15 results on '"Jon W. Lomasney"'
Search Results
2. Withdrawal: High glucose stimulates synthesis of fibronectin via a novel protein kinase C, Rap1b, and B-Raf signaling pathway
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Sumant S. Chugh, Sun Lin, Xiaomin Pan, Atul Sahai, Farhad R. Danesh, Elisabeth I. Wallner, Yashpal S. Kanwar, and Jon W. Lomasney
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Fibronectin ,biology ,Novel protein ,Chemistry ,Kinase ,High glucose ,biology.protein ,Cell Biology ,Signal transduction ,Molecular Biology ,Biochemistry ,Cell biology - Published
- 2019
3. Imprinted mesodermal specific transcript (MEST) and H19 genes in renal development and diabetes
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Christian S. Haas, Xiaomin Pan, Anil Kumar, Yashpal S. Kanwar, Jun Wada, Sun Lin, Jon W. Lomasney, and Gene Liau
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Male ,medicine.medical_specialty ,RNA, Untranslated ,Recombinant Fusion Proteins ,Mesenchyme ,Pregnancy in Diabetics ,Biology ,Kidney ,Antibodies ,Andrology ,Genomic Imprinting ,Mice ,03 medical and health sciences ,Organ Culture Techniques ,0302 clinical medicine ,Pregnancy ,Internal medicine ,mesodermal specific transcript ,Gene expression ,medicine ,Animals ,Diabetic Nephropathies ,RNA, Messenger ,Northern blot ,glucose ,030304 developmental biology ,2. Zero hunger ,Mice, Inbred ICR ,0303 health sciences ,Messenger RNA ,Expression vector ,integumentary system ,H19 ,diabetic nephropathy ,Embryogenesis ,Gene Expression Regulation, Developmental ,Proteins ,Endocrinology ,medicine.anatomical_structure ,Animals, Newborn ,Nephrology ,Hyperglycemia ,030220 oncology & carcinogenesis ,Ureteric bud ,Female ,RNA, Long Noncoding ,Genomic imprinting ,renal development - Abstract
Imprinted mesodermal specific transcript ( MEST ) and H19 genes in renal development and diabetes. Background Imprinted genes, mesodermal specific cDNA or transcript ( MEST ) and H19 , are implicated in peri-implantation embryogenesis, and their expression was assessed in embryonic kidneys undergoing glucose-induced dysmorphogenesis. Methods MEST and H19 mRNA expression was assessed by Northern blot analysis in embryonic kidneys of mice harvested at day 15 to day 19 of gestation and of 1-week-old mice obtained from hyperglycemic mothers. A full-length mouse MEST cDNA was isolated, subcloned into an expression vector, a recombinant protein prepared and an antibody raised; the latter was used to assess protein expression by immunoprecipitation and immunofluorescence microscopy in day 13 metanephric explants subjected to high glucose ambience. Also, MEST mRNA expression was assessed in high d glucose–treated explants by competitive reverse transcription-polymerase chain reaction (RT-PCR) analyses and by in situ tissue autoradiography. Results A high expression of MEST and H19 with respective transcript size of ∼2.7 and ∼2.4 kb was observed in fetal kidneys, and their expression decreased during the successive stages of gestation and was undetectable in the postnatal period. At day 13, the MEST mRNA was expressed in the mesenchyme, while H19 was expressed in the ureteric bud branches and epithelial elements of the metanephros. Their expression decreased with progression of gestation. By competitive RT-PCR and Northern blot and in situ autoradiographic analyses, both MEST and H19 expressions decreased in day 13 explants treated with high glucose and in the kidneys of fetuses obtained from diabetic mothers. The MEST protein expression was observed in the metanephric epithelial elements and ureteric bud branches instead of in the mesenchyme, and its expression decreased in glucose-treated dysmorphogenetic explants, as assessed by immunofluorescence and immunoprecipitation methods. Conclusion MEST and H19 imprinted genes are strategically located in the mammalian embryonic metanephros. They are developmentally regulated and their concomitant decreased expression in high glucose ambience or diabetic state did not follow the prevailing dogma of reciprocal inactivation/activation of imprinted genes, and such a decrease may be responsible for the perturbed epithelial:mesenchymal interactions leading to dysmorphogenesis of the mammalian metanephros.
