Vol. 61, Issue 5, 1132-1139, May 2002

Five Amino Acids of the Xenopus laevis CRF (Corticotropin-Releasing Factor) Type 2 Receptor Mediate Differential Binding of CRF Ligands in Comparison with Its Human Counterpart

Frank M. Dautzenberg, Jacqueline Higelin, Olaf Brauns, Brigitte Butscha, and Richard L. Hauger

Axovan Ltd. Innovation Center, Allschwil, Switzerland (F.M.D., B.B.); F. Hoffmann-La Roche Ltd., Pharma Division, Preclinical Research, Basel, Switzerland (J.H.); Max Planck Institute of Experimental Medicine, Department for Neuroendocrinology, Göttingen, Germany (O.B.); and University of California at San Diego and Veterans Affairs Medical Center, Department of Psychiatry, La Jolla, California (R.L.H.)

The ligand selectivity of human (hCRF_2A) and Xenopus laevis (xCRF₂) forms of the corticotropin-releasing factor type 2 (CRF₂) receptor differs. The purpose of this study was to identify amino acids in these two CRF₂ receptors conferring these differences. An amino acid triplet in the third extracellular domain (Asp²⁶²Leu²⁶³Val²⁶⁴ in hCRF_2A or Lys²⁶⁴Tyr²⁶⁵Ile²⁶⁶ in xCRF₂) was found to diverge between both receptors. When binding and signaling characteristics of receptor mutants hR2KYI and xR2DLV were assessed, the tri-amino acid motif replacement produced receptors with binding properties resembling the xCRF₂ receptor. The converse mutation created a mutant receptor with a binding pharmacology identical to the profile of the hCRF_2A receptor. This effect was most notable for xR2DLV, which possessed a binding affinity for astressin ~15-fold greater for astressin than sauvagine. In contrast, the binding profiles of the hCRF_2A receptor and hR2KYI did not differ. These data indicate that another domain of the xCRF₂ receptor mediated low-affinity binding of astressin. Two amino acids in the first extracellular domain differ in xCRF₂ (Asp⁶⁹Ser⁷⁰) and hCRF_2A (Glu⁶⁶Tyr⁶⁷) receptors. The hCRF_2A receptor mutant (hR2DS-KYI) bound astressin with a low affinity indistinguishable from the xCRF₂ receptor. Therefore, these data demonstrate that ligand selectivity differences between amphibian and human forms of the CRF_2A receptor are governed by these two motifs of the extracellular domains of the xCRF₂ receptor.