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.
Ardati
A
A
Pharma Division, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Gottowik
J
J
Henriot
S
S
Clerc
R G
RG
Kilpatrick
G J
GJ
D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism / Q000494:pharmacology*; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D011869:Radioligand Assay / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology*; D012967:Sodium Dodecyl Sulfate; D014316:Tritium / Q000378:metabolism; D054832:Urocortins
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D054832:Urocortins; D014316:Tritium; C067133:corticotropin releasing factor-binding protein; D012967:Sodium Dodecyl Sulfate; D003346:Corticotropin-Releasing Hormone