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 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.
Kraetke
Oliver
O
Department of Peptide Chemistry, Institute of Molecular Pharmacology (FMP), 13125 Berlin, Germany. kraetke@fmp-berlin.de
Holeran
Brian
B
Berger
Hartmut
H
Escher
Emanuel
E
Bienert
Michael
M
Beyermann
Michael
M
D000818:Animals; D001577:Benzophenones; D001665:Binding Sites; D002460:Cell Line; D003346:Corticotropin-Releasing Hormone / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008024:Ligands; D010446:Peptide Fragments / Q000032:analysis; D010649:Phenylalanine / Q000031:analogs & derivatives; D010777:Photochemistry; D025941:Protein Interaction Mapping / Q000379:methods*; D051381:Rats; D018019:Receptors, Corticotropin-Releasing Hormone / Q000737:chemistry*; D014162:Transfection; D054832:Urocortins; D000099052:CRF Receptor, Type 1
C488060:4-benzoylphenylalanine; D001577:Benzophenones; D003432:Cross-Linking Reagents; D008024:Ligands; D010446:Peptide Fragments; D018019:Receptors, Corticotropin-Releasing Hormone; D054832:Urocortins; D010649:Phenylalanine; D000099052:CRF Receptor, Type 1; D003346:Corticotropin-Releasing Hormone