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- 2003
4. A Novel Bifunctional Phospholipase C That Is Regulated by Gα12 and Stimulates the Ras/Mitogen-activated Protein Kinase Pathway
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Eric C. Mak, Jon W. Lomasney, Heidi E. Hamm, Jirong Ding, and Isabel Lopez
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DNA, Complementary ,Molecular Sequence Data ,Small G Protein ,Biology ,GTP-Binding Protein alpha Subunits, G12-G13 ,Biochemistry ,Phosphoinositide Phospholipase C ,Heterotrimeric G protein ,Humans ,Amino Acid Sequence ,Cloning, Molecular ,Protein kinase A ,Molecular Biology ,Diacylglycerol kinase ,Sequence Homology, Amino Acid ,Phospholipase C ,Cell Biology ,Heterotrimeric GTP-Binding Proteins ,Cell biology ,Type C Phospholipases ,Second messenger system ,ras Proteins ,Guanine nucleotide exchange factor ,Mitogen-Activated Protein Kinases ,Signal transduction ,Signal Transduction - Abstract
Three families of phospholipase C (PI-PLCbeta, gamma, and delta) are known to catalyze the hydrolysis of polyphosphoinositides such as phosphatidylinositol 4,5-bisphosphate (PIP(2)) to generate the second messengers inositol 1,4,5 trisphosphate and diacylglycerol, leading to a cascade of intracellular responses that result in cell growth, cell differentiation, and gene expression. Here we describe the founding member of a novel, structurally distinct fourth family of PI-PLC. PLCepsilon not only contains conserved catalytic (X and Y) and regulatory domains (C2) common to other eukaryotic PLCs, but also contains two Ras-associating (RA) domains and a Ras guanine nucleotide exchange factor (RasGEF) motif. PLCepsilon hydrolyzes PIP(2), and this activity is stimulated selectively by a constitutively active form of the heterotrimeric G protein Galpha(12). PLCepsilon and a mutant (H1144L) incapable of hydrolyzing phosphoinositides promote formation of GTP-Ras. Thus PLCepsilon is a RasGEF. PLCepsilon, the mutant H1144L, and the isolated GEF domain activate the mitogen-activated protein kinase pathway in a manner dependent on Ras but independent of PIP(2) hydrolysis. Our findings demonstrate that PLCepsilon is a novel bifunctional enzyme that is regulated by the heterotrimeric G protein Galpha(12) and activates the small G protein Ras/mitogen-activated protein kinase signaling pathway.
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- 2001
5. Contrast-enhanced magnetic resonance imaging of myocardium at risk
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Francis J. Klocke, Robert M. Judd, Jon W. Lomasney, Enn-Ling Chen, Raymond J. Kim, and David S. Fieno
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biology ,medicine.diagnostic_test ,business.industry ,Fissipedia ,Infarction ,Magnetic resonance imaging ,030204 cardiovascular system & hematology ,biology.organism_classification ,medicine.disease ,3. Good health ,030218 nuclear medicine & medical imaging ,03 medical and health sciences ,0302 clinical medicine ,medicine.anatomical_structure ,Ventricle ,In vivo ,medicine ,Carnivora ,business ,Nuclear medicine ,Cardiology and Cardiovascular Medicine ,Contrast-enhanced Magnetic Resonance Imaging ,Ex vivo - Abstract
OBJECTIVES We sought to determine the relationship of delayed hyperenhancement by contrast magnetic resonance imaging (MRI) to viable and nonviable myocardium within the region at risk throughout infarct healing. BACKGROUND The relationship of delayed MRI contrast enhancement patterns to injured but viable myocardium within the ischemic bed at risk has not been established. METHODS We compared in vivo and ex vivo MRI contrast enhancement to histopathologic tissue sections encompassing the entire left ventricle in dogs (n = 24) subjected to infarction with (n = 12) and without (n = 12) reperfusion at 4 h, 1 day, 3 days, 10 days, 4 weeks and 8 weeks. In vivo MR imaging was performed 30 min after contrast injection. RESULTS The sizes and shapes of in vivo myocardial regions of elevated image intensity (828 ± 132% of remote) were the same as those observed ex vivo (241 slices, r = 0.99, bias = 0.05 ± 1.6% of left ventricle [LV]). Comparison of ex vivo MRI to triphenyltetrazolim chloride–stained sections demonstrated that the spatial extent of hyperenhancement was the same as the spatial extent of infarction at every stage of healing (510 slices, lowest r = 0.95, largest bias = 1.7 ± 2.9% of LV). Conversely, hyperenhanced regions were smaller than the ischemic bed at risk defined by fluorescent microparticles at every stage of healing (239 slices, 35 ± 24% of risk region, p CONCLUSIONS Delayed contrast enhancement by MRI distinguishes between viable and nonviable regions within the myocardium at risk throughout infarct healing.
