RESUMEN
Urocortin (Ucn), the newest member of the corticotropin-releasing factor (CRF) family of peptides, has been demonstrated to have significant physiologic and behavioral effects following its peripheral and central administration, respectively. In order to assess the differences in Ucn across species, an 18-kb sheep genomic DNA fragment encoding urocortin was isolated by the hybridization screening of a lambda phage library with a probe generated from rat urocortin (rUcn) cDNA. The sheep clone contains a region that is 84% and 88% homologous to the coding region of rUcn and human Ucn (hUcn), respectively and encodes an ovine Ucn (oUcn) that is predicted to be identical to the rat peptide. Competitive binding assays demonstrated oUcn to have a high affinity (Ki=0.1 nM) for the sheep CRF-binding protein (CRF-BP) and localization studies by in situ hybridization have shown that the distribution of oUcn messenger RNA in sheep brain shares with that of rUcn in rat brain a predominant locus of expression in the Edinger-Westphal nucleus of the midbrain, though some secondary sites of expression reported in rat are not conserved. These findings demonstrate that, even across diverse species, Ucn is highly conserved with respect to its structure and pharmacology unlike CRF where significant amino acid substitutions between the rat/human and sheep peptides may underlie differences in neuroendocrine regulation.
Asunto(s)
Química Encefálica/fisiología , Hormona Liberadora de Corticotropina/genética , Genoma , ARN Mensajero/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , Unión Proteica , Ratas , Ovinos , Especificidad de la Especie , UrocortinasRESUMEN
Conserved amino acid motifs are found in numerous expressed genes. Proteins and peptides with functional relationships may be identified using probes designed to hybridize with these motifs. An oligonucleotide probe was prepared to match the sequence of the expected active region of a frog corticotropin-releasing factor-like peptide sauvagine and used to screen a sheep brain cDNA library. A novel 1331-bp cDNA encoding a putative 328-residue protein with a theoretical mass of 36 kDa was identified. The presence of a strong signal sequence indicates that it is a secreted protein. The amino- and carboxy-terminal regions are characterized by several potential phosphorylation sites and binding motifs, suggesting a role in intracellular signal transduction. Although the protein possesses a 7-residue sequence identical to that found in sauvagine, its overall primary structure most closely resembles those of the alpha-carbonic anhydrases (alpha-CAs). Moreover, the detection of the human and mouse orthologues in the EST databases, together with an evolutionary analysis, indicates that the protein represents a new member of the alpha-CA gene family, which we designate carbonic anhydrase-related protein XI (CA-RP XI), encoded by CA11 (human) and Car11 (mouse, rat). The human CA11 gene appears to be located between the secretor type alpha(1,2)-fucosyltransferase gene cluster (FUT1-FUT2-FUT2P) and the D-site binding protein gene (DBP) on chromosome 19q13.3. Despite potentially inactivating changes in the active-site residues, CA-RP XI is evolving very slowly in mammals, a property indicative of an important function, which has also been observed in the two other "acatalytic" CA isoforms, CA-RP VIII and CA-RP X, whose functions are unknown.
Asunto(s)
Anhidrasas Carbónicas/genética , Cromosomas Humanos Par 19 , Proteínas de Unión al ADN , Proteínas del Tejido Nervioso/genética , Péptidos , Factores de Transcripción/genética , Secuencia de Aminoácidos , Proteínas Anfibias , Animales , Secuencia de Bases , Biomarcadores de Tumor , Encéfalo/enzimología , Mapeo Cromosómico , Secuencia Conservada , Etiquetas de Secuencia Expresada , Humanos , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Hormonas Peptídicas , Filogenia , Biosíntesis de Proteínas , Ratas , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , OvinosRESUMEN
We report here the identification, purification and cDNA cloning of a corticotropin releasing factor (CRF) binding protein(s) (CRF-BP) from sheep brain. Native sheep and rat brain CRF-BP and recombinant rat CRF-BP were shown to be N-glycosylated. Two membrane associated forms of brain CRF-BPs of 33 and 35 kDa were purified from sheep brain homogenates after solubilization in the presence of detergent. N-Terminal sequence analysis revealed that the 35 kDa protein is proteolytically cleaved near the N-terminus giving rise to an 18 amino acid peptide and a 33 kDa CRF-BP. Both the purified 33 and 35 kDa ovine CRF-BPs could be specifically cross linked to ovine [125I]CRF and human [125I]CRF. In contrast, recombinant rat CRF-BP can only be cross-linked to human [125I]CRF. A 1.7 kb cDNA clone (Basil 7) encoding an open reading frame for a 324 amino acid CRF-BP precursor was cloned from a sheep brain lambda gtlO cDNA library and was shown to have 85% and 87% amino acid homology to the rat and human proteins, respectively. Competitive binding analysis of the recombinant sheep CRF-BP (Basil 7) expressed in CHO cells revealed that it binds human and ovine CRF with high affinity. However, the recombinant sheep CRF-BP (Basil 7) had approximately 50-fold higher affinity for human CRF than for the ovine peptide. These data present the first biochemical proof that CRF-BP is in the brain and provides evidence for the existence of different forms of CRF-BP which have evolved across species to regulate CRF.