Navegando por Palavras-chave "GPCR"
Agora exibindo 1 - 9 de 9
Resultados por página
Opções de Ordenação
- ItemSomente MetadadadosCharacterization of a conformationally sensitive TOAC spin-labeled substance P(Elsevier B.V., 2008-11-01) Shafer, Aaron M.; Nakaie, Clovis R. [UNIFESP]; Deupi, Xavier; Bennett, Vicki J.; Voss, John C.; Univ Calif Davis; Northeastern Ohio Univ Coll Med & Pharm; Universidade Federal de São Paulo (UNIFESP); Univ Autonoma BarcelonaTo probe the binding of a peptide agonist to a G-protein coupled receptor in native membranes, the spin-labeled amino acid analogue 4-amino-4-carboxy-2,2,6,6-tetramethyl-piperidino-1-oxyl (TOAC) was substituted at either position 4 or 9 within the substance P peptide (RPKPQQFFGLM-NH2), a potent agonist of the neurokinin-1 receptor. the affinity of the 4-TOAC analog is comparable to the native peptide while the affinity of the 9-TOAC derivative is similar to 250-fold lower. Both peptides activate receptor signaling, though the potency of the 9-TOAC peptide is substantially lower. the utility of these modified ligands for reporting conformational dynamics during the neurokinin-1 receptor activation was explored using EPR spectroscopy, which can determine the real-time dynamics of the TOAC nitroxides in solution. While the binding of both the 4-TOAC substance P and 9-TOAC substance P peptides to isolated cell membranes containing the neurokinin-1 receptor is detected, a bound signal for the 9-TOAC peptide is only obtained under conditions that maintain the receptor in its high-affinity binding state. in contrast, 4-TOAC substance P binding is observed by solution EPR under both low- and high-affinity receptor states, with evidence of a more strongly immobilized peptide in the presence of GDP. in addition, to better understand the conformational consequences of TOAC substitution into substance P as it relates to receptor binding and activation, atomistic models for both the 4- and 9-TOAC versions of the peptide were constructed, and the molecular dynamics calculated via simulated annealing to explore the influence of the TOAC substitutions on backbone structure. (C) 2008 Elsevier Inc. All rights reserved.
- ItemSomente MetadadadosA fluorimetric binding assay for angiotensin II and kinin receptors(Elsevier Science Inc, 2016) Martin, Renan Paulo [UNIFESP]; Filippelli-Silva, Rafael [UNIFESP]; Rodrigues, Eliete da Silva [UNIFESP]; Nakaie, Clovis Ryuichi [UNIFESP]; Shimuta, Suma Imura [UNIFESP]Angiotensin II (AngII) and kinins (bradykinin (BK) and des-Arg9-bradykinin (DBK)), are potent agents involved in the maintenance of blood pressure and several biological activities, and their better understanding is important to produce new drugs aimed to control arterial blood pressure. Previous studies on ligand-receptor binding have been based on radioactive methods, which led us to study a new method based on the fluorimetric method. A lanthanide attached to the N-terminal segment of the peptide (AngII, BK and DBK), which produces a time-resolved-fluorescent ligand, was used in a binding test with CHO cells expressing the AT1, AT2, B1 or B2 receptors in comparison with the same cell line tested with the radioactive ligand. Our findings indicated that the nonradioactive-method provided a comparable result for the angiotensin receptors. On the other hand, the kinin receptors showed a slight reduction in the binding affinity, probably due to the linkage at the N-terminal segment and/or to the lower biological stability associated to the high temperature (37 degrees C) used for the fluorimetric method, while the radioactive one was at 4 degrees C. Wecan conclude that a time-resolved fluorescence assay would provide a sensitive method as an alternative tool for receptor studies. (C) 2016 Elsevier Inc. All rights reserved.
