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

• Published in: Peptides (New York, N.Y. : 1980) Vol. 20; no. 11; pp. 1311 - 1319
• Main Authors: Rühmann, Andreas, Bonk, Ines, Köpke, Andreas K.E
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.1999
• Subjects: 5′-cyclic monophosphate; Amino Acid Sequence; Animals; Astressin; cAMP (adenosine 3; CD (circular dichroism); Cell Line; Chromatography, High Pressure Liquid; Circular Dichroism; Corticotropin-Releasing Hormone - chemistry; Corticotropin-Releasing Hormone - metabolism; CRF (corticotropin-releasing factor); CRFR1 (CRF receptor: type 1); Cyclic AMP - biosynthesis; G protein coupling; GTP-Binding Proteins - metabolism; GTPγS (guanosine 5′- O-(3-thiotriphosphate)); Guanosine 5'-O-(3-Thiotriphosphate) - pharmacology; Humans; Molecular Sequence Data; Peptide Fragments - metabolism; Protein Binding; Rats; Receptors, Corticotropin-Releasing Hormone - metabolism; Recombinant Proteins - metabolism; RPHPLC (reverse-phase HPLC); Sequence Homology, Amino Acid; Sheep; Ucn (urocortin); Urocortins; CD (circular dichroism); RPHPLC (reverse-phase HPLC); GTPγS (guanosine 5′- O-(3-thiotriphosphate)); CRF (corticotropin-releasing factor); 5′-cyclic monophosphate; Ucn (urocortin); CRFR1 (CRF receptor: type 1); cAMP (adenosine 3; Astressin; G protein coupling
• ISSN: 0196-9781; 1873-5169
• DOI: 10.1016/S0196-9781(99)00136-9

The structure-activity relationship (SAR) between the recently identified neuropeptide urocortin (Ucn) and corticotropin-releasing factor (CRF) receptor, type 1 (CRFR1), has been investigated. To this end, rat Ucn (rUcn), ovine CRF (oCRF) and chimeric peptides of rUcn and oCRF were synthesized and tested for their binding affinity and potency to stimulate cAMP production in human embryonic kidney (HEK) 293 cells stably transfected with cDNA encoding rat CRFR1 (rCRFR1). In binding studies with [ 125I-Tyr 0]oCRF or [ 3H-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 rCRFR1 coupled to G protein or uncoupled from G protein by guanosine 5′- O-(3-thiotriphosphate) (GTPγS). Consequently, rUcn was found to exert a significantly lower potency than oCRF to stimulate cAMP accumulation in transfected cells. CD spectroscopic investigations and reverse-phase HPLC (RPHPLC) retention behavior of the peptides suggested a more pronounced amphipatic α-helical character of rUcn when compared to oCRF and the chimeric peptides.