Perrin Marilyn H, Grace Christy R R, Digruccio Michael R, Fischer Wolfgang H, Maji Samir K, Cantle Jeffrey P, Smith Sean, Manning Gerard, Vale Wylie W, Riek Roland
The Clayton Foundation Laboratories for Peptide Biology, La Jolla, CA 92037, USA.
J Biol Chem. 2007 Dec 28;282(52):37529-36. doi: 10.1074/jbc.M703748200. Epub 2007 Oct 16.
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.
G蛋白偶联受体B1家族包括促肾上腺皮质激素释放因子(CRF)、生长激素释放激素、肠促胰岛素和垂体腺苷酸环化酶激活多肽受体。CRF受体2β(CRF-R2β)的第一个细胞外结构域(ECD1)在游离状态以及与抗应激素结合的复合物状态下的三维核磁共振结构包含一个寿司结构域。该结构域部分通过Asp(65)和Arg(101)之间的盐桥得以稳定。在B1家族的其他成员中,类似的残基是保守的。为了探究该受体家族中盐桥残基的重要性,我们研究了在全长CRF-R2β和分离的ECD1中突变这些残基的影响。突变D65A或D65R/R101D导致典型二硫键排列的丧失,而R101A保留了Cys(4)-Cys(6)二硫键。如通过核磁共振和1-苯胺基-8-萘磺酸盐结合所确定的,这些突变导致ECD1内错误折叠,但并未阻止细胞表面表达。CRF-R2β中的D65A突变极大地降低了结合和激活能力,但R101A替换仅有微小影响。在抗应激素与ECD1的结合上也观察到了类似效应。从复合物的三维结构推断出的Asp(65)和Arg(101)的不同相互作用,与在突变体中看到的差异效应一致。Asp(65)突变体结合能力的降低是独特的Asp(65)-Trp(71)相互作用的结果,该相互作用稳定了配体结合环。因此,盐桥的丧失导致整体折叠的破坏,但并未消除功能。由于其他B1受体中的同源突变产生类似效应,这些保守残基可能在整个受体家族中发挥类似作用。