Expression, purification, and characterization of a soluble form of the first extracellular domain of the human type 1 corticotropin releasing factor receptor.
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.
Perrin
M H
MH
The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037, USA. perrin@salk.edu
Fischer
W H
WH
Kunitake
K S
KS
Craig
A G
AG
Koerber
S C
SC
Cervini
L A
LA
Rivier
J E
JE
Groppe
J C
JC
Greenwald
J
J
Møller Nielsen
S
S
Vale
W W
WW
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002942:Circular Dichroism; D003346:Corticotropin-Releasing Hormone / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D018076:DNA, Complementary; D006801:Humans; D008969:Molecular Sequence Data; D012995:Solubility
D018076:DNA, Complementary; D003346:Corticotropin-Releasing Hormone