Photoaffinity cross-linking of the corticotropin-releasing factor receptor type 1 with photoreactive urocortin analogues

Interaction of natural peptide ligands with class 2 GPCRs, which are targets of biologically important hormones such as glucagon, secretin, and corticotropin-releasing factor (CRF), occurs with a common orientation, in that the ligand C-terminus binds to the extracellular receptor N-terminus, whereas the ligand N-terminus binds to the receptor juxtamembrane domain. N-Terminal truncation, by eight amino acids in the case of CRF, leads to antagonists, suggesting those residues constitute the receptor activating sequence. 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 receptor with Bpa residues at exclusive positions. Specific cleavage patterns of the corresponding ligand−receptor complexes, obtained using several cleavage methods in combination with SDS−PAGE for fragment size determination, showed that a Bpa group located N-terminally or in position 12 binds at the second ...