High-affinity binding of urocortin and astressin but not CRF to G protein-uncoupled CRFR1

The structure-activity relationship (SAR) between the recently identified n europeptide urocortin (Ucn) and corticotropin-releasing factor (CRF) recept or, type 1 (CRFR1), has been investigated. To this end, rat Ucn (rUcn), ovi ne CRF (oCRF) and chimeric peptides of rUcn and oCRF were synthesized and t ested for their binding affinity and potency to stimulate cAMP production i n human embryonic kidney (HEK) 293 cells stably transfected with cDNA encod ing rat CRFR1 (rCRFR1). In binding studies with [I-125-Tyr(0)]oCRF or [H-3- Leu(9)]rUcn as radioligand, it was observed that rUcn but not oCRF bound in a similar fashion as the CRF antagonist astressin with high affinity to rC RFR1 coupled to G protein or uncoupled from G protein by guanosine 5'-O-(3- thiotriphosphate) (GTP gamma S). Consequently, rUcn was found to exert a si gnificantly lower potency than oCRF to stimulate cAMP accumulation in trans fected cells. CD spectroscopic investigations and reverse-phase HPLC (RPHPL C) retention behavior of the peptides suggested a more pronounced amphipati c alpha-helical character of rUcn when compared to oCRF and the chimeric pe ptides. (C) 1999 Elsevier Science Inc. All rights reserved.