Different binding modes of amphibian and human corticotropin-releasing factor type 1 and type 2 receptors: evidence for evolutionary differences.

The binding characteristics of corticotropin-releasing factor (CRF) type 1 (CRF(1)) and type 2 (CRF(2)) receptors from human (hCRF(1) and hCRF(2alpha)) and Xenopus (xCRF(1) and xCRF(2)) were compared using four different (125)I-labeled CRF analogs, the agonists (125)I-CRF and (125)I-sauvagine, and the antagonists (125)I-astressin ((125)I-AST) and (125)I-antisauvagine-30 ((125)I-aSVG). The hCRF(2alpha) and xCRF(2) receptors bound all four radioligands with different affinities, whereas hCRF(1) did not bind (125)I-aSVG, and xCRF(1) bound neither (125)I-sauvagine nor (125)I-aSVG. Competitive binding studies using unlabeled agonists and antagonists with hCRF(1) and hCRF(2alpha) 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 (125)I-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 xCRF(1) and xCRF(2) receptors strongly depended on the nature of the radioligand used for competition. For xCRF(1), competitors showed different rank order binding profiles with (125)I-CRF compared with (125)I-AST as the displaceable ligand. Similarly, binding of competitors to the xCRF(2) receptor showed markedly different profiles with (125)I-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.