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- 2000
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6. Up-regulation of vascular endothelial growth factor mrna and angiogenesis after transmyocardial laser revascularization
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Dipen C Maun, William H. Pearce, Rodney Greene, Jon W. Lomasney, David A. Fullerton, Keith A. Horvath, and Elaine Chiu
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Pulmonary and Respiratory Medicine ,Pathology ,medicine.medical_specialty ,Vascular disease ,Angiogenesis ,business.industry ,medicine.medical_treatment ,Ischemia ,medicine.disease ,Revascularization ,Angina ,Vascular endothelial growth factor ,chemistry.chemical_compound ,chemistry ,Downregulation and upregulation ,medicine ,Immunohistochemistry ,Surgery ,Cardiology and Cardiovascular Medicine ,business - Abstract
Background. Angiogenesis has been proposed as a potential mechanism whereby transmyocardial laser revascularization (TMLR) has provided clinical relief of angina. Experimental work has found histologic evidence supporting this, as well as an improved response when angiogenic growth factors have been added to TMLR. The purpose of this study was to demonstrate that the molecular response to TMLR was an increase in the production of endogenous vascular endothelial growth factor to promote angiogenesis. Methods. Ameroid constrictors were placed on the proximal circumflex artery in 12 domestic pigs. After a chronic ischemic zone was established the animals were randomly divided into two groups. In the TMLR group the ischemic zone was treated with carbon dioxide laser. In the control group the ischemic zone was untreated. Six weeks later the animals were sacrificed, and sections from the ischemic zone and the nonischemic zone were submitted for immunohistochemical, histologic, and molecular analysis. Messenger RNA was obtained from northern blot analysis after being probed with vascular endothelial growth factor. Results. There was a twofold increase in the vascular endothelial growth factor messenger RNA in the ischemic zone of the TMLR group compared with the control group. Additionally, there was a threefold increase in the number of new blood vessels in the ischemic zone of the TMLR group compared with the control group. Conclusions. Transmyocardial laser revascularization promotes angiogenesis by upregulation of vascular endothelial growth factor. The resulting angiogenesis could be the principle mechanism for the clinical efficacy of TMLR.
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- 1999
7. Activation of Phospholipase C δ1 through C2 Domain by a Ca2+-Enzyme-Phosphatidylserine Ternary Complex
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Jon W. Lomasney, Klim King, Hwei Fang Cheng, and Steve R. Roffler
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Phosphatidylinositol 4,5-Diphosphate ,Phosphatidylserines ,Phospholipase C gamma ,Biochemistry ,Catalysis ,chemistry.chemical_compound ,Phosphatidylcholine ,Molecular Biology ,Ternary complex ,Micelles ,Phospholipids ,Phosphatidylethanolamine ,Binding Sites ,Phospholipase C ,Chemistry ,Vesicle ,Cell Biology ,Phosphatidylserine ,Phosphatidic acid ,Recombinant Proteins ,Enzyme Activation ,Isoenzymes ,Kinetics ,Amino Acid Substitution ,Models, Chemical ,Type C Phospholipases ,Liposomes ,Biophysics ,Calcium - Abstract
The concentration of free Ca(2+) and the composition of nonsubstrate phospholipids profoundly affect the activity of phospholipase C delta1 (PLCdelta1). The rate of PLCdelta1 hydrolysis of phosphatidylinositol 4,5-bisphosphate was stimulated 20-fold by phosphatidylserine (PS), 4-fold by phosphatidic acid (PA), and not at all by phosphatidylethanolamine or phosphatidylcholine (PC). PS reduced the Ca(2+) concentration required for half-maximal activation of PLCdelta1 from 5.4 to 0.5 microM. In the presence of Ca(2+), PLCdelta1 specifically bound to PS/PC but not to PA/PC vesicles in a dose-dependent and saturable manner. Ca(2+) also bound to PLCdelta1 and required the presence of PS/PC vesicles but not PA/PC vesicles. The free Ca(2+) concentration required for half-maximal Ca(2+) binding was estimated to be 8 microM. Surface dilution kinetic analysis revealed that the K(m) was reduced 20-fold by the presence of 25 mol % PS, whereas V(max) and K(d) were unaffected. Deletion of amino acid residues 646-654 from the C2 domain of PLCdelta1 impaired Ca(2+) binding and reduced its stimulation and binding by PS. Taken together, the results suggest that the formation of an enzyme-Ca(2+)-PS ternary complex through the C2 domain increases the affinity for substrate and consequently leads to enzyme activation.
