title: 125I-Tyro-sauvagine: a novel high affinity radioligand for the pharmacological and biochemical study of human corticotropin-releasing factor 2 alpha receptors.
abstract: Corticotropin-releasing factor (CRF) receptors encoded by two distinct genes have recently been identified and termed CRF1 and CRF2. CRF and the non-mammalian-related peptide sauvagine bind to and activate CRF1 receptors with high affinity and equal potency. Although CRF is significantly weaker at the CRF2 receptor, sauvagine retains its high affinity interactions with this receptor subtype. We expressed the human CRF1 and CRF2 receptor subtypes in stable cell lines and characterized 125I-Tyr0-sauvagine, a high affinity radiolabel suitable for the pharmacological and functional profiles of these proteins. 125I-Tyr0-sauvagine has high affinity (200-400 PM) for CRF1 receptors and demonstrates a pharmacological profile identical to that of 125I-Tyr0-ovine CRF-labeled CRF1 receptors. 125I-Tyr0-sauvagine binding to human CRF2 alpha receptors is saturable and of high affinity (KD = 100-300 PM) and demonstrates guanine nucleotide sensitivity typical of agonist binding to receptors. The pharmacological profile of 125I-Tyr0-sauvagine binding to CRF2 alpha receptors with respect to inhibition by CRF-related analogs is similar to the agonist profile of potencies obtained by measurements of cAMP production stimulated by these analogs in CRF2 alpha expressing cell lines and distinct from the profile of the CRF1 receptor subtype. Thus, the related nonmammalian peptides sauvagine and urotensin have high affinity and rat/ human CRF and ovine CRF have lower affinity for CRF2 receptors labeled with 125I-Tyr0-sauvagine. Because the distribution of CRF1 and CRF2 alpha receptors has been demonstrated to be distinct, suggesting selective functional roles for each receptor subtype, the ability to label CRF2 alpha receptors with 125I-Tyr0-sauvagine in vitro represents a unique opportunity for the discovery of subtype-selective nonpeptide ligands, which would presumably target different aspects of CRF-mediated disorders. We have thus identified and characterized a novel high affinity radioligand for the labeling of CRF2 receptors.
authors.affiliation: Neurocrine Biosciences, Inc., San Diego, California 92121, USA. dgrigoriadis@neurocrine.com
authors.forename: D E
authors.initials: DE
authors.lastname: Grigoriadis
authors.forename: X J
authors.initials: XJ
authors.lastname: Liu
authors.forename: J
authors.initials: J
authors.lastname: Vaughn
authors.forename: S F
authors.initials: SF
authors.lastname: Palmer
authors.forename: C D
authors.initials: CD
authors.lastname: True
authors.forename: W W
authors.initials: WW
authors.lastname: Vale
authors.forename: N
authors.initials: N
authors.lastname: Ling
authors.forename: E B
authors.initials: EB
authors.lastname: De Souza
mesh_terms: D029845:Amphibian Proteins; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D003346:Corticotropin-Releasing Hormone / Q000031:analogs & derivatives / Q000494:pharmacology*; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D006150:Guanine Nucleotides / Q000494:pharmacology; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D010446:Peptide Fragments / Q000494:pharmacology; D036361:Peptide Hormones; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D011869:Radioligand Assay; D018019:Receptors, Corticotropin-Releasing Hormone / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D014162:Transfection
chemical_list: D029845:Amphibian Proteins; C101518:CRF(2alpha) receptor; D006150:Guanine Nucleotides; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D036361:Peptide Hormones; D010455:Peptides; D018019:Receptors, Corticotropin-Releasing Hormone; D011994:Recombinant Proteins; C025727:sauvagine; D003346:Corticotropin-Releasing Hormone; D000242:Cyclic AMP