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Robert J Lefkowitz - One of the best experts on this subject based on the ideXlab platform.
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differential desensitization and phosphorylation of three cloned and transfected alpha 2 Adrenergic Receptor subtypes
Journal of Biological Chemistry, 1994Co-Authors: Hitoshi Kurose, Robert J LefkowitzAbstract:Genes encoding 3 distinct subtypes of human alpha 2-Adrenergic Receptor are known and are found, respectively, on chromosome 10, 4, and 2 (alpha 2-C10, alpha 2-C4, and alpha 2-C2 Adrenergic Receptors). All 3 Receptors inhibit adenylyl cyclase via Gi proteins. To study and compare their regulatory properties we assessed the ability of each to undergo agonist-promoted desensitization and phosphorylation. When Chinese hamster ovary cells stably expressing each of the three Receptor genes were incubated with epinephrine for 20 min, a marked decrease in sensitivity to subsequent agonist-mediated inhibition of adenylyl cyclase was observed for the alpha 2-C10 and alpha 2-C2 Receptors but not for the alpha 2-C4 Receptors. When similar incubations were performed with 32Pi-labeled cells and the Receptors were immunoprecipitated with specific antibodies, alpha 2-C10 and alpha 2-C2 Receptors were found to undergo an approximately 3-fold increase in Receptor phosphorylation after epinephrine exposure. When transfected into COS cells epinephrine also stimulated phosphorylation of alpha 2-C10 and alpha 2-C2 Receptors while having only a slight effect on alpha 2-C4 Receptors. Cotransfection of the cells with the cDNA encoding the beta-Adrenergic Receptor kinase further increased Receptor phosphorylation for alpha 2-C10 and alpha 2-C2 Receptors while having little or no effect on alpha 2-C4 Receptors. Moreover purified and reconstituted recombinant alpha 2-C10 Receptors could be phosphorylated in an agonist-dependent fashion whereas alpha 2-C4 Receptors could not. These observations suggest Receptor subtype-specific differences in susceptibility to regulatory phosphorylation and desensitization.
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cellular expression of the carboxyl terminus of a g protein coupled Receptor kinase attenuates g beta gamma mediated signaling
Journal of Biological Chemistry, 1994Co-Authors: Walter J Koch, James Inglese, Brian E Hawes, Louis M Luttrell, Robert J LefkowitzAbstract:Abstract The beta gamma subunits (G beta gamma) of heterotrimeric G proteins modulate the activity of several signal-transducing effector molecules including G protein-coupled Receptor kinases. G beta gamma binds to the carboxyl terminus of the beta-Adrenergic Receptor kinase (beta ARK) and regulates its activity. To investigate the effect of such a G beta gamma-binding domain on heterologous G beta gamma interactions, various Receptors that can stimulate phospholipase C and/or type II adenylate cyclase were coexpressed in COS-7 cells with the carboxyl terminus of beta ARK1. Phosphoinositol hydrolysis in response to activation of Receptors that stimulate phospholipase C via Gi beta gamma (alpha 2-Adrenergic and M2-muscarinic cholinergic Receptors) was markedly inhibited by the coexpressed beta ARK1 polypeptide, whereas that mediated by Gq alpha subunits (alpha 1-Adrenergic and M1-muscarinic cholinergic Receptors) was unaffected. Increased cellular cAMP levels due to stimulation of Receptors and coexpressed adenylate cyclase II displayed marked inhibition in the presence of the beta ARK1 polypeptide. Moreover, inhibition of adenylate cyclase produced by alpha 2-Adrenergic Receptor stimulation (a Gi alpha-mediated process) was unaffected, indicating that the beta ARK1 polypeptide provides a useful tool for distinguishing between G alpha and G beta gamma pathways.
