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3.
J Cell Mol Med ; 12(3): 1049-60, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18494944

RESUMO

Coronary artery smooth muscle expresses the plasma membrane Ca(2+) pump (PMCA) isoforms PMCA4 and PMCA1. We previously reported the peptide inhibitor caloxin 1b1 that was obtained by using extracellular domain 1 of PMCA4 as the target (Am J Physiol Cell.290 [2006] C1341). To engineer inhibitors with greater affinity and isoform selectivity, we have now created a phage display library of caloxin 1b1-like peptides. We screened this library by affinity chromatography with PMCA from erythrocyte ghosts that contain mainly PMCA4 to obtain caloxin 1c2. Key properties of caloxin 1c2 are (a) Ki = 2.3 +/- 0.3 microM which corresponds to a 20x higher affinity for PMCA4 than that of caloxin 1b1 and (b) it is selective for PMCA4 since it has greater than 10-fold affinity for PMCA4 than for PMCA1, 2 or 3. It had the following functional effects on coronary artery smooth muscle: (a) it increased basal tone of the de-endothelialized arteries; the increase being similar at 10, 20 or 50 microM, and (b) it enhanced the increase in the force of contraction at 0.05 but not at 1.6 mM extracellular Ca(2+) when Ca(2+) extrusion via the Na(+)-Ca(2+) exchanger and the sarco/endoplasmic reticulum Ca(2+) pump were inhibited. We conclude that PMCA4 is pivotal to Ca(2+) extrusion in coronary artery smooth muscle. We anticipate caloxin 1c2 to aid in understanding the role of PMCA4 in signal transduction and home-ostasis due to its isoform selectivity and ability to act when added extracellularly.


Assuntos
ATPase de Ca(2+) e Mg(2+)/antagonistas & inibidores , ATPase de Ca(2+) e Mg(2+)/fisiologia , Membrana Celular/fisiologia , Vasos Coronários/fisiologia , Peptídeos/administração & dosagem , Engenharia de Proteínas , Animais , Membrana Celular/efeitos dos fármacos , Vasos Coronários/efeitos dos fármacos , Mutagênese , Biblioteca de Peptídeos , Peptídeos/síntese química , Peptídeos/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Suínos
4.
J Pept Sci ; 13(9): 575-80, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17600857

RESUMO

Photoaffinity labelling is regularly used to investigate proteins, including peptidergic G protein-coupled receptors (GPCR). To this purpose benzophenone photolabels have been widely used to identify many contact residues in ligand-binding pockets. The three-dimensional binding environment of the human angiotensin II type 1 receptor hAT(1) has been determined using an iterative methionine mutagenesis strategy based on the photochemical properties and preferential incorporation of benzophenone onto methionine. This has led to the construction of a ligand-bound receptor structure. The present study investigated the effect of temperature on the accessibility of some of these contact points. The hAT(1) receptor and two representative Met mutants (H256M-hAT(1) and F293M-hAT(1)) from the iterative mutagenesis study were photolabelled with the benzophenone-ligand (125)I-[Sar(1), Bpa(8)]AngII at temperatures ranging from - 15 degrees C to 37 degrees C. Labelled receptors were partially purified and digested with cyanogen bromide to identify the contact points or segments. There were no changes in receptor contacts or labelling in the 7th transmembrane domains (TMD) of hAT(1) and F293M-hAT(1) across the temperature range. However, a temperature-dependent change in the ligand-receptor contact of H256M-hAT(1) was observed. At - 15 degrees C, H256M labelling was identical to that of hAT(1), indicating that the interaction was specific to the 7th TMD. Significant labelling changes were observed at higher temperatures and at 37 degrees C labelling occurred almost exclusively at mutated residue H256M-hAT(1) in the 6th TMD. Simultaneous competitive labelling of different areas of this target protein indicated that the ligand-receptor structure became increasingly fluctual at physiological temperatures, while a more compact, low mobility, and low energy conformation prevailed at low temperatures.


