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

Biochemistry. 2005 Nov 29;44(47):15569-77. doi: 10.1021/bi0507027.

Abstract

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 CRF(1) 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 and such in position 17 or 22 at the first extracellular receptor loop. Our results indicate that the very N-terminal ligand residues (1-11), which are responsible for receptor activation, are oriented to the juxtamembrane domain by interaction of amino acid residues 12, 17, and 22. Our findings contradict a recently proposed interaction model derived from ligand interaction with a soluble receptor N-terminus, indicating that conclusions drawn from such a reduced system may be of limited value to understand the interaction with the full-length receptor.

MeSH terms

  • Animals
  • Benzophenones
  • Binding Sites
  • Cell Line
  • Corticotropin-Releasing Hormone / chemistry*
  • Cross-Linking Reagents / chemistry*
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Ligands
  • Peptide Fragments / analysis
  • Phenylalanine / analogs & derivatives
  • Photochemistry*
  • Protein Interaction Mapping / methods*
  • Rats
  • Receptors, Corticotropin-Releasing Hormone / chemistry*
  • Transfection
  • Urocortins

Substances

  • 4-benzoylphenylalanine
  • Benzophenones
  • Cross-Linking Reagents
  • Ligands
  • Peptide Fragments
  • Receptors, Corticotropin-Releasing Hormone
  • Urocortins
  • Phenylalanine
  • CRF receptor type 1
  • Corticotropin-Releasing Hormone