LABELING OF CRF1 AND CRF2 RECEPTORS USING THE NOVEL RADIOLIGAND, [H-3] UROCORTIN

The binding of the novel radioligand, [H-3]-rat urocortin to homogenat es of rat cerebellum and homogenates of cells stably transfected with the human CRF1, rat CRF2 alpha and rat CRF2 beta receptors was examine d. In each case, specific reversible high affinity binding was observe d (K(d)s between 0.18 and 0.31 nM). The density of sites was relativel y low in the cerebellum (9 fmol/mg tissue) but high in the recombinant systems with expression levels of between 1.4 and 6.3 pmol/mg protein . Agents known to interact with CRF receptors potently competed for bi nding in each case. The pharmacological profile of binding to the reco mbinant receptors were consistent with data previously published using other radioligands. Thus, for the recombinant CRF1 receptor, binding was inhibited with similar affinity by Urocortin, sauvagine, Urotensin 1 and CRF. The non-peptidic CRF antagonists (e.g. CP 154,526 and SC 2 41) also potently inhibited binding. The CRF2 alpha, and CRF2 beta rec eptor recombinant systems had a very similar pharmacological profile w ith a clear rank order of potency for the peptide ligands (Urocortin > Sauvagine > Urotensin 1 > CRF), whereas the non-peptide CRF receptor antagonists had no measurable affinity. The pharmacological profile of specific [H-3]-urocortin binding to homogentates of rat cerebellum wa s consistent with specific labelling of a CRF1 receptor. We conclude t hat [H-3]-urocortin is a useful tool for the study of CRF receptors wi th the advantages that a filtration assay can be used, all CRF recepto rs can be labelled with the same ligand and the benefits associated wi th the low energy emitter, H-3. (C) 1997 Elsevier Science Ltd.