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constitutively active mutants of the alpha 2 Adrenergic Receptor
Journal of Biological Chemistry, 1993Co-Authors: Quen Ren, Hitoshi Kurose, Robert J Lefkowitz, Susanna CotecchiaAbstract:We have mutated a single residue, Thr373 [corrected], in the C-terminal portion of the third intracellular loop of the alpha 2C10-Adrenergic Receptor into five different amino acids. In analogy with the effect of similar mutations in the alpha 1B- and beta 2-Adrenergic Receptors, these substitutions resulted in two major biochemical modifications: 1) increased constitutive activity of the alpha 2-Adrenergic Receptor leading to agonist-independent inhibition of adenylyl cyclase and 2) increased affinity of the Receptor for binding agonist but not antagonists. The increased constitutive activity of the mutated alpha 2-Adrenergic Receptors could be inhibited by pertussis toxin, clearly indicating that it results from spontaneous ligand-independent Receptor coupling to Gi. In contrast, the increased affinity of the mutant Receptors for binding agonists was unaffected by pertussis toxin treatment, indicating that this is an inherent property of the Receptors not dependent on interaction with Gi. Coexpression of the Receptor mutants with the Receptor-specific kinase, beta ARK1, indicated that the constitutively active alpha 2-Adrenergic Receptors are substrates for beta-Adrenergic Receptor kinase (beta ARK)-mediated phosphorylation even in the absence of agonist. These findings strengthen the idea that constitutively active Adrenergic Receptors mimic the "active" state of a G protein-coupled Receptor adopting conformations similar to those induced by agonist when it binds to wild type Receptors. In addition, these results extend the notion that in the Adrenergic Receptor family the C-terminal portion of the third intracellular loop plays a general role in the processes involved in Receptor activation.
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sites in the third intracellular loop of the alpha 2a Adrenergic Receptor confer short term agonist promoted desensitization evidence for a Receptor kinase mediated mechanism
Journal of Biological Chemistry, 1992Co-Authors: Stephen B Liggett, M G Caron, Hitoshi Kurose, Jacek Ostrowski, L C Chesnut, John R Raymond, Robert J LefkowitzAbstract:Abstract To investigate the mechanisms of agonist-promoted desensitization of the alpha 2-Adrenergic Receptor (alpha 2AR), the human alpha 2AAR and a mutated form of the Receptor were expressed in CHW cells. After cells were exposed to epinephrine for 30 min, the ability of the wild type alpha 2AAR to mediate inhibition of forskolin-stimulated adenylyl cyclase was depressed by approximately 78%. To assess the role of Receptor phosphorylation during desensitization, cells were incubated with 32Pi, exposed to agonist, and alpha 2AAR purified by immunoprecipitation with a fusion protein antibody. Agonist-promoted desensitization was found to be accompanied by phosphorylation of the alpha 2AAR in vivo. The beta-Adrenergic Receptor kinase (beta ARK) is known to phosphorylate purified alpha 2AAR in vitro. We found that heparin, a beta ARK inhibitor, ablated short term agonist-induced desensitization of alpha 2AAR, while such desensitization was unaffected by inhibition of protein kinase A. To further assess the role of beta ARK, we constructed a mutated alpha 2AAR which has a portion of the third intracellular loop containing 9 serines and threonines (potential phosphorylation sites) deleted. This mutated alpha 2AAR failed to undergo short term agonist-induced desensitization. Agonist promoted in vivo phosphorylation of this mutated Receptor was reduced by 90%, consistent with the notion that Receptor phosphorylation at sites in the third intracellular loop plays a critical role in alpha 2AAR desensitization. After 24 h of agonist exposure, an even more profound desensitization of alpha 2AAR occurred, which was not accompanied by a decrease in Receptor expression. Rather, long term agonist-induced desensitization was found to be due in part to a decrease in the amount of cellular Gi, which was not dependent on Receptor third loop phosphorylation sites.
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functional interactions of recombinant alpha 2 Adrenergic Receptor subtypes and g proteins in reconstituted phospholipid vesicles
Biochemistry, 1991Co-Authors: Hitoshi Kurose, John W Regan, Marc G. Caron, Robert J LefkowitzAbstract:The functional interaction of the recombinant alpha 2 Adrenergic Receptor subtypes, alpha 2-C10 (the human platelet alpha 2 Receptor, equivalent to the alpha 2 A subtype) and alpha 2-C4 (an alpha 2 Receptor subtype cloned from a human kidney cDNA library), with G proteins was characterized in an in vitro reconstitution system. These Receptor subtypes were overexpressed in COS-7 cells and were purified to a specific activity of 1.1-3.3 nmol/mg of protein. The G proteins consisted of Gs (adenylyl cyclase stimulatory) and members of the inhibitory family, including Gi1, Gi2, and Gi3, and G0. The cloned alpha subunits of these G proteins were overexpressed in Escherichia coli and were purified to homogeneity. Prior to use, G holoproteins were prepared by mixing the alpha subunits with beta gamma subunits that had been purified from bovine brain. Following reconstitution into phospholipid vesicles, both alpha 2 Receptor subtypes could couple to the inhibitory G proteins but not to Gs, as assessed by agonist stimulation of GTPase activity. The pharmacological specificity of this interaction was preserved with respect to the two Receptor subtypes. Between the different inhibitory G proteins, the alpha 2-C10 Adrenergic Receptor subtype showed the following preference: Gi3 greater than Gi1 greater than or equal to Gi2 greater than G0. The stimulation of GTPase activity (turnover number) ranged from 6.4-fold (Gi3) to 1.5-fold (G0). The preference of G-protein interaction for the alpha 2-C4 Receptor subtype was the same as that observed for the alpha 2-C10, but the extent of activation was slightly lower. The results show that in vitro each of the alpha 2 Adrenergic Receptor subtypes can activate multiple G proteins but that clear preferences exist with respect to the individual inhibitory G-protein subtypes. Additionally, it appears that alpha 2-C10 is coupled more efficiently to G-protein activation than is alpha 2-C4.
