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

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-dimensional (3D) nuclear magnetic resonance (NMR) structure of a soluble protein corresponding to the ECD1 of CRF-R2beta (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.

Exploring the Binding Site Crevice of a Family B G Protein-Coupled Receptor, the Type 1 Corticotropin Releasing Factor Receptor

“…Another possible explanation could be that MTSEA reacts with a CRF 1 -associated protein to allosterically inhibit radioligand binding to CRF 1 . Curiously, the amino-terminal extracellular region of CRF receptors, which is essential for radioligand binding, forms a Sushi domain, which has been implicated in protein-protein interactions (Perrin et al, 2006). Substitution of serine for Cys211, Cys233, or Cys364 rendered the receptor significantly less sensitive to MTSEA.…”

Residue 17 of Sauvagine Cross-links to the First Transmembrane Domain of Corticotropin-releasing Factor Receptor 1 (CRFR1)

“…To identify the cross-linked residue we developed a method for radiosequencing of the CNBr-cleaved CRFR1 bearing Met substitution at position 114 photo-cross-linked to 125 50 for stimulation of cAMP accumulation were 8 and 1 nM, respectively (Fig. 5A).…”

International Union of Basic and Clinical Pharmacology. XCIII. The Parathyroid Hormone Receptors—Family B G Protein–Coupled Receptors

“…The ECD was produced in Escherichia coli and purified under conditions that promoted disulfide bond formation; this measure was taken because it was clear from prior mutational studies, as well from the sequence conservation patterns, that the six conserved cysteines in the ECD play a critical role in receptor function and were thought to likely form an intramolecular disulfide bond network that stabilizes the bioactive fold (Lee et al, 1994). In addition to the PTHR1, X-ray crystallography or NMR approaches have been used to obtain three-dimensional structures for the isolated ECD regions of several other family B GPCRs, including the gastric-inhibitory polypeptide receptor (Parthier et al, 2007), the CRFR1 (Grace et al, 2004(Grace et al, , 2007, the CRFR2 (Perrin et al, 2006;Pal et al, 2010), and the pituitary adenylate cyclase-activating polypeptide receptor (Kumar et al, 2011). A common protein fold for the ECD is observed for each of these ECDs, and the six conserved cysteines indeed form an intramolecular disulfide bond network that maintains the overall structural fold.…”