RESUMEN
The metalloendopeptidase EC (EP24.15) is a neuropeptide-metabolizing enzyme expressed predominantly in brain, pituitary, and testis, and is implicated in several physiological processes and diseases. Multiple putative phosphorylation sites in the primary sequence led us to investigate whether phosphorylation effects the specificity and/or the kinetics of substrate cleavage. Only protein kinase A (PKA) treatment resulted in serine phosphorylation with a stoichiometry of 1.11 +/- 0.12 mol of phosphate/mol of recombinant rat EP24.15. Mutation analysis of each putative PKA site, in vitro phosphorylation, and phosphopeptide mapping indicated serine 644 as the phosphorylation site. Phosphorylation effects on catalytic activity were assessed using physiological (GnRH, GnRH(1-9), bradykinin, and neurotensin) and fluorimetric (MCA-PLGPDL-Dnp and orthoaminobenzoyl-GGFLRRV-Dnp-edn) substrates. The most dramatic change upon PKA phosphorylation was a substrate-specific, 7-fold increase in both K(m) and k(cat) for GnRH. In both rat PC12 and mouse AtT-20 cells, EP24.15 was serine-phosphorylated, and EP24.15 phosphate incorporation was enhanced by forskolin treatment, and attenuated by H89, consistent with PKA-mediated phosphorylation. Cloning of the full-length mouse EP24.15 cDNA revealed 96.7% amino acid identity to the rat sequence, and conservation at serine 644, consistent with its putative functional role. Therefore, PKA phosphorylation is suggested to play a regulatory role in EP24.15 enzyme activity.
Asunto(s)
Metaloendopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Secuencia Conservada , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Hidrólisis , Metaloendopeptidasas/genética , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Neuropéptidos/metabolismo , Células PC12 , Fosforilación , Hipófisis/enzimología , Ratas , Alineación de Secuencia , Serina/metabolismoRESUMEN
The metalloendopeptidase EP24.15 (EC3.4.24.15) is a neuropeptide-metabolizing enzyme present in neural and endocrine tissues, presumably functioning extracellularly. Because the majority of the EP24.15 activity is identified in the soluble fraction of cellular homogenates, suggesting that the enzyme is primarily an intracellular protein, we addressed the issue of how EP24.15 arrives in the extracellular environment. We utilized a model system of neuroendocrine secretion, the AtT20 cell. According to both enzymatic activity and immunologic assays, EP24.15 was synthesized in and released from AtT20 cells. Under basal conditions and after stimulation by corticotropin-releasing hormone or the calcium ionophore A23187, EP24.15 activity accumulated in the culture medium. This secretion was not attributable to cell damage, as judged by the absence of release of cytosolic enzyme markers and the ability to exclude trypan blue dye. Pulse-chase analysis and subcellular fractionation of AtT20 cell extracts suggested that the mechanism of EP24.15 secretion is not solely via classical secretory pathways. Additionally, drugs which disrupt the classical secretory pathway, such as Brefeldin A and nocodazole, blocked A23187-stimulated EP24.15 release yet had no effect on basal EP24.15 release, suggesting differences in the basal and stimulated pathways of secretion for EP24.15. In summary, EP24.15 appears to be secreted from AtT20 pituitary cells into the extracellular milieu, where the enzyme can participate in the physiologic metabolism of neuropeptides.
Asunto(s)
Metaloendopeptidasas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Brefeldino A/farmacología , Calcimicina/antagonistas & inhibidores , Calcimicina/farmacología , Calcio/fisiología , Hormona Liberadora de Corticotropina/farmacología , Medios de Cultivo Condicionados/química , Retículo Endoplásmico/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Ionóforos/antagonistas & inhibidores , Ionóforos/farmacología , Masculino , Metaloendopeptidasas/biosíntesis , Metaloendopeptidasas/inmunología , Ratones , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/inmunología , Nocodazol/farmacología , Adenohipófisis/enzimología , Adenohipófisis/metabolismo , Neoplasias Hipofisarias/enzimología , Neoplasias Hipofisarias/metabolismo , Conejos , Ratas , Proteínas Recombinantes de Fusión/inmunología , Tasa de Secreción/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
Endopeptidase EC 3.4.24.15 (EP24.15) is a soluble, neuropeptide-degrading metalloenzyme, widely expressed in the brain, pituitary and gonads. For the physiological metabolism of neuropeptides, the enzyme should be located extracellularly, either associated with the plasma membrane or in the extracellular milieu. Western immunoblot analyses of crude cytosolic and post-nuclear membrane fractions prepared by differential centrifugation revealed a slightly smaller molecular mass ( approximately 2 kDa) for EP24.15 in the post-nuclear membrane fraction. This smaller EP24.15 species was also present in an enriched fraction of plasma membrane prepared by Percoll gradient centrifugation. To ascertain whether EP24.15 is associated with the extracellular surface of plasma membrane, two sets of experiments were carried out. First, Western immunoblot analysis of AtT-20 cells treated with the membrane-impermeable, thiol-cleavable cross-linker, 3, 3'-dithio-bis(sulpho-succinimidyl-propionate) (DTSSP), indicated an extracellular membrane association. After cross-linking and thiol-reduction, a distinct band corresponding to EP24.15 was significantly diminished under non-reducing conditions. Second, immunocytochemical studies performed at 4 degrees C on non-permeabilized AtT-20 cells (i.e., non-fixed to prevent antibody internalization), indicated that EP24.15 was expressed on the surface of the AtT-20 cells. We furthermore determined that EP24.15 enzymatic activity is present on the extracellular surface of the cell discernable from the secreted enzyme. These results suggest that the EP24.15 is associated with the extracellular surface of the AtT-20 cell plasma membrane and is enzymatically active. Taken together, the results are consistent with a putative role in the degradation of neuropeptides acting at the external cell surface.
