I-125 TYR(0)-SAUVAGINE - A NOVEL HIGH-AFFINITY RADIOLIGAND FOR THE PHARMACOLOGICAL AND BIOCHEMICAL-STUDY OF HUMAN CORTICOTROPIN-RELEASING FACTOR(2-ALPHA) RECEPTORS

Corticotropin-releasing factor (CRF) receptors encoded by two distinct genes have recently been identified and termed CRF(1) and CRF(2). CRF and the non-mammalian-related peptide sauvagine bind to and activate CRF(1) receptors with high affinity and equal potency. Although CRF is significantly weaker at the CRF(2) receptor, sauvagine retains its hi gh affinity interactions with this receptor subtype. We expressed the human CRF(1) and CRF(2) receptor subtypes in stable cell lines and cha racterized I-125-Tyr(0)-sauvagine, a high affinity radiolabel suitable for the pharmacological and functional profiles of these proteins. (1 25)ITyr(0)-sauvagine has high affinity (200-400 pM) for CRF(1) recepto rs and demonstrates a pharmacological profile identical to that of (12 5)1-Tyr(0)-ovine CRF-labeled CRF(1) receptors. I-125-Tyr(0)-sauvagine binding to human CRF(1) receptors is saturable and of high affinity (K -D = 100-300 pM) and demonstrates guanine nucleotide sensitivity typic al of agonist binding to receptors. The pharmacological profile of I-1 25-Tyro-sauvagine binding to CRF(2 alpha) receptors with respect to in hibition by CRF-related analogs is similar to the agonist profile of p otencies obtained by measurements of cAMP production stimulated by the se analogs in CRF(2 alpha) expressing cell lines and distinct from the profile of the CRF(1) receptor subtype. Thus, the related nonmammalia n peptides sauvagine and urotensin have high affinity and rat/human CR F and ovine CRF have lower affinity for CRF(2) receptors labeled with I-125-Tyro-sauvagine. Because the distribution of CRF(1) and CRF(2 alp ha) receptors has been demonstrated to be distinct, suggesting selecti ve functional roles for each receptor subtype, the ability to label CR F(2 alpha), receptors with I-125-Tyr-0-sauvagine in vitro represents a unique opportunity for the discovery of subtype-selective nonpeptide ligands, which would presumably target different aspects of CRF-mediat ed disorders. We have thus identified and characterized a novel high a ffinity radioligand for the labeling of CRF(2) receptors.