Expression, purification, and characterization of a soluble form of the first extracellular domain of the human type 1 corticotropin releasing factor receptor. Academic Article uri icon

abstract

  • The first extracellular domain (ECD-1) of the corticotropin releasing factor (CRF) type 1 receptor, (CRFR1), is important for binding of CRF ligands. A soluble protein, mNT-CRFR1, produced by COS M6 cells transfected with a cDNA encoding amino acids 1--119 of human CRFR1 and modified to include epitope tags, binds a CRF antagonist, astressin, in a radioreceptor assay using [(125)I-d-Tyr(0)]astressin. N-terminal sequencing of mNT-CRFR1 showed the absence of the first 23 amino acids of human CRFR1. This result suggests that the CRFR1 protein is processed to cleave a putative signal peptide corresponding to amino acids 1--23. A cDNA encoding amino acids 24--119 followed by a FLAG tag, was expressed as a thioredoxin fusion protein in Escherichia coli. Following thrombin cleavage, the purified protein (bNT-CRFR1) binds astressin and the agonist urocortin with high affinity. Reduced, alkylated bNT-CRFR1 does not bind [(125)I-D-Tyr(0)]astressin. Mass spectrometric analysis of photoaffinity labeled bNT-CRFR1 yielded a 1:1 complex with ligand. Analysis of the disulfide arrangement of bNT-CRFR1 revealed bonds between Cys(30) and Cys(54), Cys(44) and Cys(87), and Cys(68) and Cys(102). This arrangement is similar to that of the ECD-1 of the parathyroid hormone receptor (PTHR), suggesting a conserved structural motif in the N-terminal domain of this family of receptors.

published proceedings

  • J Biol Chem

altmetric score

  • 9

author list (cited authors)

  • Perrin, M. H., Fischer, W. H., Kunitake, K. S., Craig, A. G., Koerber, S. C., Cervini, L. A., ... Vale, W. W

citation count

  • 80

complete list of authors

  • Perrin, MH||Fischer, WH||Kunitake, KS||Craig, AG||Koerber, SC||Cervini, LA||Rivier, JE||Groppe, JC||Greenwald, J||Møller Nielsen, S||Vale, WW

publication date

  • June 2001