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To investigate the structure-function relationship of urocortin (Ucn), ten Ucn-related peptides lacking Cor N-terminal amino acid residues were prepared by solid phase technology. ACTH release and cAMP accumulation induced by these synthetic peptides were examined in the mouse anterior pituitary tumor cell line, AtT-20. Among ten Ucn-related peptides tested, Ucn(3—40) was found to be nearly equipotent to Ucn in the activities inducing ACTH release and cAMP accumulation, suggesting that two N-terminal amino acids may not be crucial for these actions of Ucn. On the other hand, Ucn(23—40) and Ucn(28-40) caused extremely low ACTH release. ACTH release was extremely low when AtT-20 was stimulated by synthetic peptides lacking six amino acids in the C-terminal region of Ucn, such as Ucn(]34), Ucn(3-34), Ucn(5--34), and Ucn(8-34); and by synthetic peptides lacking C-terminal region of Ucn, Ucn(23—34) and Ucn(1—Z8). These results indicate that the C-terminal hexapeptide region of Ucn is of extreme importance for the activity of Ucn to cause ACTH release. Little, if any, cAMP accumulation was observed in the presence of Ucn(5-34), Ucn(8—34), Ucn(23--34) or Ucn(]-18), supporting clearly that the C-terminal 35-40 sequence of Ucn may be responsible for CRF/Ucn receptor binding. Urocortin (Ucn), which was originally cloned from rat midbrain region (18), consists of 40amino acid residues having 45% sequence identity with human/rat CRF and 63% sequence identity with carp and sucker urotensin I. Subsequently, Ucn has been found to bind with high affinity for both CRF-type l and CRF-type 2 receptors and the CRF-binding protein (2, 6, 8-10, 16-18). Ucn has been shown to be more potent than CRF in stimulating ACTH release from anterior pituitary cells, and l0 times more potent than CRF in stimulating cAMP accumulation in cells expressCorrespondence to: Dr Minoru Hoshino at the above address. Fax: +81-54-264-5909, E-mail: hoshino@u-shizuoka-ken.ac.jp Abbreviations: All amino acids are ofthe L-configuration. Boc, z‘ert-butyloxycarbonyl; DCM, dichloromethan; DMF, N, N-dimethylformamide; FAB, fast atom bombardment; HPLC, high performance liquid chromatography; PAM, phenyl acetamidomethyl; TFA, trifluoroacetic acid; Tos, tosyl; BOP, benzotriazol-lyl-oxytris-(dimethylamino)phosphonium hexafluorophosphate; HOBt, l-hydroxy benzotriazole; DIEA, diisopropylethylamine; cAMP, cyclic adenosine monophosphate; ACTH, adrenocorticotropic hormone; tBu, tert-butyl; CRF, corticotropin~releasing factor; Fmoc, 9-fluorenylmethoxycarbonyl; HATU, 0-(7azabenzotriazol-l-yl)—l,l,3,3-tetramethyluronium hexafluorophosphate; OtBu, tert-butyl ester; Pbf, 2,2,4,6, 7»-pentamethyldihydrobenzofuran-5-sulfonyl; Trt, trityl; Ucn, urocortin

ACTH-releasing Activity and cAMP Accumulation of Rat Urocortin-related Peptides in Pituitary Corticotropic Cells