Rühmann A, Bonk I, Lin C R, Rosenfeld M G, Spiess J
Department of Molecular Neuroendocrinology, Max Planck Institute for Experimental Medicine, Hermann-Rein-Strasse 3, D-37075 Göttingen, Germany.
Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15264-9. 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 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.
基于人/大鼠促肾上腺皮质激素释放因子（h/rCRF）、绵羊促肾上腺皮质激素释放因子（oCRF）、大鼠尿皮质素（rUcn）或蛙皮素（Svg）的氨基酸序列，合成了不同的截短型和构象受限类似物，并测试了它们从稳定转染了编码大鼠促肾上腺皮质激素释放因子受体1型（rCRFR1）或小鼠促肾上腺皮质激素释放因子受体2β型（mCRFR2β）的cDNA的人胚肾（HEK）293细胞膜匀浆中置换[125I-Tyr0]oCRF或[125I-Tyr0]Svg的能力。此外，还测定了促肾上腺皮质激素释放因子拮抗剂抑制表达rCRFR1（HEK-rCRFR1细胞）或mCRFR2β（HEK-mCRFR2β细胞）的转染HEK 293细胞中oCRF或Svg刺激的cAMP产生的效力。与对rCRFR1（Kd = 5.7 +/- 1.6 nM）和mCRFR2β（Kd = 4.0 +/- 2.3 nM）表现出相似亲和力的阿斯特辛相比，[DPhe11,His12]Svg(11-40)、[DLeu11]Svg(11-40)、[DPhe11]Svg(11-40)和Svg(11-40)与mCRFR2β的结合亲和力分别比对rCRFR1高110倍、80倍、68倍和54倍。rUcn的截短类似物对mCRFR2β的结合显示出适度的偏好（2至7倍）。与这些结合实验的结果一致，名为抗蛙皮素-30的[DPhe11, His12]Svg(11-40)是抑制表达mCRFR2β的HEK细胞中激动剂诱导的腺苷酸环化酶活性的最有效和选择性最强的配体。
