Impact of N-Terminal Domains for Corticotropin-Releasing Factor (CRF) Receptor-Ligand Interactions
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- 作者：Jana Klose ; Klaus Fechner ; Michael Beyermann ; Eberhard Krause ; Norbert Wendt ; Michael Bienert ; Rainer Rudolph ; and Sven Rothemund
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：February 8, 2005
- 年：2005
- 卷：44
- 期：5
- 页码：1614 - 1623
- 全文大小：257K
- 年卷期：v.44,no.5(February 8, 2005)
- ISSN：1520-4995
文摘
The large extracellular N-terminal domains (NTs) of class B G protein-coupled receptors serveas major ligand binding sites. However, little is known about the ligand requirements for interactionswith these receptor domains. Recently, we have shown that the most potent CRF receptor agonist urocortin1 (Ucn1) has two segregated receptor binding sites Ucn1(1-21) and Ucn1(32-40). For locating the receptordomains interacting with these two sites, we have investigated the binding of appropriate Ucn1 analoguesto the receptor N-termini compared to the corresponding full-length receptors. For this purpose receptorNTs of CRF(rat) subtypes 1 and 2(
) without their signal sequences were overexpressed in Escherichiacoli and folded in vitro. For CRF2(a)-rNT, which bears five cysteine residues (C2-C6), the disulfidearrangement C2-C5 and C4-C6 was found, leaving C3 free. This is consistent with the disulfide patternof CRF1-rNT, which has six cysteines and in which C1 is paired with C3. Binding studies of N-terminallytruncated 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 receptorNT. Since the binding of Ucn1 to the juxtamembrane domain has been shown to be segregated frombinding to the receptor N-terminus [Hoare et al. (2004) Biochemistry 43, 3996-4011], a third bindingdomain 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.
) without their signal sequences were overexpressed in Escherichiacoli and folded in vitro. For CRF2(a)-rNT, which bears five cysteine residues (C2-C6), the disulfidearrangement C2-C5 and C4-C6 was found, leaving C3 free. This is consistent with the disulfide patternof CRF1-rNT, which has six cysteines and in which C1 is paired with C3. Binding studies of N-terminallytruncated 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 receptorNT. Since the binding of Ucn1 to the juxtamembrane domain has been shown to be segregated frombinding to the receptor N-terminus [Hoare et al. (2004) Biochemistry 43, 3996-4011], a third bindingdomain 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.