Abstract

The binding characteristics of corticotropin-releasing factor (CRF) type 1 (CRF₁) and type 2 (CRF₂) receptors from human (hCRF₁ and hCRF_2α) andXenopus (xCRF₁ and xCRF₂) were compared using four different ¹²⁵I-labeled CRF analogs, the agonists ¹²⁵I-CRF and ¹²⁵I-sauvagine, and the antagonists ¹²⁵I-astressin (¹²⁵I-AST) and¹²⁵I-antisauvagine-30 (¹²⁵I-aSVG). The hCRF_2α and xCRF₂ receptors bound all four radioligands with different affinities, whereas hCRF₁ did not bind ¹²⁵I-aSVG, and xCRF₁ bound neither¹²⁵I-sauvagine nor ¹²⁵I-aSVG. Competitive binding studies using unlabeled agonists and antagonists with hCRF₁ and hCRF_2α receptors revealed that most agonists exhibited higher affinity in displacing agonist radioligands compared with displacement of antagonist radioligands. Exceptions were the agonists human and rat urocortin, which displayed high-affinity binding in the presence of either ¹²⁵I-labeled agonist or antagonist ligands. In contrast, the affinities of antagonists were independent of the nature of the radioligand. We also found that, in contrast to the mammalian CRF receptors, the affinity of ligand binding to xCRF₁ and xCRF₂ receptors strongly depended on the nature of the radioligand used for competition. For xCRF₁, competitors showed different rank order binding profiles with ¹²⁵I-CRF compared with ¹²⁵I-AST as the displaceable ligand. Similarly, binding of competitors to the xCRF₂ receptor showed markedly different profiles with¹²⁵I-CRF as the competed ligand compared with the other radioligands. These data demonstrate that amphibian CRF receptors have distinctly different binding modes compared with their mammalian counterparts.