Assuntos
Receptor Tipo 1 de Angiotensina/química , Receptor Tipo 1 de Angiotensina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação/genética , Células COS , Chlorocebus aethiops , Humanos , Ligantes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Marcadores de Fotoafinidade , Estrutura Terciária de Proteína , Receptor Tipo 1 de Angiotensina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica
5.
J Phys Chem B ; 111(7): 1620-4, 2007 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-17256896

RESUMO

We report the mass spectrometric measurement of anions desorbed by 3-24 eV electron impact on thin films of formamide-1-d (DCONH2) and on the self-assembled monolayer (SAM) of two different Lys amide molecules used as a molecular model of the peptide backbone. In the present SAM configuration, the amides are elevated from a gold substrate by hydrocarbon chains to remove the effects of the metal substrate. Electron irradiation produces H- and D- from the formamide-1-d film and H-, CH3-, O-, and OH- from the SAM Lys amides. Below 13 eV, the dependence of the anion yields on the incident electron energy exhibits structures indicative of the dissociative electron attachment process, which is responsible for molecular fragmentation via the initial formation of core-excited anions. Above 13 eV, anion desorption is dominated principally by non-resonant dipolar dissociation. Our results suggest that the sensitivity of the peptide backbone to secondary electrons produced by ionizing radiation depends on the chemical environment (i.e., the amino acids sequence).


Assuntos
Elétrons , Peptídeos/química , Radiação , Acetamidas/química , Formamidas/química , Ouro/química , Hidrocarbonetos/química , Lisina/química , Espectrometria de Massas
11.
Can J Physiol Pharmacol ; 80(5): 418-25, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-12056548

RESUMO

UNLABELLED: We have applied photoaffinity labelling methods combined with site-directed mutagenesis towards the two principal angiotensin II (AnglI) receptors AT1 and AT2 in order to determine contact points between AngII and the two receptors. We have first identified the receptor contact points between an N- and a C-terminal residue of the AngII molecule and the AT1 receptor and constructed with this stereochemical restriction a molecular model of AT1. A similar approach with a modified procedure of photoaffinity labelling has allowed us now to determine contact points also in the AT2 receptor. Molecular modelling of AT2 on the rhodopsin scaffold and energy minimisation of AngII binding into this AT2 model produced a model strikingly similar to the AT11 structure. Superposition of the experimentally obtained contact points of AngII with AT2 upon this model revealed excellent congruence between the experimental and modelling results. CONCLUSIONS: (i) athough AT1 and AT2 have quite low sequence homology, they both bind AngII with similar affinity and in an almost identical fashion, as if the ligand dictates the way it has to be bound, and (ii) in its bound form, AngII adopts an extended conformation in both AT1 and AT2, contrary to all previous predictions.


Assuntos
Angiotensina II/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Angiotensina/metabolismo , Sequência de Aminoácidos/fisiologia , Angiotensina II/química , Angiotensina II/genética , Animais , Bovinos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina/química , Receptores de Angiotensina/genética
12.
Circ Res ; 90(8): 844-9, 2002 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-11988484

RESUMO

Growth hormone-releasing peptides (GHRPs) are known as potent growth hormone secretagogues whose actions are mediated by the ghrelin receptor, a G protein-coupled receptor cloned from pituitary libraries. Hexarelin, a hexapeptide of the GHRP family, has reported cardiovascular activity. To identify the molecular target mediating this activity, rat cardiac membranes were labeled with a radioactive photoactivatable derivative of hexarelin and purified using lectin affinity chromatography and preparative gel electrophoresis. A binding protein of M(r) 84 000 was identified. The N-terminal sequence determination of the deglycosylated protein was identical to rat CD36, a multifunctional glycoprotein, which was expressed in cardiomyocytes and microvascular endothelial cells. Activation of CD36 in perfused hearts by hexarelin was shown to elicit an increase in coronary perfusion pressure in a dose-dependent manner. This effect was lacking in hearts from CD36-null mice and hearts from spontaneous hypertensive rats genetically deficient in CD36. The coronary vasoconstrictive response correlated with expression of CD36 as assessed by immunoblotting and covalent binding with hexarelin. These data suggest that CD36 may mediate the coronary vasospasm seen in hypercholesterolemia and atherosclerosis.