Gabsang Lee - One of the best experts on this subject based on the ideXlab platform.
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large scale screening using familial dysautonomia induced pluripotent stem cells identifies compounds that rescue ikbkap expression
Nature Biotechnology, 2012Co-Authors: Gabsang Lee, Hyesoo Kim, Christina N Ramirez, Nadja Zeltner, Becky Liu, Constantin Radu, Bhavneet Bhinder, Yong Jun Kim, In Young ChoiAbstract:Patient-specific induced pluripotent stem cells (iPSCs) represent a novel system for modeling human genetic disease and could develop into a key drug discovery platform. We recently reported disease-specific phenotypes in iPSCs from familial dysautonomia (FD) patients. FD is a rare but fatal genetic disorder affecting neural crest lineages. Here we demonstrate the feasibility of performing a primary screen in FD-iPSC derived neural crest precursors. Out of 6,912 compounds tested we characterized 8 hits that rescue expression of IKBKAP, the gene responsible for FD. One of those hits, SKF-86466, is shown to induce IKBKAP transcription via modulation of intracellular cAMP levels and PKA dependent CREB phosphorylation. SKF-86466 also rescues IKAP protein expression and the disease-specific loss of autonomic neuron marker expression. Our data implicate Alpha-2 Adrenergic Receptor activity in regulating IKBKAP expression and demonstrate that small molecule discovery in an iPSC-based disease model can identify candidate drugs for potential therapeutic intervention.
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large scale screening using familial dysautonomia induced pluripotent stem cells identifies compounds that rescue ikbkap expression
Nature Biotechnology, 2012Co-Authors: Gabsang Lee, Hyesoo Kim, Christina N Ramirez, Nadja Zeltner, Becky Liu, Constantin Radu, Bhavneet Bhinder, Yong Jun Kim, In Young ChoiAbstract:Patient-specific induced pluripotent stem cells (iPSCs) represent a novel system for modeling human genetic disease and could provide a source of cells for large-scale drug-discovery screens. Here we demonstrate the feasibility of performing a primary screen in neural crest precursors derived from iPSCs that were generated from individuals with familial dysautonomia (FD), a rare, fatal genetic disorder affecting neural crest lineages. We tested 6,912 small-molecule compounds and characterized eight that rescued expression of IKBKAP, the gene responsible for FD. One of the hits, SKF-86466, was found to induce IKBKAP transcription through modulation of intracellular cAMP levels and PKA-dependent CREB phosphorylation. SKF-86466 also rescued IKAP protein expression and the disease-specific loss of autonomic neuronal marker expression. Our data implicate Alpha-2 Adrenergic Receptor activity in regulating IKBKAP expression and demonstrate that small-molecule discovery using an iPSC-based disease model can identify candidate drugs for potential therapeutic intervention.
Lee E Limbird - One of the best experts on this subject based on the ideXlab platform.
