title: Impact of N-terminal domains for corticotropin-releasing factor (CRF) receptor-ligand interactions.
abstract: The large extracellular N-terminal domains (NTs) of class B G protein-coupled receptors serve as major ligand binding sites. However, little is known about the ligand requirements for interactions with these receptor domains. Recently, we have shown that the most potent CRF receptor agonist urocortin 1 (Ucn1) has two segregated receptor binding sites Ucn1(1-21) and Ucn1(32-40). For locating the receptor domains interacting with these two sites, we have investigated the binding of appropriate Ucn1 analogues to the receptor N-termini compared to the corresponding full-length receptors. For this purpose receptor NTs of CRF(rat) subtypes 1 and 2(alpha) without their signal sequences were overexpressed in Escherichia coli and folded in vitro. For CRF2(a)-rNT, which bears five cysteine residues (C2-C6), the disulfide arrangement C2-C5 and C4-C6 was found, leaving C3 free. This is consistent with the disulfide pattern of CRF1-rNT, which has six cysteines and in which C1 is paired with C3. Binding studies of N-terminally truncated or C-terminally modified Ucn1 analogues demonstrate that it is the C-terminal part, Ucn1(11-40), that binds to receptor NT, indicating a two-domain binding mechanism for Ucn binding to receptor NT. Since the binding of Ucn1 to the juxtamembrane domain has been shown to be segregated from binding to the receptor N-terminus [Hoare et al. (2004) Biochemistry 43, 3996-4011], a third binding domain should exist, probably comprising residues 8-10 of Ucn, which particularly contribute to a high-affinity binding to full-length receptors but not to receptor NT.
authors.affiliation: Research Institute of Molecular Pharmacology, Robert-Roessle-Strasse 13, D-13125 Berlin, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Klose
authors.forename: Klaus
authors.initials: K
authors.lastname: Fechner
authors.forename: Michael
authors.initials: M
authors.lastname: Beyermann
authors.forename: Eberhard
authors.initials: E
authors.lastname: Krause
authors.forename: Norbert
authors.initials: N
authors.lastname: Wendt
authors.forename: Michael
authors.initials: M
authors.lastname: Bienert
authors.forename: Rainer
authors.initials: R
authors.lastname: Rudolph
authors.forename: Sven
authors.initials: S
authors.lastname: Rothemund
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003346:Corticotropin-Releasing Hormone / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051381:Rats; D018019:Receptors, Corticotropin-Releasing Hormone / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D054832:Urocortins; D000099052:CRF Receptor, Type 1
chemical_list: D004220:Disulfides; D008024:Ligands; D010446:Peptide Fragments; D018019:Receptors, Corticotropin-Releasing Hormone; D011994:Recombinant Proteins; D054832:Urocortins; C499434:corticotropin-releasing factor receptor type 2a, rat; D000099052:CRF Receptor, Type 1; D003346:Corticotropin-Releasing Hormone