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Titolo:
Functional and protein chemical characterization of the N-terminal domain of the rat corticotropin-releasing factor receptor 1
Autore:
Hofmann, BA; Sydow, S; Jahn, O; Van Werven, L; Liepold, T; Eckart, K; Spiess, J;
Indirizzi:
Max Planck Inst Expt Med, Dept Mol Neuroendocrinol, D-37073 Gottingen, Germany Max Planck Inst Expt Med Gottingen Germany D-37073 73 Gottingen, Germany
Titolo Testata:
PROTEIN SCIENCE
fascicolo: 10, volume: 10, anno: 2001,
pagine: 2050 - 2062
SICI:
0961-8368(200110)10:10<2050:FAPCCO>2.0.ZU;2-C
Fonte:
ISI
Lingua:
ENG
Soggetto:
CONSERVED EXTRACELLULAR CYSTEINES; MESSENGER-RNA EXPRESSION; FACTOR CRF RECEPTOR; DISULFIDE BOND; HORMONE RECEPTOR; TUPAIA-BELANGERI; XENOPUS-LAEVIS; LIGAND-BINDING; UROTENSIN-I; CLONING;
Keywords:
corticotropin-releasing factor; CRF receptor; human embryonic kidney cells; scintillation proximity assay; amino-terminal domain; binding domain; disulfide bond structure; glycosylation structure;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
60
Recensione:
Indirizzi per estratti:
Indirizzo: Eckart, K Max Planck Inst Expt Med, Dept Mol Neuroendocrinol, Hermann ReinStr 3, D-37073 Gottingen, Germany Max Planck Inst Expt Med Hermann Rein Str 3 Gottingen Germany D-37073
Citazione:
B.A. Hofmann et al., "Functional and protein chemical characterization of the N-terminal domain of the rat corticotropin-releasing factor receptor 1", PROTEIN SCI, 10(10), 2001, pp. 2050-2062

Abstract

Rat corticotropin-releasing factor receptor 1 (rCRFR 1) was produced either in transfected HEK 293 cells as a complex glycosylated protein or in the presence of the mannosidase I inhibitor kifunensine as a high mannose glycosylated protein. The altered glycosylation did not influence the biologicalfunction of rCRFR1 as demonstrated by competitive binding of rat urocortin(rUcn) or human/rat corticotropin-releasing factor (h/rCRF) and agonist-induced CAMP accumulation. The low production rate of the N-terminal domain of rCRFR1 (rCRFR1-NT) by transfected HEK 293 cells, was increased by a factor of 100 in the presence of kifunensine. The product, rCRFR1-NT-Kif, bound rUen specifically (K-D = 27 nM) and astressin (K-I = 60 nM). This affinity was 10-fold lower than the affinity of full length rCRFR1. However, it was sufficiently high for rCRFR1-NT-Kif to serve as a model for the N-terminal domain of rCRFR1. With protein fragmentation, Edman degradation, and mass spectrometric analysis, evidence was found for the signal peptide cleavage site C-terminally to Thr(23) and three disulfide bridges between precursor residues 30 and 54, 44 and 87, and 68 and 102. Of all putative N-glycogylation sites in positions 32, 38, 45, 78, 90, and 98, all Asn residues except for Asn(32) were glycosylated to a significant extent. No O-glycosylation was observed.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 05/04/20 alle ore 09:37:22