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the three alpha 2 Adrenergic Receptor subtypes achieve basolateral localization in madin darby canine kidney ii cells via different targeting mechanisms
Journal of Biological Chemistry, 1996Co-Authors: Magdalena Wozniak, Lee E LimbirdAbstract:Abstract The present studies examined the localization of the α- and α-Adrenergic Receptor (AR) subtypes in polarized Madin-Darby canine kidney cells (MDCK II) and the mechanisms by which this is achieved. Previously we demonstrated that the αAR subtype is directly delivered to lateral subdomain of MDCK II cells. Surface biotinylation strategies demonstrated that the αAR, like the αAR, achieves 85-90% basolateral localization at steady-state. However, in contrast to the αAR, this polarization occurs after initial random insertion of the αAR into both apical and basolateral surfaces followed by selective retention on the lateral subdomain (ton the apical surface is 15-30 min; ton the basolateral surface is 8-10 h). The αAR also is enriched on the basolateral surface at steady-state and, like the αAR, is directly delivered there. Morphological evaluation of the epitope-tagged αAR, αAR, and αAR subtypes by laser confocal microscopy not only corroborated the biochemically-defined basolateral localization of all three αAR subtypes but also revealed that the αAR uniquely exists in an intracellular compartment(s) as well. Immunofluorescence due to intracellular αAR partially overlaps that due to calnexin, a marker for endoplasmic reticulum, as well as that due to mannosidase II, a marker for the trans-Golgi network. Taken together, the present findings demonstrate that the αAR, αAR, and αAR subtypes, which possess highly homologous structures and ultimately achieve similar polarization to the lateral surface of MDCK II cells, nonetheless manifest distinct trafficking itineraries.
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the alpha 2a Adrenergic Receptor is targeted directly to the basolateral membrane domain of madin darby canine kidney cells independent of coupling to pertussis toxin sensitive gtp binding proteins
Journal of Biological Chemistry, 1993Co-Authors: Jeffrey R Keefer, Lee E LimbirdAbstract:The alpha 2-Adrenergic Receptor (alpha 2-AR) is a member of the seven transmembrane-spanning G-protein-coupled Receptor superfamily. In the kidney, the alpha 2-AR is most abundant in the epithelial cells of the proximal tubule where it is important in enhancing Na+ reabsorption via the modulation of Na+/H+ exchange. Radioligand binding and physiological studies suggest that the alpha 2-AR residues primarily on the basolateral surface of these proximal tubule cells in vivo. To investigate the mechanisms underlying alpha 2-AR polarization in epithelial cells, we permanently expressed wild-type and an epitope-tagged version of the alpha 2A-AR in Madin-Darby canine kidney (MDCK) cells. Using a steady-state surface biotinylation assay, we observe that 80-90% of the alpha 2A-AR in MDCK cell clones is located on the basolateral membrane domain. Immunolocalization studies confirm the biotinylation results and demonstrate that the alpha 2A-AR is actually confined primarily to the lateral domain of the basolateral surface. Metabolic labeling experiments suggest that basolateral polarization of the alpha 2A-AR is achieved by direct targeting of the Receptor to the basolateral domain. Targeting of the alpha 2A-AR to the basolateral surface is not perturbed by pertussis toxin-treatment of MDCK cells, suggesting that coupling of the alpha 2A-AR to GTP-binding proteins is not important for Receptor polarization.
Hitoshi Kurose - One of the best experts on this subject based on the ideXlab platform.
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differential desensitization and phosphorylation of three cloned and transfected alpha 2 Adrenergic Receptor subtypes
Journal of Biological Chemistry, 1994Co-Authors: Hitoshi Kurose, Robert J LefkowitzAbstract:Genes encoding 3 distinct subtypes of human alpha 2-Adrenergic Receptor are known and are found, respectively, on chromosome 10, 4, and 2 (alpha 2-C10, alpha 2-C4, and alpha 2-C2 Adrenergic Receptors). All 3 Receptors inhibit adenylyl cyclase via Gi proteins. To study and compare their regulatory properties we assessed the ability of each to undergo agonist-promoted desensitization and phosphorylation. When Chinese hamster ovary cells stably expressing each of the three Receptor genes were incubated with epinephrine for 20 min, a marked decrease in sensitivity to subsequent agonist-mediated inhibition of adenylyl cyclase was observed for the alpha 2-C10 and alpha 2-C2 Receptors but not for the alpha 2-C4 Receptors. When similar incubations were performed with 32Pi-labeled cells and the Receptors were immunoprecipitated with specific antibodies, alpha 2-C10 and alpha 2-C2 Receptors were found to undergo an approximately 3-fold increase in Receptor phosphorylation after epinephrine exposure. When transfected into COS cells epinephrine also stimulated phosphorylation of alpha 2-C10 and alpha 2-C2 Receptors while having only a slight effect on alpha 2-C4 Receptors. Cotransfection of the cells with the cDNA encoding the beta-Adrenergic Receptor kinase further increased Receptor phosphorylation for alpha 2-C10 and alpha 2-C2 Receptors while having little or no effect on alpha 2-C4 Receptors. Moreover purified and reconstituted recombinant alpha 2-C10 Receptors could be phosphorylated in an agonist-dependent fashion whereas alpha 2-C4 Receptors could not. These observations suggest Receptor subtype-specific differences in susceptibility to regulatory phosphorylation and desensitization.