Assuntos
Antígenos CD36/fisiologia , Coração/fisiologia , Oligopeptídeos/farmacologia , Vasoconstritores/farmacologia , Animais , Sítios de Ligação , Antígenos CD36/genética , Antígenos CD36/isolamento & purificação , Membrana Celular/química , Coração/efeitos dos fármacos , Immunoblotting , Camundongos , Camundongos Knockout , Miocárdio/química , Oligopeptídeos/metabolismo , Técnicas de Cultura de Órgãos , Marcadores de Fotoafinidade/química , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Ratos Sprague-Dawley , Receptores de Neuropeptídeos/isolamento & purificação , Receptores de Hormônios Reguladores de Hormônio Hipofisário/isolamento & purificação , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/metabolismo
14.
Am J Physiol Cell Physiol ; 280(4): C1027-30, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11245619

RESUMO

Plasma membrane (PM) Ca2+ pump is a Ca+-Mg2+-ATPase that expels Ca2+ from cells to help them maintain low concentrations of cytosolic Ca2+ . There are no known extracellularly acting PM Ca2+ pump inhibitors, as digoxin and ouabain are for Na+ pump. In analogy with digoxin, we define caloxins as extracellular PM Ca2+ pump inhibitors and describe caloxin 2A1. Caloxin 2A1 is a peptide obtained by screening a random peptide phage display library for binding to the second extracellular domain (residues 401-413) sequence of PM Ca2+ pump isoform 1b. Caloxin 2A1 inhibits Ca2+-Mg2+-ATPase in human erythrocyte leaky ghosts, but it does not affect basal Mg2+-ATPase or Na+-K+-ATPase in the ghosts or Ca2+-Mg2+-ATPase in the skeletal muscle sarcoplasmic reticulum. Caloxin 2A1 also inhibits Ca2+-dependent formation of the 140-kDa acid-stable acylphosphate, which is a partial reaction of this enzyme. Consistent with inhibition of the PM Ca2+ pump in vascular endothelium, caloxin 2A1 produces an endothelium-dependent relaxation that is reversed by N(G)-nitro-L-arginine methyl ester. Thus caloxin 2A1 is a novel PM Ca2+ pump inhibitor selected for binding to an extracellular domain.


Assuntos
ATPases Transportadoras de Cálcio/antagonistas & inibidores , Membrana Eritrocítica/enzimologia , Proteínas de Membrana/metabolismo , Peptídeos/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Cálcio/metabolismo , Endotélio Vascular/enzimologia , Homeostase/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas de Membrana/genética , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/genética , Transdução de Sinais/fisiologia
15.
Methods Mol Med ; 51: 297-303, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-21331725

RESUMO

Photoaffinity labeling is a useful method to covalently bind two interacting moieties whether they be substrate and enzyme or ligand and receptor. Irreversibly labeling any particular molecule is a practical way of detecting the latter throughout the course of a characterization or a purification procedure.

16.
J Cardiovasc Pharmacol ; 36(5 Suppl 1): S362-6, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11078421

RESUMO

The gene expression and levels of endothelins (ETs) are increased in various animal models of lipopolysaccharide-(LPS) induced septic shock as well as in patients with endotoxaemia (ENDO). A positive correlation was reported between the expression and production of ETs, and the severity of haemodynamic and haematological disturbances, organ injury and circulatory failure in ENDO. Previous studies using ET(A)- and/or ET(B)-receptor antagonists exacerbated the effects of LPS in anaesthetized and conscious rats. We investigated the effect of a selective neutral endopeptidase (NEP) (CGS 24592) or a mixed NEP/endothelin-converting enzyme (ECE) (CGS 26303) inhibitor in LPS-induced ENDO in anaesthetized Sprague-Dawley rats. Four hours post-LPS injection, blood pressure was 39% lower in the presence of CGS 26303, compared to control-saline or LPS-injected rats. In rats treated with CGS 26303, white blood cells and platelet counts decreased, whereas lymphocytes increased. In addition, progressive liver dysfunction, characterized by increases in plasma bilirubin and alanine transferase, became even more apparent (higher than in those injected with LPS). Plasma creatinine and blood urea were similar to those of the LPS-injected group. Similar results were observed with CGS 24592. Thus, these inhibitors enhanced some, but not all, of the LPS-induced deleterious effects.