Asunto(s)
Metaloendopeptidasas/metabolismo , Animales , Western Blotting , Membrana Celular/metabolismo , Inmunohistoquímica , Ratones , Neuropéptidos/metabolismo , Células Tumorales CultivadasRESUMEN
Three major isoforms of the receptor protein tyrosine phosphatase HPTPzeta/RPTPbeta (RPTPzeta/beta) have been previously identified, two with identical transmembrane and intracellular catalytic domains that differ by virtue of a long cysteine-free extracellular region, and a soluble proteoglycan called phosphacan that lacks the transmembrane and carboxy-terminal catalytic domains. To determine whether these RPTPzeta/beta variants are produced by alternative mRNA splicing of a common primary transcript, we performed genomic Southern analysis and characterized several rat cDNA and genomic RPTPzeta/beta clones. These studies indicated that the three major transcripts which encode phosphacan and the two RPTPzeta/beta phosphatase variants are encoded by a single gene, and further that additional alternative mRNA splicing is likely to result in the deletion of a 7 amino acid insert from the intracellular juxtamembrane region of both long and short phosphatase isoforms. Simultaneous quantitation of the three major isoforms by RNase protection analysis indicated that the mRNA encoding phosphacan had the highest relative abundance in the CNS while that encoding the short phosphatase isoform was most abundant relative to the other RPTPzeta/beta variants in the PNS. Following peripheral nerve crush, all RPTPzeta/beta mRNAs, including phosphacan and the phosphatase variants with and without the 21 base insert, were significantly induced in the distal segments of the sciatic nerve with a time course that correlated well with the response of Schwann cells to this injury.
Asunto(s)
Empalme Alternativo/fisiología , Proteínas Tirosina Fosfatasas/genética , Nervio Ciático/lesiones , Secuencia de Aminoácidos , Animales , Southern Blotting , Proteoglicanos Tipo Condroitín Sulfato/genética , Clonación Molecular , Exones , Femenino , Regulación Enzimológica de la Expresión Génica , Datos de Secuencia Molecular , Compresión Nerviosa , Proteoglicanos/fisiología , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Nervio Ciático/enzimología , Transcripción Genética/fisiologíaRESUMEN
Endopeptidase EC 3.4.24.15 (EP24.15) is a thermolysin-like metalloendopeptidase involved in the regulated metabolism of a number of neuropeptides. Unlike other thermolysin-like peptidases EP24.15 displays a unique thiol activation, a mechanism that is not clearly understood. In this study we show that both recombinant and tissue-derived EP24.15 are activated up to 8-fold by low concentrations (0.1 mM) of dithiothreitol. Additionally, under non-reducing conditions, recombinant and native EP24.15 forms multimers that can be returned to the monomeric form by reduction. We have also shown that competitive inhibitor binding occurs only to the monomeric form, which indicates that catalytic site access is restricted in the multimeric forms. Through systematic site-directed mutagenesis we have identified that cysteine residues 246, 253, and possibly 248 are involved in the formation of these multimers. Furthermore, both a double mutant (C246S/C253S) and a triple mutant (C246S/C248S/C253S) are fully active in the absence of reducing agents, as measured by both inhibitor binding and hydrolysis. The formation and disruption of disulfide bonds involving these cysteine residues may be a mechanism by which EP24.15 activity is regulated through changes in intra- and extracellular redox potential.
Asunto(s)
Metaloendopeptidasas/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Catálisis , Cisteína/metabolismo , Disulfuros/metabolismo , Activación Enzimática , Luz , Masculino , Metaloendopeptidasas/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismo , Dispersión de Radiación , Testículo/enzimologíaRESUMEN
Ionotropic excitatory amino acid (EAA) receptors are divided pharmacologically into three categories termed NMDA, AMPA/kainate, and high affinity kainate receptors. Each of these receptor subtypes is composed of a specific subset of subunits termed GluR1-4 (AMPA/kainate), GluR5-7, KA1-2 (high affinity kainate), and NMDAR1, 2 A-D (NMDA). Although colocalization of NMDA and non-NMDA receptors has been previously demonstrated electrophysiologically in rat, comprehensive analyses of subunit specific colocalization patterns have not been possible until the advent of appropriate antibodies. The present study investigates such immunocytochemical colocalization of several EAA receptor subunits within individual cells as well as dendritic spines in the monkey hippocampus. Double-label immunohistochemical experiments using antibodies which are specific for GluR2(4), GluR5-7, and NMDAR1 demonstrated that virtually all projection neurons in each subfield of the hippocampus contain subunits from the AMPA/kainate, kainate, and NMDA receptor families. In addition, confocal microscopy has demonstrated that individual spines may contain subunits representative of multiple EAA receptor families. Furthermore, detailed regional, cellular, and ultrastructural distribution patterns of the EAA receptor subunits GluR2 and GluR4 in monkey hippocampus are presented based on the use of a monoclonal antibody (mAb), 3A11, which was generated against the putative extracellular N-terminal domain of GluR2. Since this antibody recognizes only GluR2 in Western blots, and GluR2 as well as GluR4 in fixed transiently transfected cells, it has been designated anti-GluR2(4). Immunocytochemical labeling with mAb 3A11 revealed pyramidal cell somata and dendrites in each field of the hippocampus, as well as granule cells and polymorphic hilar cells in the dentate gyrus. Small cells with the morphologic characteristics of astroglia were also immunolabeled for GluR2(4) within the alveus and fimbria. Immunoreactivity at the ultrastructural level was localized to postsynaptic densities on dendritic spines and shafts and within the somatodendritic cytoplasm in all major hippocampal regions, as well as in a subset of dentate granule cell axons within the mossy fiber projection.