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- 1999
8. A Single Amino Acid Substitution in the Pleckstrin Homology Domain of Phospholipase C δ1 Enhances the Rate of Substrate Hydrolysis
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Paul Andrew Bromann, Evan E. Boetticher, and Jon W. Lomasney
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Phosphatidylinositol 4,5-Diphosphate ,Stereochemistry ,Inositol 1,4,5-Trisphosphate ,Phosphatidylinositols ,Biochemistry ,Michaelis–Menten kinetics ,Structure-Activity Relationship ,chemistry.chemical_compound ,Enzyme activator ,Humans ,Point Mutation ,Phosphatidylinositol ,Molecular Biology ,Phospholipase C ,biology ,Chemistry ,Hydrolysis ,Blood Proteins ,Cell Biology ,Phosphoproteins ,Enzyme structure ,Enzyme assay ,Enzyme Activation ,Pleckstrin homology domain ,Dissociation constant ,Kinetics ,Type C Phospholipases ,biology.protein - Abstract
The pleckstrin homology (PH) domain has been postulated to serve as an anchor for enzymes that operate at a lipid/water interface. To understand further the relationship between the PH domain and enzyme activity, a phospholipase C (PLC) delta1/PH domain enhancement-of-activity mutant was generated. A lysine residue was substituted for glutamic acid in the PH domain of PLC delta1 at position 54 (E54K). Purified native and mutant enzymes were characterized using a phosphatidylinositol 4,5-bisphosphate (PI(4, 5)P2)/dodecyl maltoside mixed micelle assay and kinetics measured according to the dual phospholipid model of Dennis and co-workers (Hendrickson, H. S., and Dennis, E. A. (1984) J. Biol. Chem. 259, 5734-5739; Carmen, G. M., Deems, R. A., and Dennis, E. A. (1995) J. Biol. Chem. 270, 18711-18714). Our results show that both PLC delta1 and E54K bind phosphatidylinositol bisphosphate cooperatively (Hill coefficients, n = 2.2 +/- 0.2 and 2.0 +/- 0.1, respectively). However, E54K shows a dramatically increased rate of (PI(4, 5)P2)-stimulated PI(4,5)P2 hydrolysis (interfacial Vmax for PLC delta1 = 4.9 +/- 0.3 micromol/min/mg and for E54K = 31 +/- 3 micromol/min/mg) as well as PI hydrolysis (Vmax for PLC delta1 = 27 +/- 3.4 nmol/min/mg and for E54K = 95 +/- 12 nmol/min/mg). In the absence of PI(4,5)P2 both native and mutant enzyme hydrolyze PI at similar rates. E54K also has a higher affinity for micellar substrate (equilibrium dissociation constant, Ks = 85 +/- 36 microM for E54K and 210 +/- 48 microM for PLC delta1). Centrifugation binding assays using large unilamelar phospholipid vesicles confirm that E54K binds PI(4,5)P2 with higher affinity than native enzyme. E54K is more active even though the interfacial Michaelis constant (Km) for E54K (0.034 +/- 0.01 mol fraction PI(4,5)P2) is higher than the Km for native enzyme (0.012 +/- 0.002 mol fraction PI(4,5)P2). D-Inositol trisphosphate is less potent at inhibiting E54K PI(4,5)P2 hydrolysis compared with native enzyme. These results demonstrate that a single amino acid substitution in the PH domain of PLC delta1 can dramatically enhance enzyme activity. Additionally, the marked increase in Vmax for E54K argues for a direct role of PH domains in regulating catalysis by allosteric modulation of enzyme structure.
