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 (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-glycosylation 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.
Hofmann
B A
BA
Department of Molecular Neuroendocrinology, Max Planck Institute for Experimental Medicine, D-37073 Göttingen, Germany.
Sydow
S
S
Jahn
O
O
van Werven
L
L
Liepold
T
T
Eckart
K
K
Spiess
J
J
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D004220:Disulfides / Q000737:chemistry*; D017353:Gene Deletion; D006031:Glycosylation; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000502:physiology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D018019:Receptors, Corticotropin-Releasing Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000099052:CRF Receptor, Type 1
D004220:Disulfides; D008024:Ligands; D021382:Protein Sorting Signals; D018019:Receptors, Corticotropin-Releasing Hormone; D000099052:CRF Receptor, Type 1