Distinct Structural and Functional Roles of Conserved Residues in the First Extracellular Domain of Receptors for Corticotropin-releasing Factor and Related G-protein-coupled Receptors

Abstract

The G-protein-coupled receptor B1 family includes corticotropin-releasing factor (CRF), growth hormone-releasing hormone, incretin, and pituitary adenylate cyclase-activating polypeptide receptors. The three-dimensional NMR structure of the first extracellular domain (ECD1) of CRF receptor 2␤ (CRF-R2␤), free and complexed with astressin, comprises a Sushi domain. This domain is stabilized in part by a salt bridge between Asp 65 and Arg 101 . Analogous residues are conserved in other members of the B1 family. To address the importance of the salt bridge residues within this receptor family, we studied the effects of mutating the residues in full-length CRF-R2␤ and isolated ECD1. Mutation D65A or D65R/R101D resulted in loss of the canonical disulfide arrangement, whereas R101A retained the The B1 family of G-protein-coupled receptors (GPCRs) 4 comprises 15 genes in human, including receptors for corticotropin-releasing factor (CRF), growth hormone-releasing hormone (GHRH), vasoactive intestinal peptide (VIP), parathyroid hormone, pituitary adenylate cyclase-activating polypeptide, incretins (GIP and GLP), secretin, calcitonin, and glucagon and are characterized by relatively large first extracellular domains (ECD1s). In particular, the CRF system includes two distinct receptors, CRF-R1 and CRF-R2, as well as the peptide ligands CRF, urocortins (Ucns) 1, 2, and 3 in mammals, and the related peptides, (fish) urotensin and (frog) sauvagine. In addition, high affinity antagonist peptides have been synthesized, most notably astressin, which binds with equally high affinity to both receptors (1). Many studies have shown that the ECD1s of the CRF receptors constitute major ligand-binding sites. Chimeric receptors expressing the ECD1s bind CRF family ligands with high affinity, as do soluble proteins corresponding to the ECD1s (2-9). Studies of other B1 receptors report similar data (10 -16).A significant advance in understanding the structural determinants of ligand recognition for this receptor subfamily follows upon the determination of the three-dimensional NMR structure of the ECD1 of the type 2␤ CRF receptor, both free (17) and in complex with a peptide antagonist, astressin (18). In both forms, the ECD1 folds into a short consensus repeat (19) or Sushi domain, which contains two antiparallel ␤-sheets, three disulfide bonds, and a salt bridge between aspartic acid 65

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

“…6). This model is consistent with the NMR structure of the CRFR2 N-terminal extracellular domain (28), with the functional data showing full binding capacity of the N-terminal extracellular domain (15,28,30,31,40,41,45,48,50), and with the cross-linking data of Bpa-substituted UCN1 at positions 17 and 22, which showed crosslinking to the first extracellular loop (26). However, the model does not fit the cross-linking data of Bpa-substituted at position 0 and 12, which showed cross-linking to the second extracellular loop (26).…”

“…Cross-linking with Bpa-substituted UCN1 analogs showed that a Bpa group located at positions 0 or 12 cross-links to the second extracellular receptor loop, whereas Bpa at positions 17 or 22 cross-links to the first extracellular receptor loop (26); these data suggest that the N terminus of UCN1 binds closer to the CRFR1 extracellular surface. It is also possible that UCN1 binding to CRFR1 expressed in mammalian cells as shown by UCN1 cross-linking (26) is different from the binding of the free N-terminal extracellular domain of CRFR1, which is shown to be both necessary and sufficient for binding N-terminally truncated CRF, sauvagine, and UCN1 based analogs (15,28,30,31,40,41,45,48,50).…”

“…Recently, the extracellular domain of the PTH1R, fused to the bacterial mannose binding protein and bound to PTH (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34), was crystallized (51). The crystal structure showed an interesting pattern with the PTH1R extracellular domain engulfing PTH (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34) as a "hot dog in a bun" (51).…”

“…The extracellular cysteine residues form disulfide bridges that stabilize the structure of the N terminus (15,16). The N-linked glycosylation moieties are important for processing through the Golgi system and cell surface expression (17).…”

Molecular Recognition of Corticotropin-releasing Factor by Its G-protein-coupled Receptor CRFR1

“…The same packing interactions of these conserved residues are observed in all crystal structures of class B GPCR ECDs solved to date (30 -32). We see no evidence for a salt bridge between Arg 85 and Asp 49 as proposed for the equivalent residues in the mCRFR2␤ ECD (29,47).…”