title: Structural requirements for peptidic antagonists of the corticotropin-releasing factor receptor (CRFR): development of CRFR2beta-selective antisauvagine-30.
abstract: Different truncated and conformationally constrained analogs of corticotropin-releasing factor (CRF) were synthesized on the basis of the amino acid sequences of human/rat CRF (h/rCRF), ovine CRF (oCRF), rat urocortin (rUcn), or sauvagine (Svg) and tested for their ability to displace [125I-Tyr0]oCRF or [125I-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 (rCRFR1), or mouse CRF receptor, type 2beta (mCRFR2beta). Furthermore, the potency of CRF antagonists to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRFR1 (HEK-rCRFR1 cells) or mCRFR2beta (HEK-mCRFR2beta cells) was determined. In comparison with astressin, which exhibited a similar affinity to rCRFR1 (Kd = 5.7 +/- 1.6 nM) and mCRFR2beta (Kd = 4.0 +/- 2.3 nM), [DPhe11,His12]Svg(11-40), [DLeu11]Svg(11-40), [DPhe11]Svg(11-40), and Svg(11-40) bound, respectively, with a 110-, 80-, 68-, and 54-fold higher affinity to mCRFR2beta than to rCRFR1. The truncated analogs of rUcn displayed modest preference (2- to 7-fold) for binding to mCRFR2beta. In agreement with the results of these binding experiments, [DPhe11, His12]Svg(11-40), named antisauvagine-30, was the most potent and selective ligand to suppress agonist-induced adenylate cyclase activity in HEK cells expressing mCRFR2beta.
authors.affiliation: Department of Molecular Neuroendocrinology, Max Planck Institute for Experimental Medicine, Hermann-Rein-Strasse 3, D-37075 Göttingen, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Rühmann
authors.forename: I
authors.initials: I
authors.lastname: Bonk
authors.forename: C R
authors.initials: CR
authors.lastname: Lin
authors.forename: M G
authors.initials: MG
authors.lastname: Rosenfeld
authors.forename: J
authors.initials: J
authors.lastname: Spiess
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D003346:Corticotropin-Releasing Hormone / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D003432:Cross-Linking Reagents; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D007668:Kidney; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051381:Rats; D018019:Receptors, Corticotropin-Releasing Hormone / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
chemical_list: D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D018019:Receptors, Corticotropin-Releasing Hormone; D011994:Recombinant Proteins; C116607:antisauvagine 30; D003346:Corticotropin-Releasing Hormone; D000242:Cyclic AMP