Assuntos
Ácido Aspártico Endopeptidases/antagonistas & inibidores , Endotoxemia/fisiopatologia , Lipopolissacarídeos/toxicidade , Neprilisina/antagonistas & inibidores , Organofosfonatos/farmacologia , Fenilalanina/análogos & derivados , Inibidores de Proteases/farmacologia , Tetrazóis/farmacologia , Equilíbrio Ácido-Base/efeitos dos fármacos , Animais , Contagem de Células Sanguíneas , Enzimas Conversoras de Endotelina , Hemodinâmica/efeitos dos fármacos , Masculino , Metaloendopeptidases , Fenilalanina/farmacologia , Ratos , Ratos Sprague-Dawley
17.
Arzneimittelforschung ; 50(6): 526-9, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10918944

RESUMO

Four sets of angiotensin II (AngII) analogues with position 5 modifications, two agonist series with either Asp or Sar in position 1 and L-Phe in position 8, and two antagonist series with again Asp or Sar in position 1 and Leu in position 8 were synthesized. Modifications in positions 5 were introduced successively: Ile, Nle, Met, S-ethyl Cys, S-n-propyl-Cys, S-n-butyl Cys, S-t-butyl Cys and S-benzyl Cys in all four series. The study was undertaken in order to investigate the 5-position residue of AngII by replacing the hydrophobic side-chain by another containing an electrophilic moiety. The analogues were synthesised by solid phase synthesis using the Boc/Bzl or Fmoc/But strategy. All analogues were evaluated by their binding properties to the AT1 receptor on bovine adrenocortical membranes (bAT1). The results indicate that AngII analogues bind, irrespective of their agonistic or antagonistic nature or of their position 1 modification, in a similar manner and that position 5 modifications without beta-branching behave in an additive manner towards their affinity.


Assuntos
Angiotensina II/análogos & derivados , Angiotensina II/farmacologia , Córtex Suprarrenal/efeitos dos fármacos , Córtex Suprarrenal/metabolismo , Angiotensina I/metabolismo , Angiotensina II/síntese química , Animais , Bovinos , Cromatografia em Gel , Técnicas In Vitro , Membranas/efeitos dos fármacos , Membranas/metabolismo , Coelhos , Receptores de Angiotensina/efeitos dos fármacos , Receptores de Angiotensina/metabolismo , Relação Estrutura-Atividade
18.
Biochemistry ; 39(32): 9662-70, 2000 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-10933782

RESUMO

An angiotensin II (AngII) peptidic analogue in which the third residue (valine) was substituted with the photoreactive p-benzoyl-L-phenylalanine (Bpa) was used to identify ligand-binding sites of the human AT(1) receptor. High-affinity binding of the analogue, (125)I-[Bpa(3)]AngII, to the AT(1) receptor heterologously expressed in COS-7 cells enabled us to efficiently photolabel the receptor. Chemical and enzymatic digestions of the (125)I-[Bpa(3)]AngII-AT(1) complex were performed, and receptor fragments were analyzed in order to define the region of the receptor with which the ligand interacts. Results show that CNBr hydrolysis of the photolabeled receptor gave a glycosylated fragment which, after PNGase-F digestion, migrated as a 11.4 kDa fragment, circumscribing the labeled domain between residues 143-243 of the AT(1) receptor. Digestion of the receptor-ligand complex with Endo Lys-C or trypsin followed by PNGase-F treatment yielded fragments of 7 and 4 kDa, defining the labeling site of (125)I-[Bpa(3)]AngII within residues 168-199 of the AT(1) receptor. Photolabeling of three mutant receptors in which selected residues adjacent to residue 168 were replaced by methionine within the 168-199 fragment (I172M, T175M, and I177M) followed by CNBr cleavage revealed that the bound photoligand (125)I-[Bpa(3)]AngII forms a covalent bond with the side chain of Met(172) of the second extracellular loop of the AT(1) receptor. These data coupled with previously obtained results enable us to propose a model whereby AngII adopts an extended beta-strand conformation when bound to the receptor and would orient itself within the binding domain by having its N-terminal portion interacting with the second extracellular loop and its C-terminus interacting with residues of the seventh transmembrane domain.


