RESUMO
From three-dimensional models of its receptors, residues which bind the carboxy-terminus of endothelin were predicted. This site is in a pocket consisting of five putative transmembrane helices and includes a histidine in the sixth helix. This residue is either phenylalanine or asparagine in cationic neurotransmitter receptors. The histidine alkylating agent diethylpyrocarbonate potently inhibited binding of [125I]endothelin-1 to its receptors in bovine cerebellum, where a single population of endothelin ETB receptors was shown to exist. From the absence of pH sensitivity of inhibition above pH 5 and the reversal by hydroxylamine of inhibition, diethylpyrocarbonate is concluded to inhibit by histidine modification. Diethylpyrocarbonate inhibited ligand binding to several receptors with the potency order endothelin ETB > or = bombesin > or = dopamine D2 > or = m2 muscarinic > alpha 1-adrenoceptor > or = m 1 muscarinic > 5-HT2. This is consistent with histidine in the binding site of endothelin (and some other peptidergic) receptors and the proposed model.
Assuntos
Cerebelo/metabolismo , Endotelinas/metabolismo , Receptores de Endotelina/metabolismo , Receptores de Neurotransmissores/metabolismo , Sequência de Aminoácidos , Animais , Asparagina/química , Sítios de Ligação/efeitos dos fármacos , Bovinos , Dietil Pirocarbonato/farmacologia , Histidina/química , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Ratos , Receptores da Bombesina , Receptores Dopaminérgicos/química , Receptores Dopaminérgicos/metabolismo , Receptores de Endotelina/química , Receptores da Neurocinina-2 , Receptores de Neurotransmissores/química , Receptores de Serotonina/química , Receptores de Serotonina/metabolismo , Análise de SequênciaRESUMO
The structure of porcine neuropeptide Y in 0.05 M CD3COOD/D2O was determined by nuclear magnetic resonance spectroscopy. Nuclear Overhauser spectra yielded 377 distances which define a helical segment formed by residues 11-36. An additional set of 24 distances were interpreted as intermolecular distances within a dimer. A combination of distance geometry calculations, energy minimization and molecular dynamics yielded a model of the dimer having antiparallel packing of two curved helical units whose hydrophobic sides form a well defined core. The N-terminus (residues 1-9) appears as an unstructured mobile segment. Large changes in the intrinsic fluorescence intensity of neuropeptide Y tyrosine residues allowed the determination of the dimer dissociation constant as 1.6 +/- 0.6 microM at pH 2-8 in aqueous buffers and also indicated the enclosure of several tyrosine residues in the hydrophobic environment of the interface region in the dimeric species. Fluorescence anisotropy data reveals the slow rotation of such shielded residues.