Photoaffinity Cross-Linking of the Corticotropin-Releasing Factor Receptor Type 1 with Photoreactive Urocortin Analogues
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- 作者：Oliver Kraetke ; Brian Holeran ; Hartmut Berger ; Emanuel Escher ; Michael Bienert ; and Michael Beyermann
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：November 29, 2005
- 年：2005
- 卷：44
- 期：47
- 页码：15569 - 15577
- 全文大小：297K
- 年卷期：v.44,no.47(November 29, 2005)
- ISSN：1520-4995
文摘
Interaction of natural peptide ligands with class 2 GPCRs, which are targets of biologicallyimportant hormones such as glucagon, secretin, and corticotropin-releasing factor (CRF), occurs with acommon orientation, in that the ligand C-terminus binds to the extracellular receptor N-terminus, whereasthe ligand N-terminus binds to the receptor juxtamembrane domain. N-Terminal truncation, by eight aminoacids in the case of CRF, leads to antagonists, suggesting those residues constitute the receptor activatingsequence. Here, we identified by photoaffinity cross-linking using p-benzoyl-L-phenylalanine (Bpa)analogues of urocortin (Ucn) the most affine CRF receptor agonist, interaction domains of CRF1 receptorwith Bpa residues at exclusive positions. Specific cleavage patterns of the corresponding ligand-receptorcomplexes, obtained using several cleavage methods in combination with SDS-PAGE for fragment sizedetermination, showed that a Bpa group located N-terminally or in position 12 binds at the second andsuch in position 17 or 22 at the first extracellular receptor loop. Our results indicate that the very N-terminalligand residues (1-11), which are responsible for receptor activation, are oriented to the juxtamembranedomain by interaction of amino acid residues 12, 17, and 22. Our findings contradict a recently proposedinteraction model derived from ligand interaction with a soluble receptor N-terminus, indicating thatconclusions drawn from such a reduced system may be of limited value to understand the interactionwith the full-length receptor.