The Three-Dimensional Structure of the N-Terminal Domain of Corticotropin-Releasing Factor Receptors

Sushi Domains and the B1 Family of G Protein–Coupled Receptors


Address for correspondence: Marilyn H. Perrin, The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 N. Torrey Pines Road, La Jolla, CA 92037. Voice: 858-453-4100, ext. 1497; fax: 858-552-1546.


Abstract: The corticotropin-releasing factor (CRF) receptors, CRF-R1 and CRF-R2, belong to the B1 subfamily of G protein–coupled Receptors (GPCRs), including receptors for secretin, growth hormone-releasing hormone (GHRH), vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), calcitonin, parathyroid hormone (PTH), glucagon, and glucagon-like peptide-1 (GLP-1). The peptide ligand family comprises CRF, Ucn 1, 2, and 3. CRF plays the major role in integrating the response to stress. Additionally, the ligands exhibit many effects on muscle, pancreas, heart, and the GI, reproductive, and immune systems. CRF-R1 has higher affinity for CRF than does CRF-R2 while both receptors bind Ucn 1 equally. CRF-R2 shows specificity for Ucns 2 and 3. A major binding domain of the CRFRs is the N terminus/first extracellular domain (ECD1). Soluble proteins corresponding to the ECD1s of each receptor bind CRF ligands with nanomolar affinities. Our three-dimentional (3D) nuclear magnetic resonance (NMR) structure of a soluble protein corresponding to the ECD1 of CRF-R2β (1) identified its structural fold as a Sushi domain/short consensus repeat (SCR), stabilized by three disulfide bridges, two tryptophan residues, and an internal salt bridge (Asp65–Arg101). Disruption of the bridge by D65A mutation abrogates ligand recognition and results in loss of the well-defined disulfide pattern and Sushi domain structure. NMR analysis of the ECD1 in complex with astressin identified key amino acids involved in ligand recognition. Mutation of some of these residues in the full-length receptor reduces its affinity for CRF ligands. A structure-based sequence comparison shows conservation of key amino acids in all the B1 subfamily receptors, suggesting a corresponding conservation of a Sushi domain structural fold of their ECD1s.