Labrou N E, Bhogal N, Hurrell C R, Findlay J B
School of Biochemistry & Molecular Biology, The University of Leeds, Leeds, LS2 9JT United Kingdom.
J Biol Chem. 2001 Oct 12;276(41):37944-9. doi: 10.1074/jbc.M106330200. Epub 2001 Aug 6.
We report the use of thiol chemistry to define specific and reversible disulfide interactions of Cys-substituted NK2 receptor mutants with analogues of neurokinin A (NKA) containing single cysteine substitutions. The NKA analogues were N-biotinylated to facilitate the rapid detection of covalent analogue-receptor interactions utilizing streptavidin reactivity. N-biotinyl-[Tyr1,Cys9]NKA, N-biotinyl-[Tyr1,Cys10]NKA were both found to reversibly disulfide bond to the NK2 receptor mutant Met297 --> Cys. This is consistent with the improved affinities of these particular analogues for the Met297 --> Cys receptor as compared with those for the wild-type and Met297 --> Leu receptors. In our three-dimensional model, Met297 occupies the equivalent position in helix 7 to the retinal binding Lys296 in rhodopsin. Binding of the NK2 receptor antagonist [3H]SR 48968 and of 125I-NKA was used to characterize additional receptor mutants. It seems that the aromatic residues Trp99 (helix 3), His198 (helix 5), Tyr266, His267, and Phe270 play an important role in NKA binding as structural determinants. The existence of overlapping SR 48968 and NKA binding sites is also evident. These data suggest that the peptide binding site of the NK2R is at least in part formed by residues buried deep within the transmembrane bundle and that this intramembranous binding domain may correspond to the binding sites for substantially smaller endogenous GPCR ligands.
我们报道了利用硫醇化学来定义半胱氨酸取代的NK2受体突变体与含有单个半胱氨酸取代的神经激肽A(NKA)类似物之间特定且可逆的二硫键相互作用。将NKA类似物进行N-生物素化,以利用链霉亲和素反应性促进对共价类似物-受体相互作用的快速检测。发现N-生物素基-[Tyr1,Cys9]NKA、N-生物素基-[Tyr1,Cys10]NKA均与NK2受体突变体Met297→Cys形成可逆的二硫键。这与这些特定类似物对Met297→Cys受体的亲和力相较于对野生型和Met297→Leu受体的亲和力有所提高是一致的。在我们的三维模型中,Met297在螺旋7中占据的位置与视紫红质中视网膜结合的Lys296相当。使用NK2受体拮抗剂[3H]SR 48968和125I-NKA的结合来表征其他受体突变体。似乎芳香族残基Trp99(螺旋3)、His198(螺旋5)、Tyr266、His267和Phe270作为结构决定因素在NKA结合中起重要作用。SR 48968和NKA结合位点重叠的存在也很明显。这些数据表明,NK2R的肽结合位点至少部分由深埋在跨膜束中的残基形成,并且这种膜内结合域可能对应于实质上更小的内源性GPCR配体的结合位点。