- ItemSomente MetadadadosFunctional characterization of heterotrimeric G-proteins in rat diaphragm muscle(Elsevier B.V., 2011-02-15) Andrade-Lopes, Ana Luiza [UNIFESP]; Pires-Oliveira, Marcelo [UNIFESP]; Sandro Menezes-Rodrigues, Francisco [UNIFESP]; Godinho, Rosely Oliveira [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Seven-transmembrane receptors mediate diverse skeletal muscle responses for a wide variety of stimuli, via activation of heterotrimeric G-proteins. Herein we evaluate the expression and activation of rat diaphragm or cultured skeletal muscle G-proteins using [(35)S]GTP gamma S. Total membrane G alpha subunit content was 4-7 times higher in rat primary cultured myotubes and L6 cell line than in diaphragm (32.6 +/- 1.2 fmol/mg protein) and 7-27% of them were in the active conformational state. Immunoprecipitation assay showed equal expression of diaphragm G alpha s, G alpha q and G alpha i/o. Addition of GDP allowed the measurement of G-protein activation by different GPCR, including adrenoceptor, adenosine, melatonin and muscarinic receptors. Diaphragm denervation resulted in a marked increase in both total and active state G-protein levels. Together, the results show that [(35)S]GTP gamma S binding assay is a sensitive and valuable method to evaluate GPCR activity in skeletal muscle cells, which is of particular interest for pharmacological analysis of drugs with potential use in the management of respiratory muscle failure. (C) 2010 Elsevier B.V. All rights reserved.
- ItemAcesso aberto (Open Access)Heavier-than-air flying machines are impossible(Elsevier B.V., 2004-04-30) Oliveira, Laerte [UNIFESP]; Hulsen, T.; Hulsik, D. L.; Paiva, Antonio Cechelli de Mattos [UNIFESP]; Vriend, Gerrit; KUN; Universidade Federal de São Paulo (UNIFESP); Univ UtrechtMany G protein-coupled receptor (GPCR) models have been built over the years. the release of the structure of bovine rhodopsin in August 2000 enabled us to analyze models built before that period to learn more about the models we build today. We conclude that the GPCR modelling field is riddled with 'common knowledge' similar to Lord Kelvin's remark in 1895 that heavier-than-air flying machines are impossible, and we summarize what we think are the (im)possibilities of modelling GPCRs using the coordinates of bovine rhodopsin as a template. Associated WWW pages: http://www.gpcr.org/ articles/2003_mod/. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosInteraction of a non-peptide agonist with angiotensin II AT(1) receptor mutants(Natl Research Council Canada, 2002-05-01) Costa-Neto, Claudio Miguel; Miyakawa, Ayumi A.; Pesquero, João Bosco [UNIFESP]; Oliveira, Laerte; Hjorth, Siv A.; Schwartz, Thue W.; Paiva, Antonio Cechelli de Mattos [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ CopenhagenTo identify residues of the rat AT(1A) angiotensin II receptor involved with signal transduction and binding of the non-peptide agonist L-162,313 (5,7-dimethyl-2-ethyl-3-[[4-[2(n-butyloxycarbonylsulfonamido)-5-isobutyl-3-thienyl]phenyl]methyl]imidazol[4,5,6]-pyridine) we have performed ligand binding and inositol phosphate turnover assays in COS-7 cells transiently transfected with the wild-type and mutant forms of the receptor. Mutant receptors bore modifications in the extracellular region: T88H, Y92H, G196I, G196W, and D278E. Compound L-162,313 displaced [I-125]-Sar(1),Leu(8)-AngII from the mutants G196I and G196W with IC50 values similar to that of the wild-type. the affinity was, however, slightly affected by the D278E mutation and more significantly by the T88H and Y92H mutations. in inositol phosphate turnover assays, the ability of L-162,313 to trigger the activation cascade was compared with that of angiotensin II. These assays showed that the G196W mutant reached a relative maximum activation exceeding that of the wild-type receptor; the efficacy was slightly reduced in the G196I mutant and further reduced in the T88H, Y92H, and D278E mutants. Our data suggest that residues of the extracellular domain of the AT(1) receptor are involved in the binding of the non-peptide ligand, or in a general receptor activation phenomenon that involves conformational modifications for a preferential binding of agonists or antagonists.
