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 2beta (CRF-R2beta), 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-R2beta and isolated ECD1. Mutation D65A or D65R/R101D resulted in loss of the canonical disulfide arrangement, whereas R101A retained the Cys(4)-Cys(6) disulfide bond. The mutations resulted in misfolding within the ECD1 as determined by NMR and 1-anilino-8-naphthalenesulfonate binding but did not prevent cell surface expression. The D65A mutation in CRF-R2beta greatly reduced binding and activation, but the R101A substitution had only a small effect. Similar effects were seen on astressin binding to the ECD1. The different interactions of Asp(65) and Arg(101), deduced from the three-dimensional structure of the complex, are consistent with the differential effects seen in the mutants. The reduction in binding of Asp(65) mutants is a consequence of a distinct Asp(65)-Trp(71) interaction, which stabilizes the ligand-binding loop. Hence, loss of the salt bridge leads to disruption of the overall fold but does not abolish function. Because homologous mutations in other B1 receptors produce similar effects, these conserved residues may play similar roles in the entire receptor family.
Highlights
The overall fold but does not abolish function
To gain further understanding of the role of the amino acids in the salt bridge, we have studied the structure, binding, and signaling capabilities of a series of mutant corticotropin-releasing factor (CRF)-R2s in which the salt bridge is either disrupted or “reversed.” we have characterized the corresponding mutants in the isolated ECD1
Structural Characterization of the Salt Bridge Asp65–Arg101— Fig. 1 shows a schematic representation of the NMR core structure of ECD1 from CRF-R2 as determined by Grace et al [18], highlighting the salt bridge formed by Asp65 and Arg101 and the hydrogen bond between Asp65 and Trp71
Summary
The overall fold but does not abolish function. Because homologous mutations in other B1 receptors produce similar effects, these conserved residues may play similar roles in the entire receptor family. 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. To gain further understanding of the role of the amino acids in the salt bridge, we have studied the structure, binding, and signaling capabilities of a series of mutant CRF-R2s in which the salt bridge is either disrupted or “reversed.” we have characterized the corresponding mutants in the isolated ECD1 These studies shed light on the structural and functional roles played by the two highly conserved amino acid residues involved in the salt bridge of the CRF receptor system and in the B1 subfamily of GPCRs
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