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

Rat corticotropin‐releasing factor receptor 1 (rCRFR1) 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 rUcn specifically (KD = 27 nM) and astressin (KI = 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 Thr23 and three disulfide bridges between precursor residues 30 and 54, 44 and 87, and 68 and 102. Of all putative N‐glycosylation sites in positions 32, 38, 45, 78, 90, and 98, all Asn residues except for Asn32 were glycosylated to a significant extent. No O‐glycosylation was observed.