- ItemSomente MetadadadosThe interaction of Class B G protein-coupled receptors with their hormones(Harwood Acad Publ Gmbh, 1998-01-01) Horn, Florence; Bywater, Robert; Krause, G.; Kuipers, Wilma; Oliveira, Laerte [UNIFESP]; Paiva, Antonio Cechelli de Mattos [UNIFESP]; Sander, Chris; Vriend, Gerrit [UNIFESP]; European Mol Biol Lab; Novo Nordisk AS; Forschungsinst Mol Pharmakol; Solvay Duphar Med Chem; Universidade Federal de São Paulo (UNIFESP); European Bioinformat InstIn common with many G protein-coupled receptors, dysfunction in members of the Class B or glucagon-like receptors can elicit a wide spectrum of disease related activities. Consequently, they an potential targets in many different areas of pharmacological research. Unlike the class A or rhodopsin-like receptors, for which at least some structural similarity to bacteriorhodopsin has been detected, absolutely no structural information is available for the Class B G protein-coupled receptors,We present a computational study that exploits the experimental work performed by evolution to indicate residues that are potentially involved in ligand binding in the Class B G protein-coupled receptors.We perform an analysis of mutations that occurred in a correlated fashion between the receptors and their peptidic ligands, The inference that the residues detected in this manner are involved in a direct interaction between the receptor and the ligand is in good agreement with the mutation studies that have already been published.
- ItemSomente MetadadadosA low resolution model for the interaction of G proteins with G protein-coupled receptors(Oxford Univ Press, 1999-12-01) Oliveira, Laerte [UNIFESP]; Paiva, Antonio Cechelli de Mattos [UNIFESP]; Vriend, Gerrit; European Mol Biol Lab; Universidade Federal de São Paulo (UNIFESP)A model is presented for the interaction between G proteins and G protein-coupled receptors, the model is based on the fact that this interaction shows Little specificity and thus conserved parts of the G proteins have to interact with conserved parts of the receptors. These parts are a conserved negative residue in the G protein, a fully conserved arginine in the receptor and a series of residues that are not conserved but always hydrophobic like the hydrophobic side of the C-terminal helix of the G protein and the hydrophobic side of a helix in the C-terminal domain of the receptor. Other, mainly cytosolic, factors determine the specificity and regulation of this interaction. the relation between binding and activation will be shown. ii large body of experimental evidence supports this model. Despite the fact that the model does not provide atomic resolution, it can be used, to explain some experimental data that would otherwise seem inexplicable, and it suggests experiments for its falsification or verification.
- ItemSomente MetadadadosReceptors coupling to G proteins: Is there a signal behind the sequence?(Wiley-Blackwell, 2000-12-01) Horn, F.; van der Wenden, E. M.; Oliveira, Laerte [UNIFESP]; IJzerman, A. P.; Vriend, Gerrit; CMBI; European Mol Biol Lab; Leiden Univ; Universidade Federal de São Paulo (UNIFESP)Upon the binding of their ligands, G protein-coupled receptors couple to the heterotrimeric G proteins to transduce a signal. One receptor family may couple to a single G protein subtype and another family to several ones. Is there a signal in the receptor sequence that can give an indication of the G protein subtype selectivity? We used a sequence analysis method on biogenic amine and adenosine receptors and concluded that a weak signal can be detected in receptor families where specialization for coupling to a given G protein occurred during a recent divergent evolutionary process. Proteins 2000;41:448-459. (C) 2000 Wiley-Liss, Inc.
- ItemSomente MetadadadosA role for transmembrane domains V and VI in ligand binding and maturation of the angiotensin II AT1 receptor(Walter de Gruyter & Co, 2006-03-01) Pignatari, G. C.; Rozenfeld, R.; Ferro, E. S.; Oliveira, L.; Paiva, ACM; Devi, L. A.; Universidade Federal de São Paulo (UNIFESP); CUNY Mt Sinai Sch Med; Universidade de São Paulo (USP)Several studies have proposed that angiotensin II (Ang II) binds to its receptor AT1 through interactions with residues in helices V and VI, suggesting that the distance between these helices is crucial for ligand binding. Based on a 3D model of AT1 in which the C-terminus of Ang II is docked, we identified the hydrophobic residues of TM V and VI pointing towards the external face of the helices, which may play a role in the structure of the binding pocket and in the structural integrity of the receptor. We performed a systematic mutagenesis study of these residues and examined the binding, localization, maturation, and dimerization of the mutated receptors. We found that mutations of hydrophobic residues to alanine in helix V do not alter binding, whereas mutations to glutamate lead to loss of binding without a loss in cell surface expression, suggesting that the external face of helix V may not directly participate in binding, but may rather contribute to the structure of the binding pocket. in contrast, mutations of hydrophobic residues to glutamate in helix VI lead to a loss in cell surface expression, suggesting that the external surface of helix VI plays a structural role and ensures correct folding of the receptor.