Assuntos
Angiotensina II/metabolismo , Receptores de Angiotensina/metabolismo , Sequência de Aminoácidos , Angiotensina II/análogos & derivados , Sítios de Ligação , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Fenilalanina/análogos & derivados , Marcadores de Fotoafinidade , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
19.
Biochim Biophys Acta ; 1497(1): 94-102, 2000 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-10838163

RESUMO

A complementary DNA for the angiotensin II (AngII) type 1 (AT(1)) receptor from Mustela putorius furo (ferret) was isolated from a ferret atria cDNA library. The cDNA encodes a protein (fAT(1)) of 359 amino acids having high homologies (93-99%) to other mammalian AT(1) receptor counterparts. When fAT(1) was expressed in COS-7 cells and photoaffinity labeled with the photoactive analogue (125)I-¿Sar(1), Bpa(8)AngII, a protein of 100 kDa was detected by autoradiography. The formation of this complex was specific since it was abolished in the presence of the AT(1) non-peptidic antagonist L-158,809. Functional analysis indicated that the fAT(1) receptor efficiently coupled to phospholipase C as demonstrated by an increase in inositol phosphate production following stimulation with AngII. Binding studies revealed that the fAT(1) receptor had a high affinity for the peptide antagonist ¿Sar(1), Ile(8)AngII (K(d) of 5. 8+/-1.4 nM) but a low affinity for the AT(1) selective non-peptidic antagonist DuP 753 (K(d) of 91+/-15.6 nM). Interestingly, when we substituted Thr(163) with an Ala residue, which occupies this position in many mammalian AT(1) receptors, we restored the high affinity of this receptor for Dup 753 (11.7+/-5.13 nM). These results suggest that position 163 of the AT(1) receptor does not contribute to the overall binding of peptidic ligands but that certain non-peptidic antagonists such as Dup 753 are clearly dependent on this position for efficient binding.


Assuntos
Furões/genética , Losartan/metabolismo , Receptores de Angiotensina/genética , 1-Sarcosina-8-Isoleucina Angiotensina II/análogos & derivados , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Angiotensina II/análogos & derivados , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Antagonistas de Receptores de Angiotensina , Animais , Sequência de Bases , Sítios de Ligação , Ligação Competitiva/efeitos dos fármacos , Células COS , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , DNA Recombinante/genética , DNA Recombinante/metabolismo , Imidazóis/farmacologia , Radioisótopos do Iodo , Losartan/farmacologia , Dados de Sequência Molecular , Plasmídeos , Ligação Proteica , Piridinas/farmacologia , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Treonina/genética , Treonina/metabolismo
20.
Recept Channels ; 7(1): 41-52, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10800775

RESUMO

The inositol 1,4,5-trisphosphate (InsP3) receptor forms a tetrameric channel responsible for the release of Ca2+ from intracellular stores. In the present study we showed that the experimental approach used to separate bound and free ligands may discriminate between two populations of InsP3 binding sites in bovine adrenal cortex microsomes. A large population of low affinity sites and a small population of high affinity sites were detected with centrifugation and filtration approaches, respectively. Both populations were found in the supernatant and the cytoskeleton fractions of Triton X-100 solubilized microsomes. After treatment of microsomes with thimerosal, an alkylating reagent known to increase InsP3 receptor affinity, the filtration and the centrifugation approaches yielded identical results. With selective anti-InsP3 receptor antibodies, we showed that types 1, 2 and 3 InsP3 receptors are present in intact microsomes and in the cytoskeleton fraction. Binding studies on immunoprecipitated receptors revealed that anti-type 1 antibody recognizes a large population of low affinity sites whereas anti-type 2 antibody recognizes a small population of high affinity sites. Our results indicate that the three types of InsP3 receptors are expressed at different levels in the bovine adrenal cortex. The presence of different types of InsP3 receptors with different ligand binding affinities and their association with the cytoskeleton offer a convenient way for the cell to simultaneously regulate its intracellular Ca2+ concentration and reorganize the spatial distribution of its Ca2+ stores.


Assuntos
Córtex Suprarrenal/metabolismo , Canais de Cálcio/classificação , Inositol 1,4,5-Trifosfato/metabolismo , Receptores Citoplasmáticos e Nucleares/classificação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Western Blotting , Canais de Cálcio/metabolismo , Bovinos , Linhagem Celular , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Testes de Precipitina , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo
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