Labelling of CRF 1 and CRF 2 receptors using the novel radioligand, [ 3H]-urocortin

Abstract

The binding of the novel radioligand, [ 3H]-rat urocortin to homogenates of rat cerebellum and homogenates of cells stably transfected with the human CRF 1, rat CRF 2α and rat CRF 2β receptors was examined. In each case, specific reversible high affinity binding was observed ( K ds between 0.18 and 0.31 nM). The density of sites was relatively 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 binding in each case. The pharmacological profile of binding to the recombinant receptors were consistent with data previously published using other radioligands. Thus, for the recombinant CRF 1 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 241) also potently inhibited binding. The CRF 2α and CRP 2β receptor recombinant systems had a very similar pharmacological profile with 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 [ 3H]-urocortin binding to homogentates of rat cerebellum was consistent with specific labelling of a CRF 1 receptor. We conclude that [ 3H]-urocortin is a useful tool for the study of CRF receptors with the advantages that a filtration assay can be used, all CRF receptors can be labelled with the same ligand and the benefits associated with the low energy emitter, 3H.