Kitanovic S, Yuen T, Flanagan C A, Ebersole B J, Sealfon S C
Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine New York, New York 10029, USA.
Mol Endocrinol. 2001 Mar;15(3):390-7. doi: 10.1210/mend.15.3.0611.
The pattern of side-chain conservation at the cytoplasmic side of the third transmembrane domain of rhodopsin family G protein-coupled receptors, Asp/Glu-Arg-Tyr/X-X-X-Ile/Val, defines a structural "arginine cage" domain. Previous computational and mutagenesis studies of the GnRH receptor indicated an important contribution of local interactions to the function of this domain. We have investigated the functional importance of the intrahelical position and orientation of the arginine cage using insertional mutagenesis. Introduction of a single Ala proximal to the conserved Asp-Arg of this domain caused loss of detectable ligand binding. Inserting a second Ala, however, restored high-affinity agonist binding. Further insertion of three or four Ala residues at this site generated receptors that bound agonist with an affinity 3- to 10-fold higher than that of the wild-type receptor. Loss of detectable coupling to inositol phosphate turnover in all these mutant receptors confirms that the structure required in this region for efficient signaling is highly constrained. In contrast, the recovery of agonist binding with the progressive insertion of two to four Ala residues indicates that specific orientations of this segment can stabilize high-affinity receptor conformations that are uncoupled from signal transduction.
视紫红质家族G蛋白偶联受体第三个跨膜结构域胞质侧的侧链保守模式，即天冬氨酸/谷氨酸-精氨酸-酪氨酸/ X-X-X-异亮氨酸/缬氨酸，定义了一个结构上的“精氨酸笼”结构域。先前对促性腺激素释放激素（GnRH）受体的计算和诱变研究表明，局部相互作用对该结构域的功能有重要贡献。我们使用插入诱变研究了精氨酸笼的螺旋内位置和取向的功能重要性。在该结构域保守的天冬氨酸-精氨酸附近引入单个丙氨酸会导致可检测到的配体结合丧失。然而，插入第二个丙氨酸可恢复高亲和力激动剂结合。在此位点进一步插入三个或四个丙氨酸残基产生的受体，其结合激动剂的亲和力比野生型受体高3至10倍。所有这些突变受体中与肌醇磷酸周转的可检测偶联丧失，证实了该区域有效信号传导所需的结构受到高度限制。相反，随着两到四个丙氨酸残基的逐步插入，激动剂结合得以恢复，这表明该片段的特定取向可以稳定与信号转导解偶联的高亲和力受体构象。