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- 1997
9. Cloning and Identification of Amino Acid Residues of Human Phospholipase Cδ1 Essential for Catalysis
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Chih-Lin Chen, Meei.Jyh Jiang, Klim King, Hwei-Fang Cheng, Li-Ping Wong, Su-Min Liu, and Jon W. Lomasney
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Phosphatidylinositol 4,5-Diphosphate ,DNA, Complementary ,Molecular Sequence Data ,Restriction Mapping ,Mutant ,Phospholipid ,Saccharomyces cerevisiae ,Biology ,Phospholipase ,Polymerase Chain Reaction ,Biochemistry ,chemistry.chemical_compound ,Phosphatidylinositol Phosphates ,Escherichia coli ,medicine ,Animals ,Humans ,Point Mutation ,Inositol ,Amino Acid Sequence ,Phosphatidylinositol ,Cloning, Molecular ,Binding site ,Molecular Biology ,Aorta ,Conserved Sequence ,DNA Primers ,Gene Library ,chemistry.chemical_classification ,Binding Sites ,Base Sequence ,Sequence Homology, Amino Acid ,Cell Biology ,Trypsin ,Molecular biology ,Recombinant Proteins ,Rats ,Isoenzymes ,Molecular Weight ,Kinetics ,Enzyme ,chemistry ,Type C Phospholipases ,Mutagenesis, Site-Directed ,Calcium ,Drosophila ,lipids (amino acids, peptides, and proteins) ,medicine.drug - Abstract
In vitro single point mutagenesis, inositol phospholipid hydrolysis, and substrate protection experiments were used to identify catalytic residues of human phosphatidylinositide-specific phospholipase C delta 1 (PLC delta 1) isolated from a human aorta cDNA library. Invariant amino acid residues containing a functional side chain in the highly conserved X region were changed by in vitro mutagenesis. Most of the mutant enzymes were still able to hydrolyze inositol phospholipid with activity ranging from 10 to 100% of levels in the wild type enzyme. Exceptions were mutants with the conversion of Arg338 to Leu (R338L), Glu341 to Gly (E341G), or His356 to Leu (H356L), which made the enzyme severely defective in hydrolyzing inositol phospholipid. Phospholipid vesicle binding experiments showed that these three cleavage-defective mutant forms of PLC delta 1 could specifically bind to phosphatidylinositol 4,5-bisphosphate (PIP2) with an affinity similar to that of wild type enzyme. Western blotting analysis of trypsin-treated enzyme-PIP2 complexes revealed that a 67-kDa major protein fragment survived trypsin digestion if the wild type enzyme, E341G, or H356L mutant PLC delta 1 was preincubated with 7.5 microM PIP2, whereas if it was preincubated with 80 microM PIP2, the size of major protein surviving was comparable to that of intact enzyme. However, mutant enzyme R338L was not protected from trypsin degradation by PIP2 binding. These observations suggest that PLC delta 1 can recognize PIP2 through a high affinity and a low affinity binding site and that residues Glu341 and His356 are not involved in either high affinity or low affinity PIP2 binding but rather are essential for the Ca(2+)-dependent cleavage activity of PLC.
