Pharmacological characterisation of the recombinant human CRF binding protein using a simple assay

We present the pharmacological characterisation of the recombinant human corticotropin releasing factor binding protein (hCRF-BP) using a simple assay. In this assay we employed [3H]urocortin as the radioligand and, as a means to separate bound and free ligand, adsorption to activated charcoal. Using this method, approximately 60-70% of total binding was specific. Kinetic analysis revealed that association of specific [3H]urocortin binding was monophasic and slow and that the binding was irreversible. Saturation analysis showed a single saturable site of relatively high density (94 fmol per 10 microl of medium from cells transfected with the recombinant CRF binding protein). The apparent Kd for [3H]urocortin binding of 0.25 nM is similar to previously reported affinities of rat urocortin for hCRF-BP. A range of CRF-related peptides potently competed for specific [3H]urocortin binding. The rank order of potency of these agents was human/rat CRF = urotensin 1 > human urocortin > CRF6-33 > sauvagine > ovine CRF. The non-peptide CRF1 receptor antagonists CP 154,526 (N-butyl-N-[2,5-dimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]p yri midin-4-yl]-N-ethylamine) and SC 241 ([3-(2-bromo-4-isopropyl-phenyl)-5-methyl-3H-[1,2,3]triazo lo[4,5-d]pyrimidin-7-yl]-bis-(2-methoxy-ethyl)-amine) were not active at the highest concentration tested (10(-6) M). We conclude that this is a simple and accurate assay for characterisation of the pharmacology of the recombinant CRF-BP. This assay should assist with further study of the pharmacology and function of the CRF-BP.