RESUMEN
Sequential proteolytic processing of the Amyloid Precursor Protein (APP) by beta- and gamma-secretases generates the 4-kDa amyloid (A beta) peptide, a key component of the amyloid plaques seen in Alzheimer's disease (AD). We and others have recently reported the identification and characterisation of an aspartic proteinase, Asp2 (BACE), as beta-secretase. Here we describe the characterization of a second highly related aspartic proteinase, Asp1 as a second beta-secretase candidate. Asp1 is expressed in brain as detected at the mRNA level and at the protein level. Transient expression of Asp1 in APP-expressing cells results in an increase in the level of beta-secretase-derived soluble APP and the corresponding carboxy-terminal fragment. Paradoxically there is a decrease in the level of soluble A beta secreted from the cells. Asp1 colocalizes with APP in the Golgi/endoplasmic reticulum compartments of cultured cells. Asp1, when expressed as an Fc fusion protein (Asp1-Fc), has the N-terminal sequence ALEP..., indicating that it has lost the prodomain. Asp1-Fc exhibits beta-secretase activity by cleaving both wild-type and Swedish variant (KM/NL) APP peptides at the beta-secretase site.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/análisis , Precursor de Proteína beta-Amiloide/química , Animales , Ácido Aspártico Endopeptidasas/química , Sitios de Unión/fisiología , Células COS , Clonación Molecular , Endopeptidasas , Femenino , Glicoproteínas/análisis , Humanos , Masculino , Proteínas de la Membrana/análisis , Datos de Secuencia Molecular , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
GPR10 is a novel G-protein coupled receptor that is the human orthologue of rat Unknown Hypothalamic Receptor-1 (UHR-1). Human prolactin-releasing peptide (PrRP) has been identified as an endogenous ligand for GPR10, and occurs as 31 and 20 amino acid forms. The present study characterizes the binding of [(125)I]-PrRP-20 to HEK293 cells stably expressing GPR10 receptors. Specific binding of [(125)I]-PrRP-20 was saturable, and analysis suggested evidence of both high and low affinity sites, with K:(D:) values of 0.026+/-0.006 and 0.57+/-0.14 nM respectively, and B(max) values of 3010+/-400 and 8570+/-2240 fmol mg protein(-1) respectively. Kinetic studies were unable to distinguish two sites, but single site analysis of association and dissociation data produced a K:(D:) of 0.012 nM. Competition studies revealed that human and rat PrRP-20 and PrRP-31 all display high affinity for GPR10. A range of other drugs which are known ligands at receptors which share limited homology with GPR10 were also tested. None of the drugs tested, including the RF-amide neuropeptide FF, demonstrated any affinity for GPR10. Human PrRP-20 failed to alter basal or forskolin-stimulated levels of intracellular cyclic AMP in HEK293-GPR10 cells, suggesting that GPR10 does not couple via either G(s) or G(i). Functional studies using measurements of intracellular calcium confirmed that human and rat PrRP-20 and PrRP-31 are all potent, full agonists at the GPR10 receptor. The response was blocked both by thapsigargin, indicating mobilization of intracellular Ca(2+) stores. These studies indicate that [(125)I]-PrRP-20 is a specific, high affinity radioligand for GPR10. The availability of this radioligand binding assay will be a valuable tool for the investigation of the key features involved in PrRP binding and studies on the localization and function of GPR10.
Asunto(s)
Hormonas Hipotalámicas/metabolismo , Neuropéptidos/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G , Secuencia de Aminoácidos , Animales , Unión Competitiva , Calcio/metabolismo , Células Cultivadas , AMP Cíclico/biosíntesis , Humanos , Hormonas Hipotalámicas/farmacología , Radioisótopos de Yodo , Cinética , Datos de Secuencia Molecular , Neuropéptidos/farmacología , Hormona Liberadora de Prolactina , RatasRESUMEN
0-4-Dimethylcarbamoylbenzyl-L-tyrosine, N (epsilon)-4-dimethylcarbamoylbenzyloxycarbonyl-L-lysine, and simple derivatives, have been prepared. The protecting groups are markedly more stable to trifluoroacetic acid than are the unsubstituted benzylanalogues. N-t-Butoxycarbonyl-O-4-dimethylcarbamoylbenzyl-L-serine (cyclohexylammonium salt) is also described.