| Abstract
| - 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.
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