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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.
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