Subject(s)
Adenylyl Cyclases/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Protein beta Subunits , GTP-Binding Protein gamma Subunits , GTP-Binding Proteins/metabolism , Heterotrimeric GTP-Binding Proteins , Calmodulin/metabolism , Escherichia coli , Protein Conformation , Recombinant Proteins/metabolism , Structure-Activity Relationship
13.
Science ; 283(5406): 1328-31, 1999 Feb 26.
Article in English | MEDLINE | ID: mdl-10037603

ABSTRACT

The alpha subunit (Gsalpha) of the stimulatory heterotrimeric guanosine triphosphate binding protein (G protein) Gs activates all isoforms of mammalian adenylyl cyclase. Adenylyl cyclase (Type V) and its subdomains, which interact with Gsalpha, promoted inactivation of the G protein by increasing its guanosine triphosphatase (GTPase) activity. Adenylyl cyclase and its subdomains also augmented the receptor-mediated activation of heterotrimeric Gs and thereby facilitated the rapid onset of signaling. These findings demonstrate that adenylyl cyclase functions as a GTPase activating protein (GAP) for the monomeric Gsalpha and enhances the GTP/GDP exchange factor (GEF) activity of receptors.


Subject(s)
Adenylyl Cyclases/metabolism , GTP Phosphohydrolases/metabolism , GTP-Binding Protein alpha Subunits, Gs/metabolism , Signal Transduction , Adenylyl Cyclases/chemistry , Adenylyl Cyclases/genetics , Animals , Cell Line , GTPase-Activating Proteins , Guanine Nucleotide Exchange Factors , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Guanosine Triphosphate/metabolism , Magnesium/pharmacology , Proteins/metabolism , Receptors, Adrenergic, beta-2/metabolism , Recombinant Proteins/metabolism
14.
Proc Natl Acad Sci U S A ; 94(18): 9602-7, 1997 Sep 02.
Article in English | MEDLINE | ID: mdl-9275169

ABSTRACT

Using the full-length and two engineered soluble forms (C1-C2 and Cla-C2) of type V adenylyl cyclase (ACV), we have investigated the role of an intramolecular interaction in ACV that modulates the ability of the alpha subunit of the stimulatory GTP-binding protein of AC (Gsalpha) to stimulate enzyme activity. Concentration-response curves with Gsalpha suggested the presence of high and low affinity sites on ACV, which interact with the G protein. Activation of enzyme by Gsalpha interaction at these two sites was most apparent in the C1a-C2 form of ACV, which lacks the C1b region (K572-F683). Yeast two-hybrid data demonstrated that the C1b region interacted with the C2 region and its 64-aa subdomain, C2I. Using peptides corresponding to the C2I region of ACV, we investigated the role of the C1b/C2I interaction on Gsalpha-mediated stimulation of C1-C2 and full-length ACV. Our data demonstrate that a 10-aa peptide corresponding to L1042-T1051 alters the profile of the activation curves of full-length and C1-C2 forms of ACV by different Gsalpha concentrations to mimic the activation profile observed with C1a-C2 ACV. The various peptides used in our studies did not alter forskolin-mediated stimulation of full-length and C1-C2 forms of ACV. We conclude that the C1b region of ACV interacts with the 10-aa region (L1042-T1051) in the C2 domain of the enzyme to modulate Gsalpha-elicited stimulation of activity.


Subject(s)
Adenylyl Cyclases/metabolism , GTP-Binding Proteins/metabolism , Adenylyl Cyclases/chemistry , Amino Acid Sequence , Enzyme Activation , Escherichia coli , Molecular Sequence Data , Protein Binding , Recombinant Proteins/chemistry
15.
Proc Natl Acad Sci U S A ; 94(7): 2915-20, 1997 Apr 01.
Article in English | MEDLINE | ID: mdl-9096321

ABSTRACT

Type V adenylyl cyclase (ACV) belongs to the family of Ca2+-inhibited cyclases. We have generated two soluble forms of the enzyme containing the C1 or C1a region (which lacks the C-terminal 112 amino acids) linked to the C2 domain and compared their regulation with the full-length ACV. All three forms of ACV were stimulated by the alpha subunit of the stimulatory G protein Gs (G(s alpha)) and forskolin. However, the synergistic stimulation by both these activators was markedly enhanced in the soluble enzymes. Moreover, the alpha subunit of the inhibitory G protein Gi (G(i alpha)) inhibited all forms of the enzyme, indicating that the regions for G(s alpha) and G(i alpha) interaction are preserved in the soluble forms. Ca2+ inhibited forskolin-stimulated adenylyl cyclase (AC) activity of the full-length and C1-C2 forms of ACV but did not alter the activity of the C1a-C2 form. Maximal stimulation of AC activity by combination of G(s alpha) and forskolin obliterated the Ca2+-mediated inhibition of the full-length and C1-C2 forms of ACV. In 45Ca2+ overlay experiments, the C1-C2 but not the C1a-C2 soluble ACV bound Ca2+. Moreover, proteins corresponding to the C1a and C2 domains did not bind calcium. On the other hand, the proteins corresponding to C1 and its C-terminal 112 amino acids (C1b) bound 45Ca2+. To our knowledge, this is the first report of nonchimeric soluble forms of AC in which regulation by G(s alpha) and G(i alpha) is preserved. Moreover, we demonstrate that the 112 amino acid C1b region of ACV is responsible for the binding of Ca2+ and inhibition of enzyme activity.


Subject(s)
Adenylyl Cyclases/metabolism , Animals , Calcium/metabolism , Cell Line , GTP-Binding Proteins/metabolism , Recombinant Proteins/metabolism , Solubility , Spodoptera
16.
J Biol Chem ; 272(9): 5413-20, 1997 Feb 28.
Article in English | MEDLINE | ID: mdl-9038141

ABSTRACT

Previously, we have demonstrated that epidermal growth factor (EGF) can stimulate adenylyl cyclase activity via activation of Gs in the heart. Moreover, we have recently shown that Gsalpha is phosphorylated by the EGF receptor protein tyrosine kinase and that the juxtamembrane region of the EGF receptor can stimulate Gs directly. Therefore, employing isolated cardiac membranes, the two-hybrid assay, and in vitro association studies with purified EGF receptor and Gsalpha we have investigated Gsalpha complex formation with the EGF receptor and elucidated the region in the receptor involved in this interaction. In isolated cardiac membranes, immunoprecipitation of EGF receptor was accompanied by co-immunoprecipitation of Gsalpha. In the yeast two-hybrid assay, the cytosolic domain of the EGF receptor and the N-terminal 64 amino acids of this region (Met644-Trp707) associated with Gsalpha. However, interactions of these regions of the EGF receptor with constitutively active Gsalpha were diminished in the two-hybrid assay. Employing purified proteins, our studies demonstrate that the EGF receptor, directly and stoichiometrically, associates with Gsalpha (1 mol of Gsalpha/mol of EGF receptor). This association was not altered in the presence or absence of ATP and therefore, was independent of tyrosine phosphorylation of either of the proteins. Peptides corresponding to the juxtamembrane region of the receptor decreased association of the EGF receptor with Gsalpha. However, neither the C-terminally truncated EGF receptor (Delta1022-1186) nor a peptide corresponding to residues 985-996 of the receptor altered association with Gsalpha, thus indicating the selectivity of the G protein interaction with the juxtamembrane region. Interestingly, peptides corresponding to N and C termini of Gsalpha did not alter the association of Gsalpha with the EGF receptor. Consistent with the findings from the two-hybrid assay where constitutively active Gsalpha poorly associated with the EGF receptor, in vitro experiments with purified proteins also demonstrated that activation of Gsalpha by guanosine 5'-3-O-(thio)triphosphate decreased the association of G protein with the EGF receptor. Thus we conclude that the juxtamembrane region of the EGF receptor, directly and stoichiometrically, associates with Gsalpha and that upon activation of Gsalpha this association is decreased.


Subject(s)
Cytosol/metabolism , ErbB Receptors/metabolism , GTP-Binding Protein alpha Subunits, Gs/metabolism , Oncogene Proteins/metabolism , Adenosine Triphosphate/metabolism , Animals , Electrophoresis, Polyacrylamide Gel , Epidermal Growth Factor/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Male , Myocardium/cytology , Myocardium/metabolism , Rats , Rats, Sprague-Dawley , Saccharomyces cerevisiae
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