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- 1995
10. Lipid-mediated Regulation of G Protein-coupled Receptor Kinases 2 and 3
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Judy Ptasienski, Evan E. Boetticher, Shubhik K. DebBurman, M. Marlene Hosey, Jon W. Lomasney, and Jeffrey L. Benovic
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G-Protein-Coupled Receptor Kinase 3 ,Macromolecular Substances ,G protein ,Recombinant Fusion Proteins ,Membrane lipids ,Protein Serine-Threonine Kinases ,Spodoptera ,Biology ,Transfection ,Binding, Competitive ,Biochemistry ,chemistry.chemical_compound ,GTP-Binding Proteins ,Animals ,Humans ,Phosphatidylinositol ,Molecular Biology ,Phospholipids ,Glutathione Transferase ,G protein-coupled receptor ,G protein-coupled receptor kinase ,Kinase ,Cell Membrane ,Autophosphorylation ,Receptor Protein-Tyrosine Kinases ,Cell Biology ,Cyclic AMP-Dependent Protein Kinases ,Receptors, Muscarinic ,Cell biology ,Enzyme Activation ,Pleckstrin homology domain ,Kinetics ,chemistry ,beta-Adrenergic Receptor Kinases - Abstract
G protein-coupled receptor-mediated signaling is attenuated by a process referred to as desensitization, wherein agonist-dependent phosphorylation of receptors by G protein-coupled receptor kinases (GRKs) is proposed to be a key initial event. However, mechanisms that activate GRKs are not fully understood. In one scenario, beta gamma-subunits of G proteins (G beta gamma) activate certain GRKs (beta-adrenergic receptor kinases 1 and 2, or GRK2 and GRK3), via a pleckstrin homology domain in the COOH terminus. This interaction has been proposed to translocate cytosolic beta-adrenergic receptor kinases (beta ARKs) to the plasma membrane and facilitate interaction with receptor substrates. Here, we report a novel finding that membrane lipids modulate beta ARK activity in vitro in a manner that is analogous and competitive with G beta gamma. Several lipids, including phosphatidylserine (PS), stimulated, whereas phosphatidylinositol 4,5-bisphosphate inhibited, the ability of these GRKs to phosphorylate agonist-occupied m2 muscarinic acetylcholine receptors. Furthermore, both PS and phosphatidylinositol 4,5-bisphosphate specifically bound to beta ARK1, whereas phosphatidylcholine, a lipid that did not modulate beta ARK activity, did not bind to beta ARK1. The lipid regulation of beta ARKs did not occur via a modulation of its autophosphorylation state. PS- and G beta gamma-mediated stimulation of beta ARK1 was compared and found strikingly similar; moreover, their effects together were not additive (except at initial stages of reaction), which suggests that PS and G beta gamma employed a common interaction and activation mechanism with the kinase. The effects of these lipids were prevented by two well known G beta gamma-binding proteins, phosducin and GST-beta ARK-(466-689) fusion protein, suggesting that the G beta gamma-binding domain (possibly the pleckstrin homology domain) of the GRKs is also a site for lipid:protein interaction. We submit the intriguing possibility that both lipids and G proteins co-regulate the function of GRKs.
- Published
- 1995
11. Distribution of α2-adrenergic receptor subtype gene expression in rat brain
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Diane M. Hayden-Hixson, Mika Scheinin, Robert T. Fremeau, Robert J. Lefkowitz, Marc G. Caron, Uta B. Schambra, and Jon W. Lomasney
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Male ,Central nervous system ,Gene Expression ,Hippocampus ,Alpha (ethology) ,In situ hybridization ,Biology ,Sulfur Radioisotopes ,Rats, Sprague-Dawley ,Cellular and Molecular Neuroscience ,Receptors, Adrenergic, alpha-2 ,Postsynaptic potential ,Gene expression ,medicine ,Animals ,Molecular Biology ,In Situ Hybridization ,Brain ,RNA Probes ,Rats ,Antisense Elements (Genetics) ,medicine.anatomical_structure ,Organ Specificity ,Cerebral cortex ,Autoradiography ,Locus coeruleus ,Neuroscience - Abstract
alpha 2-Adrenergic receptors in brain are important presynaptic modulators of central noradrenergic function (autoreceptors) and postsynaptic mediators of many of the widespread effects of catecholamines and related drugs. alpha 2-Adrenergic agonists are currently used as antihypertensives and preanesthetic agents, but new subtype-selective alpha 2-adrenoceptor agonists and antagonists have additional therapeutic application potential. Three genes encoding specific alpha 2-adrenoceptor subtypes (alpha 2A, alpha 2B, and alpha 2C) have been isolated and characterized. RNA blotting indicates that all three are expressed in rat brain. This study used in situ hybridization with 35S-labeled RNA probes to map the distribution of alpha 2-adrenoceptor subtype gene expression in rat brain. alpha 2A mRNA was most abundant in the locus coeruleus, but was also widely distributed in the brain stem, cerebral cortex, septum, hypothalamus, hippocampus and amygdala. alpha 2B mRNA was observed only in the thalamus. alpha 2C mRNA was mainly localized to the basal ganglia, olfactory tubercle, hippocampus, and cerebral cortex. These mRNA distributions largely agree with previous findings on the alpha 2-adrenoceptor distributions in the rat brain, but suggest that the localization patterns for each receptor subtype are unique. The expression of alpha 2A mRNA in noradrenergic neurons indicates that this subtype mediates presynaptic autoreceptor functions. Furthermore, the localization of alpha 2A mRNA in noradrenergic projection areas suggests that this receptor may also have an important role in mediating postsynaptic effects. The precise physiological and pharmacological roles of the alpha 2-adrenoceptor subtypes are still largely unknown, but it is expected that in situ hybridization coupled to various methods to identify the transmitter phenotypes of the subtype-expressing neurons will help to clarify these important issues in the near future.