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constitutively active mutants of the alpha 2 Adrenergic Receptor
Journal of Biological Chemistry, 1993Co-Authors: Quen Ren, Hitoshi Kurose, Robert J Lefkowitz, Susanna CotecchiaAbstract:We have mutated a single residue, Thr373 [corrected], in the C-terminal portion of the third intracellular loop of the alpha 2C10-Adrenergic Receptor into five different amino acids. In analogy with the effect of similar mutations in the alpha 1B- and beta 2-Adrenergic Receptors, these substitutions resulted in two major biochemical modifications: 1) increased constitutive activity of the alpha 2-Adrenergic Receptor leading to agonist-independent inhibition of adenylyl cyclase and 2) increased affinity of the Receptor for binding agonist but not antagonists. The increased constitutive activity of the mutated alpha 2-Adrenergic Receptors could be inhibited by pertussis toxin, clearly indicating that it results from spontaneous ligand-independent Receptor coupling to Gi. In contrast, the increased affinity of the mutant Receptors for binding agonists was unaffected by pertussis toxin treatment, indicating that this is an inherent property of the Receptors not dependent on interaction with Gi. Coexpression of the Receptor mutants with the Receptor-specific kinase, beta ARK1, indicated that the constitutively active alpha 2-Adrenergic Receptors are substrates for beta-Adrenergic Receptor kinase (beta ARK)-mediated phosphorylation even in the absence of agonist. These findings strengthen the idea that constitutively active Adrenergic Receptors mimic the "active" state of a G protein-coupled Receptor adopting conformations similar to those induced by agonist when it binds to wild type Receptors. In addition, these results extend the notion that in the Adrenergic Receptor family the C-terminal portion of the third intracellular loop plays a general role in the processes involved in Receptor activation.
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sites in the third intracellular loop of the alpha 2a Adrenergic Receptor confer short term agonist promoted desensitization evidence for a Receptor kinase mediated mechanism
Journal of Biological Chemistry, 1992Co-Authors: Stephen B Liggett, M G Caron, Hitoshi Kurose, Jacek Ostrowski, L C Chesnut, John R Raymond, Robert J LefkowitzAbstract:Abstract To investigate the mechanisms of agonist-promoted desensitization of the alpha 2-Adrenergic Receptor (alpha 2AR), the human alpha 2AAR and a mutated form of the Receptor were expressed in CHW cells. After cells were exposed to epinephrine for 30 min, the ability of the wild type alpha 2AAR to mediate inhibition of forskolin-stimulated adenylyl cyclase was depressed by approximately 78%. To assess the role of Receptor phosphorylation during desensitization, cells were incubated with 32Pi, exposed to agonist, and alpha 2AAR purified by immunoprecipitation with a fusion protein antibody. Agonist-promoted desensitization was found to be accompanied by phosphorylation of the alpha 2AAR in vivo. The beta-Adrenergic Receptor kinase (beta ARK) is known to phosphorylate purified alpha 2AAR in vitro. We found that heparin, a beta ARK inhibitor, ablated short term agonist-induced desensitization of alpha 2AAR, while such desensitization was unaffected by inhibition of protein kinase A. To further assess the role of beta ARK, we constructed a mutated alpha 2AAR which has a portion of the third intracellular loop containing 9 serines and threonines (potential phosphorylation sites) deleted. This mutated alpha 2AAR failed to undergo short term agonist-induced desensitization. Agonist promoted in vivo phosphorylation of this mutated Receptor was reduced by 90%, consistent with the notion that Receptor phosphorylation at sites in the third intracellular loop plays a critical role in alpha 2AAR desensitization. After 24 h of agonist exposure, an even more profound desensitization of alpha 2AAR occurred, which was not accompanied by a decrease in Receptor expression. Rather, long term agonist-induced desensitization was found to be due in part to a decrease in the amount of cellular Gi, which was not dependent on Receptor third loop phosphorylation sites.
