Structural Requirements for Peptidic Antagonists of the Corticotropin-Releasing Factor Receptor (CRFR): Development of CRFR2β -selective Antisauvagine-30

• Published in: Proceedings of the National Academy of Sciences - PNAS 1998-12, Vol.95 (26), p.15264-15269
• Main Authors: Ruhmann, Andreas, Bonk, Ines, Lin, Chijen R., Rosenfeld, Michael G., Spiess, Joachim
• Format: Article
• Language: English
• Subjects: Agonists; Amino acids; Biochemistry; Biological Sciences; Complementary DNA; Endocrinology; Ligands; Post hoc; Rats; Receptors; Statistical significance
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.95.26.15264

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 2β (mCRFR2β ). 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 mCRFR2β (HEK-mCRFR2β cells) was determined. In comparison with astressin, which exhibited a similar affinity to rCRFR1 (Kd= 5.7 ± 1.6 nM) and mCRFR2β (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 mCRFR2β than to rCRFR1. The truncated analogs of rUcn displayed modest preference (2- to 7-fold) for binding to mCRFR2β . 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 mCRFR2β .