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J Biol Chem. 2007 Dec 28;282(52):37529-36. Epub 2007 Oct 16.

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.

Author information

1
The Clayton Foundation Laboratories for Peptide Biology, La Jolla, CA 92037, USA. perrin@salk.edu

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.

PMID:
17940290
DOI:
10.1074/jbc.M703748200
[Indexed for MEDLINE]
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