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functional interactions of recombinant alpha 2 Adrenergic Receptor subtypes and g proteins in reconstituted phospholipid vesicles
Biochemistry, 1991Co-Authors: Hitoshi Kurose, John W Regan, Marc G. Caron, Robert J LefkowitzAbstract:The functional interaction of the recombinant alpha 2 Adrenergic Receptor subtypes, alpha 2-C10 (the human platelet alpha 2 Receptor, equivalent to the alpha 2 A subtype) and alpha 2-C4 (an alpha 2 Receptor subtype cloned from a human kidney cDNA library), with G proteins was characterized in an in vitro reconstitution system. These Receptor subtypes were overexpressed in COS-7 cells and were purified to a specific activity of 1.1-3.3 nmol/mg of protein. The G proteins consisted of Gs (adenylyl cyclase stimulatory) and members of the inhibitory family, including Gi1, Gi2, and Gi3, and G0. The cloned alpha subunits of these G proteins were overexpressed in Escherichia coli and were purified to homogeneity. Prior to use, G holoproteins were prepared by mixing the alpha subunits with beta gamma subunits that had been purified from bovine brain. Following reconstitution into phospholipid vesicles, both alpha 2 Receptor subtypes could couple to the inhibitory G proteins but not to Gs, as assessed by agonist stimulation of GTPase activity. The pharmacological specificity of this interaction was preserved with respect to the two Receptor subtypes. Between the different inhibitory G proteins, the alpha 2-C10 Adrenergic Receptor subtype showed the following preference: Gi3 greater than Gi1 greater than or equal to Gi2 greater than G0. The stimulation of GTPase activity (turnover number) ranged from 6.4-fold (Gi3) to 1.5-fold (G0). The preference of G-protein interaction for the alpha 2-C4 Receptor subtype was the same as that observed for the alpha 2-C10, but the extent of activation was slightly lower. The results show that in vitro each of the alpha 2 Adrenergic Receptor subtypes can activate multiple G proteins but that clear preferences exist with respect to the individual inhibitory G-protein subtypes. Additionally, it appears that alpha 2-C10 is coupled more efficiently to G-protein activation than is alpha 2-C4.
D B Bylund - One of the best experts on this subject based on the ideXlab platform.
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alpha 2 Adrenergic Receptor development in rat cns an autoradiographic study
Neuroscience, 2004Co-Authors: H K Happe, D B Bylund, Cynthia L Coulter, M E Gerety, J D Sanders, M Orourke, L C MurrinAbstract:Abstract During development norepinephrine plays a role in determining the morphologic organization of the CNS and the density and future responsiveness of Adrenergic Receptors. α-2 Adrenergic Receptors, one of three Adrenergic Receptor types, regulate important adult CNS functions and may have a distinct role during development. We examined α-2 Receptor distribution and density in the rat brain at postnatal days 1, 5, 10, 15, 21, 28 and in adults using the antagonist [ 3 H]RX821002 for autoradiography. Binding kinetics and pharmacology for α-2 Adrenergic Receptors were the same in adults and neonates. There was an overall increase in α-2 Receptor levels during postnatal development with great variability in pattern and timing of Receptor density changes among brain regions. Three major patterns were apparent. First, in many regions Receptor density increased during postnatal development, generally reaching adult levels around postnatal day 15. Within this group there was variability in timing between regions and there were several regions with Receptor densities higher than adult levels during the postnatal period. Second, there were regions with very high levels of Receptors at birth and little or no change in density during the postnatal period. Third, some regions demonstrated decreasing or transient expression of α-2 Adrenergic Receptors in the course of postnatal development, including white matter regions, cerebellum and many brainstem nuclei, suggesting specific roles for α-2 Receptors during development. This study investigates the development of α-2 Adrenergic Receptors in the rat CNS. It demonstrates there is region-specific regulation of α-2 Receptor development and identifies brain regions where these Receptors may play a specific and critical role in the regulation normal development.
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alpha 2 Adrenergic Receptor stimulation of phospholipase a2 and of adenylate cyclase in transfected chinese hamster ovary cells is mediated by different mechanisms
Molecular Pharmacology, 1991Co-Authors: S B Jones, S P Halenda, D B BylundAbstract:The effect of alpha 2-Adrenergic Receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-Adrenergic Receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic Receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipase A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-Adrenergic Receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism.