Functional and protein chemical characterization of the N-terminal domain of the rat corticotropin-releasing factor receptor 1

被引:36
作者
Hofmann, BA [1 ]
Sydow, S [1 ]
Jahn, O [1 ]
Van Werven, L [1 ]
Liepold, T [1 ]
Eckart, K [1 ]
Spiess, J [1 ]
机构
[1] Max Planck Inst Expt Med, Dept Mol Neuroendocrinol, D-37073 Gottingen, Germany
关键词
corticotropin-releasing factor; CRF receptor; human embryonic kidney cells; scintillation proximity assay; amino-terminal domain; binding domain; disulfide bond structure; glycosylation structure;
D O I
10.1110/ps.12101
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 biological function 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.
引用
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页码:2050 / 2062
页数:13
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