- Published
- 1994
12. Pharmacologic characterization of cloned α1-adrenoceptor subtypes: selective antagonists suggest the existence of a fourth subtype
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Jon W. Lomasney and Debra A. Schwinn
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Pharmacology ,Spiperone ,Binding Sites ,Niguldipine ,Receptors, Adrenergic, alpha ,Biology ,biology.organism_classification ,Binding, Competitive ,In vitro ,HeLa ,chemistry.chemical_compound ,chemistry ,Biochemistry ,Cell culture ,Cell surface receptor ,medicine ,Benoxathian ,Receptor ,Adrenergic alpha-Agonists ,Adrenergic alpha-Antagonists ,medicine.drug - Abstract
In membranes prepared from Cos-7 or HeLa cells expressing one of three individual cloned alpha 1-adrenoceptor subtypes, competition with 2-[(beta-(4-hydroxy-3-[125I]iodophenyl)ethylaminomethyl]-tetralone ([125I]HEAT) by the selective compounds [+]-niguldipine, 5-methyl-urapidil, and benoxathian reveals high affinity for the cloned alpha 1C-adrenoceptor subtype and low affinity for both the cloned alpha 1A-adrenoceptor and alpha 1B-adrenoceptor. Competition with [125I]HEAT by spiperone revealed high affinity for the cloned alpha 1C-adrenoceptor, intermediate affinity for the cloned alpha 1B-adrenoceptor, and low affinity for the cloned alpha 1A-adrenoceptor. Combining pharmacological properties previously described for alpha 1-adrenoceptor subtypes in rat membranes and here described from cloned receptors, these data suggest the existence of a fourth distinct alpha 1-adrenoceptor subtype.
- Published
- 1992
13. Molecular cloning and expression of the cDNA for the alpha 1A-adrenergic receptor. The gene for which is located on human chromosome 5
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Robert J. Lefkowitz, Michael J. Brownstein, Wah Ying Leung, Debra A. Schwinn, Teresa L. Yang-Feng, Marc G. Caron, Jon W. Lomasney, Susanna Cotecchia, and Wulfing Lorenz
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Estrogen-related receptor alpha ,Thyroid hormone receptor alpha ,Retinoic acid receptor alpha ,Interleukin 5 receptor alpha subunit ,Liver X receptor alpha ,5-HT5A receptor ,Cell Biology ,Biology ,Molecular Biology ,Biochemistry ,Alpha-1A adrenergic receptor ,Molecular biology ,Interleukin 10 receptor, alpha subunit - Abstract
Pharmacological and molecular cloning studies have demonstrated heterogeneity of alpha 1-adrenergic receptors. We have now cloned two alpha 1-adrenergic receptors from a rat cerebral cortex cDNA library, using the hamster alpha 1B-adrenergic receptor as a probe. The deduced amino acid sequence of clone RA42 encodes a protein of 560 amino acids whose putative topology is similar to that of the family of G-protein-coupled receptors. The primary structure though most closely resembles that of an alpha 1-adrenergic receptor, having approximately 73% amino acid identity in the putative transmembrane domains with the previously isolated hamster alpha 1B receptor. Analysis of the ligand binding properties of RA42 expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique alpha 1-adrenergic receptor pharmacology. High affinity for the antagonist WB4101 and agonists phenylephrine and methoxamine suggests that cDNA RA42 encodes the alpha 1A receptor subtype. Northern blot analysis of various rat tissues also shows the distribution expected of the alpha 1A receptor subtype with abundant expression in vas deferens followed by hippocampus, cerebral cortex, aorta, brainstem, heart and spleen. The second alpha 1-adrenergic receptor cloned represents the rat homolog of the hamster alpha 1B subtype. Expression of mRNA for this receptor is strongly detected in liver followed by heart, cerebral cortex, brain stem, kidney, lung, and spleen. This study provides definitive evidence for the existence of three alpha 1-adrenergic receptor subtypes.
