Characterization of a sheep brain corticotropin releasing factor binding protein.
We report here the identification, purification and cDNA cloning of a corticotropin releasing factor (CRF) binding protein(s) (CRF-BP) from sheep brain. Native sheep and rat brain CRF-BP and recombinant rat CRF-BP were shown to be N-glycosylated. Two membrane associated forms of brain CRF-BPs of 33 and 35 kDa were purified from sheep brain homogenates after solubilization in the presence of detergent. N-Terminal sequence analysis revealed that the 35 kDa protein is proteolytically cleaved near the N-terminus giving rise to an 18 amino acid peptide and a 33 kDa CRF-BP. Both the purified 33 and 35 kDa ovine CRF-BPs could be specifically cross linked to ovine [125I]CRF and human [125I]CRF. In contrast, recombinant rat CRF-BP can only be cross-linked to human [125I]CRF. A 1.7 kb cDNA clone (Basil 7) encoding an open reading frame for a 324 amino acid CRF-BP precursor was cloned from a sheep brain lambda gtlO cDNA library and was shown to have 85% and 87% amino acid homology to the rat and human proteins, respectively. Competitive binding analysis of the recombinant sheep CRF-BP (Basil 7) expressed in CHO cells revealed that it binds human and ovine CRF with high affinity. However, the recombinant sheep CRF-BP (Basil 7) had approximately 50-fold higher affinity for human CRF than for the ovine peptide. These data present the first biochemical proof that CRF-BP is in the brain and provides evidence for the existence of different forms of CRF-BP which have evolved across species to regulate CRF.
Behan
D P
DP
Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, San Diego, CA 92037, USA.
Cepoi
D
D
Fischer
W H
WH
Park
M
M
Sutton
S
S
Lowry
P J
PJ
Vale
W W
WW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D016466:CHO Cells / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins; D012756:Sheep
D002352:Carrier Proteins; D018076:DNA, Complementary; D011994:Recombinant Proteins; C067133:corticotropin releasing factor-binding protein; D003346:Corticotropin-Releasing Hormone