- Published
- 1991
14. Molecular cloning and expression of the cDNA for a novel alpha 1-adrenergic receptor subtype
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Robert J. Lefkowitz, Teresa L. Yang-Feng, Susanna Cotecchia, Debra A. Schwinn, Marc G. Caron, Robert T. Fremeau, Jon W. Lomasney, Pamela J. Szklut, and Wulfing Lorenz
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Thyroid hormone receptor alpha ,Retinoic acid receptor alpha ,Interleukin 5 receptor alpha subunit ,Liver X receptor alpha ,5-HT5A receptor ,Cell Biology ,Biology ,Alpha-1D adrenergic receptor ,Molecular Biology ,Biochemistry ,Molecular biology ,Alpha-2B adrenergic receptor ,Interleukin 10 receptor, alpha subunit - Abstract
A novel alpha 1-adrenergic receptor subtype has been cloned from a bovine brain cDNA library. The deduced amino acid sequence is that of a 466-residue polypeptide. The structure is similar to that of the other adrenergic receptors as well as the larger family of G protein-coupled receptors that have a presumed seven-membrane-spanning domain topography. The greatest sequence identity of this receptor protein is with the previously cloned hamster alpha 1B-adrenergic receptor being approximately 72% within the presumed membrane-spanning domains. Localization on different human chromosomes provides evidence that the bovine cDNA is distinct from the hamster alpha 1B-adrenergic receptor. The bovine cDNA clone expressed in COS7 cells revealed 10-fold higher affinity for the alpha 1-adrenergic antagonists WB4101 and phentolamine and the agonist oxymetazoline as compared with the alpha 1B receptor, results similar to pharmacologic binding properties described for the alpha 1A receptor. Despite these similarities in pharmacological profiles, the bovine alpha 1-adrenergic receptor is sensitive to inhibition by the alkylating agent chloroethylclonidine unlike the alpha 1A-adrenergic receptor subtype. In addition, a lack of expression in tissues where the alpha 1A subtype exists suggests that this receptor may actually represent a novel alpha 1-adrenergic receptor subtype not previously appreciated by pharmacological criteria.
- Published
- 1990
15. Mammalian alpha 1-adrenergic receptor. Purification and characterization of the native receptor ligand binding subunit
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Jon W. Lomasney, L. M.F. Leeb-Lundberg, John W. Regan, J. F. Debernardis, Robert J. Lefkowitz, M G Caron, and Susanna Cotecchia
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chemistry.chemical_classification ,Gel electrophoresis ,Chemistry ,Stereochemistry ,Protein subunit ,Peptide ,Cell Biology ,Ligand (biochemistry) ,Biochemistry ,Receptor–ligand kinetics ,chemistry.chemical_compound ,Digitonin ,Receptor ,Molecular Biology ,Alpha-1 adrenergic receptor - Abstract
alpha 1-Adrenergic receptors from a cultured smooth muscle cell line (DDT1 MF-2) have been solubilized with digitonin and purified to apparent homogeneity by sequential chromatography on a biospecific affinity support (Sepharose-A55453 (4-amino-6,7-dimethoxy-2-[4-[5-(4-amino-3-phenyl) pentanoyl]-1-piperazinyl]-quinazoline), an alpha 1 receptor-selective antagonist), a wheat germ agglutinin-agarose gel, and a high performance steric exclusion liquid chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveals a peptide with an apparent Mr = 80,000 that co-migrates with the peptide labeled by the specific alpha 1-adrenergic receptor photoaffinity probe 4-amino-6,7-dimethoxy-2-[4-[5-(4-azido-3-[125I]iodophenyl)pentanoyl] -1-piperazinyl] quinazoline. The specific activity (approximately 13,600 pmol of ligand binding/mg of protein) of purified receptor preparations is consistent with that expected for a pure peptide of Mr = 80,000 containing a single ligand binding site. Overall yields approximate 14% of initial crude particulate binding. The purified receptor preparations bind agonist and antagonist ligands with appropriate alpha 1-adrenergic specificity, stereoselectivity, and affinity. Peptide maps of the pure alpha 1-adrenergic receptor and the pure human platelet alpha 2-adrenergic receptor (Regan, J.W., Nakata, H., DeMarinis, R.M., Caron, M.G., and Lefkowitz, R.J. (1986) J. Biol. Chem. 261, 3894-3900) using several different proteases suggest that these two receptors show little if any structural homology.
- Published
- 1986
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